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Structure-function studies of murine gamma interferon

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Title:
Structure-function studies of murine gamma interferon a synthetic peptide and antibody approach
Alternate title:
Synthetic peptide and antibody approach
Creator:
Jarpe, Michael Andrew, 1962-
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English
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v, 89 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Amino acids ( jstor )
Antibodies ( jstor )
Epitopes ( jstor )
Interferons ( jstor )
Molecules ( jstor )
Monoclonal antibodies ( jstor )
Proteins ( jstor )
Receptors ( jstor )
Truncation ( jstor )
Tumors ( jstor )
Amino Acid Sequence ( mesh )
Antibodies, Monoclonal -- physiology ( mesh )
Department of Pathology and Laboratory Medicine thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pathology and Laboratory Medicine -- UF ( mesh )
Interferon Type II -- physiology ( mesh )
Molecular Sequence Data ( mesh )
Muridae ( mesh )
Peptides -- physiology ( mesh )
Research ( mesh )
Structure-Activity Relationship ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 76-88.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Michael Andrew Jarpe.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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STRUCTURE-FUNCTION STUDIES OF MURINE GAMMA INTERFERON: A
SYNTHETIC PEPTIDE AND ANTIBODY APPROACH













BY


MICHAEL ANDREW JARPE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1990




STRUCTURE-FUNCTION STUDIES OF MURINE GAMMA INTERFERON: A
SYNTHETIC PEPTIDE AND ANTIBODY APPROACH
BY
MICHAEL ANDREW JARPE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990


ACKNOWLEDGEMENTS
I wish to acknowledge the following individuals who were
instrumental in helping me at some point in my life as a
student. First of all, thanks go to Howard for his patience
and scientific philosophy and for not getting sick of my face
before this was complete. Thanks belong to my committee
members, John Dankert, Paul Hargrave, Lindsey Hutt-Fletcher,
and Brough Peck, for their helpful guidance through this long
process called graduate school. I credit Allen G. Harmsen
for giving me my first taste of science. I thank my parents,
Jay and Marion, for bringing me up the way they did and my
bothers and sister for not killing me on long car trips.
Thank go to Myron, Carol, Sarah, Jeff, Mark, Rob, Jeanne,
Russell, Lori, Steve, Steve, and Doug, my comrades in arms,
who have helped me along the way by calming me down when
things got too serious or livening things up when they became
boring. And finally, I thank my wife, Alyssa, for without
her love and support (financial and otherwise) this whole
ordeal would not have been worth it.
ii


TABLE OF CONTENTS
ease
ACKNOWLEDGEMENTS ii
ABSTRACT iv
CHAPTERS
1. INTRODUCTION
Interferons: Historical Perspective 1
IFN-y Function 5
Structure/Function Studies of IFN-y 8
The Synthetic Peptide Approach 11
Summary and Objective 14
2. STRUCTURE OF AN EPITOPE IN A REGION OF THE IFN-y
MOLECULE THAT IS INVOLVED IN RECEPTOR
INTERACTION
Introduction 16
Material and Methods 17
Results and Discussion 24
3. TOPOLOGY OF RECEPTOR BINDING DOMAINS OF MOUSE IFN-y
THAT INCLUDE THE N-AND C-TERMINI.
Introduction 4 6
Materials and Methods 47
Results and Discussion 52
4. SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS 71
LIST OF REFERENCES 7 6
BIOGRAPHICAL SKETCH 89
iii


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STRUCTURE/FUNCTION STUDIES OF MURINE GAMMA INTERFERON: A
SYNTHETIC PEPTIDE AND ANTIBODY APPROACH
By
Michael Andrew Jarpe
August, 1990
Chairman: Howard M. Johnson
Major Department: Pathology and Laboratory Medicine
Gamma Interferon (IFN-y) is an important
immunoregulatory lymphokine that affects a wide variety of
immune functions. I have undertaken studies to elucidate the
structural basis for IFN-y function. These
structure/function studies of IFN-y have yielded data on the
location of functional domains.
A monoclonal antibody (mAb) specific for the N-terminus
of mouse IFN-y was shown to neutralize IFN-y function by
blocking receptor binding. I mapped the epitope specificity
of this monoclonal antibody to residues 3 through 14.
Additionally, residues at positions 3, 4, and 5, are
particularly important in the structure of the epitope, since
peptides that lacked these residues had reduced ability to
interact with the mAb. Furthermore, peptide analogs that
iv


replaced these residues with nonconservative substitutions
lost antibody binding activity. Tyrosine in position 14 was
also found to be critical, since peptides lacking it also
lost binding activity. The epitope specificity for this mAb
encompasses the 12 residues between 3 and 14 and is linear
and discontinuous. Because this mAb blocks IFN-y receptor
binding, this epitope may be involved in receptor
interaction.
Antibodies raised against five overlapping synthetic
peptides that encompass the entire sequence of mouse IFN-y
were tested for their ability to inhibit IFN-y function.
Only antisera raised against the N-terminal and the C-
terminal peptides could neutralize IFN-y by blocking receptor
binding. Further mapping studies showed that the C-terminal
specific antiserum was directed to several neutralizing
epitopes within the region. The antibody neutralization data
suggest that IFN-y contains two binding domains, one in the
N-terminus and one in the C-terminus. A three dimensional
model of IFN-y has been constructed utilizing both functional
data, predictive algorithms for secondary and tertiary
structure, and comparison to the known structure of IL-2.
The molecule is predicted to form six a-helices divided by
five turns and to form a four-helix bundle motif. The N-
terminus and C-terminus form two receptor binding domains and
are found close together. This model provides a working base
for future studies.
v


CHAPTER 1
INTRODUCTION
Interferons: Historical Perspective
Interferon (IFN) was first discovered in 1957 by Isaacs
and Lindenmann (1) as a substance that had the ability to
protect host cells against viral infection. The term
interferon has subsequently come to describe a class of
proteins that fall into three distinct groups designated as
IFN-a, IFN-P, and IFN-y. IFN-a and IFN-fJ are classically
produced in cells induced by viruses or polyribonucleotides
(2). IFN-y is classically produced by T-lymphocytes and
natural killer (NK) cells in response to antigen or mitogen
stimulation (2).
Genes for all three classes of IFNs have been cloned and
sequenced. IFN-a and IFN~P have about 29% sequence homology
while IFN-y has little homology to IFN-a or IFN~p. IFN-a and
IFN~P have been shown to exist in most vertebrates; however,
IFN-y has been shown to exist only in mammals (3). IFN-a is
actually a family of 23 genes each without introns and 15 of
these genes encode for full length proteins of between 165
and 172 amino acid residues (4). There are three IFN~P
genes, one without introns coding for a protein of 166 amino
acid residues and two with four introns each (5-10). The
1


2
genes containing introns are called IFN-J32 or IL-6 (5) IFN-
(32 has been shown to be a B-cell differentiation factor
(originally called BSF-2) that can induce B-cell
proliferation and enhance antibody production (6,8).
Although IFN-p2 activity can be neutralized by antiserum to
IFN-{3, IFN-f}2 bears little homology to the other IFN-fi
molecules and shows little antiviral activity, therefore its
classification as an IFN is debatable (11).
IFN-y has been cloned in human (12,13), mouse (14), rat
(15), and bovine (16) and is encoded by a gene with three
introns and it appears that there is only one IFN-ygene
present in the genome. A strong species specificity exists
with IFN-y (11). Table 1-1 illustrates the amino acid
sequence homology between the sequences of the four known
IFN-y proteins from human, cattle, rat and mouse. The
sequence analysis was performed on a microVAX using sequence
data from the GenBank database and the program GAP. GAP
aligns sequences and produces a best fit by inserting gaps
within the sequences. The program reports the percentage of
residues that are identical between sequences and also
reports the percentage similarity. Similarity is defined as
the percentage of identical residues plus the percentage of
residues that are conservative substitutions. In most of the
comparisons the percentage identity is low. For example,
when human IFN-y and mouse IFN-y are compared, there is a 40%
identity (Table I). However, the percentage similarity is
67%. Sixty-seven percent similarity indicates there are many


3
conservative amino acid substitutions. This same trend
appears in all of the comparisons. The lack of homology may
account for the species specificity. Based on the degree of
similarity, it is likely that the proteins are structurally
similar. This suggests that information concerning the
location of functional domains in one species may be
applicable to other species.
A receptor for IFN-y has been identified, cloned and
sequenced in both human and mouse (17-21) and shown to exist
on chromosome six in the human (22). The deduced amino acid
sequence yields a protein of 451 and 472 amino acids for
mouse and human, respectively (17-21). There is about 55%
overall homology between the mouse and human proteins (18-
21). A predicted transmembrane domain divides the protein in
half and both domains bear no appreciable homology to any
known receptors (17-21). The external domain comprises the
binding domain and when expressed on the surface of a cell
exhibits identical binding characteristics as the native
receptor which suggest that there is only one binding protein
for IFN-y (17-21). The expressed protein has no function
when IFN-y binds to it (17-21). However, if the short arm of
human chromosome 22 is present in hamster/human hybrid cells
in addition to the receptor, function is restored, suggesting
that there is some other factor required for functional
activity in addition to binding of IFN-y (23) The
requirements of the receptor for binding and the requirements


4
Table 1-1. Amino acid sequence homology between known IFN-y
proteins from different species.
Species
Human
Bovine
Rat
Bovine
62(76)
-
-
Rat
37(60)
45(68)
-
Mouse
40(67)
43(67)
86(95)
Values are percent
amino acid homology.
Values in parentheses
are
percent homology plus percent conservative substitutions.


5
of other as yet undiscovered factors for receptor function
need further study.
IfN-y Function
IFN-y is a glycoprotein of approximately 20,000 daltons
that possesses many diverse activities. IFN-y was
classically described as a product of white blood cells
stimulated with phytohaemagglutinin, a T-cell mitogen, that
possessed antiviral activity (24). Later, this product was
shown to be different from the type I interferons (IFN-a and
IFN-f}) in its instability at pH 2 (24) IFN-y was also found
to be antigenically different from type I IFNs (25). These
characteristics, in part, classify it as type II IFN or
immune IFN. The observation that IFN had immunomodulatory
activities was initially made with IFN-a and IFN-[} when it
was shown that they had a suppressive effect on antibody
production (26-31). IFN was also shown to regulate delayed
type hypersensitivity (32-36), cellular immunity (32-36), and
natural killer cytotoxic activity (37-40). IFN-y was shown
to possess immunomodulatory activities, some in common to and
others unique from IFN-a and IFN-p (41). In fact, the
immunoregulatory potency of IFN-y is much greater than that
of IFN-a and IFN-p (42) .
IFN-y has the ability to modulate antibody production by
B lymphocytes both positively and negatively (43-49). IFN-y
was shown to suppress in vitro antibody response, in mice,


6
before clonal expansion of B-cells had occurred (44,45,47).
Conversely, IFN-y had a positive effect when added to B-
lymphocytes that had already undergone clonal expansion by
inducing antibody secretion and terminal differentiation
(43, 46, 48,49) .
The increased expression of a variety of cell surface
molecules can be achieved with IFN-y. IFN-y can increase the
expression of interleukin-2 receptors on lymphocytes (50).
IFN-y can increase surface expression of Fc receptors on
macrophages which are involved in antibody binding and
antibody dependent cell cytotoxicity (51,52). Class-I major
histocompatibilty antigen expression is increased by IFN-y
(53,54). Class-I expression is required for T-cell
recognition for the killing of virally infected cells.
Class-II molecule expression, which is critical for antigen
presentation and the induction of an immune response, is also
increased by IFN-y (55,56).
Another immunomodulatory function of IFN-y, and possibly
one that has received the most attention, is the activation
of a variety of cytotoxic cells that have the ability to kill
tumor cells. Both NK cell activity and cytotoxic T-cell
activity are increased by IFN-y (37-40,57). The tumoricidal
activity of macrophages has been shown to be augmented by
factor(s) liberated from antigen or mitogen stimulated
lymphocytes and has been termed macrophage activating factor
or MAF (58-62). The process of activation actually occurs in
a series of steps, beginning with priming of macrophages by


7
MAF then subsequent triggering by a small amount of
lipopolysaccharide (LPS) (60,61) Macrophages so treated
have a greatly enhanced ability to kill tumor cells in vitro.
MAF activity has been shown to be mainly, if not exclusively,
due to IFN-y (59,60,63). Recombinant or purified natural
IFN-y has potent MAF activity, and IFN-y neutralizing
antibodies can remove MAF activity completely from MAF
containing cultures (60, 63). Both IFN-a and IFN-|3 have MAF
activity (58) although they are much less potent when
compared with IFN-y (64).
The in vivo properties of IFN-y have been studied
primarily in the context of its potential anti-cancer
properties. Studies have shown, at least in experimental
animal systems, that IFN-y has a direct role in host tumor
defense (65,66). In a mouse tumor model where tumors can be
implanted that either progress or spontaneously regress when
the dose of tumor cells is varied, higher levels of IFN-y
were observed in regressing tumors (65). Additionally, when
an IFN-y neutralizing monoclonal antibody (mAb) was injected
intraperitoneally into mice that possessed regressing tumors,
the tumors were converted to progressing tumors. A non
neutralizing mAb that bound IFN-y but did not neutralize its
function had no effect on tumor growth. Others have shown
similar results using direct tumor injection of IFN-y
neutralizing mAbs in similar tumor models (66). These
finding suggest that IFN-y may be functioning in host tumor
defense under normal circumstances, but when administered in


8
clinical trials, IFN-y seems to be ineffective in treating
tumors (67-74). The reason for its ineffectivity is unknown.
Structure/Function Studies of IFN-y
When the genes for human and mouse IFN-y were cloned in
the early 1980s and the primary sequence was determined, it
was thought that an understanding of the mechanism for IFN-y
function would soon follow. To the contrary, the
structure/function relationship of IFN-y has proven to be
more complicated than first thought. Studies designed to
explore this area have involved the use of a variety of
techniques and can be divided into approaches using molecular
biology, antibodies, synthetic peptides, and combinations
thereof. Some of these studies have yielded conflicting
results.
Some studies have shown that IFN-y may exist as
homodimer (75-77) or larger multimer (78); however, the exact
role of these multimer structures is unclear. It has been
shown that IFN-y isolated in its monomer form has activity
(75). Recombinant IFN-y expressed in E. coli lacks
glycosylation but retains full activity (12,14) which shows
that glycosylation is not required for IFN-y activity. It
has been postulated that glycosylation of IFN-y provides
protection of the mature protein against proteolysis (75).
Initially, the amino (N) terminus of IFN-y was thought to be
the amino acid sequence CYS-TYR-CYS which left the


9
possibility of disulfide bond formation (12,14). This
possibility was rejected when N-terminal sequencing of human
natural IFN-y showed that the N-terminus was pyroglutamic
acid (75). Therefore, no disulfide bonding occurs in IFN-y.
Structure/function studies have been carried out with
IFN-y using truncated molecules generated through mutation of
cDNA clones or through limited proteolysis of recombinant
IFN-y (79-83). Human and mouse IFN-y lacking the N-terminal
eight or nine amino acid residues have been shown to have
significantly reduced functional activity and reduced
receptor binding activity (79-80). This abrogation of
activity in human IFN-y was shown to correlate to a
significant loss in a-helical structure (80). Recombinant
human IFN-y lacking portions of its C-terminus had either a
significant reduction in activity (81,83) or no change in
activity (82). Human recombinant IFN-y treated with
proteases that removed 11 (81) or 13 (83) residues from the
C-terminus showed a significant decrease in IFN-y function
and receptor binding activity. Conversely, a mutant
recombinant IFN-y from E. coli that lacked the 23 C-terminal
residues retained full activity (82). The reason for this
discrepancy remains unclear and may have something to do with
proteolytic attack altering more than just the C-terminus,
although in each proteolytic experiment N-terminal sequencing
confirmed that the N-terminus was not altered (81,83).
The use of antibodies, both polyclonal and monoclonal,
to probe for functional sites along the IFN-y molecule has


10
proven useful in the study of the structure/function
relationship of IFN-y. Most of these studies hint at a two
or multiple functional domain model of IFN-y; however,
controversy surrounds the issue of where these domains lie.
Some evidence exists that suggests that there are different
functional domains for different IFN-y functions such as
antiviral activity and macrophage priming activity (84);
however, this has yet to be confirmed. One study using 21
neutralizing mAb to human IFN-y supports a multi-domain model
(85). These mAb were divided into two groups using antibody-
antibody competition assays with each group having a
different epitope specificity, suggesting more than one
functional domain. The location of these epitopes is unknown
as they have not been mapped. Other studies have either
localized mAb epitopes or raised site directed antibodies to
synthetic peptides of the IFN-y molecule. Antibodies raised
against a synthetic peptide corresponding to the first 20
residues of human IFN-y (the first three residues were CYS-
TYR-CYS) neutralized IFN-y activity, suggesting that the N-
terminal region of IFN-y was involved in function (86). A
polyclonal antiserum raised against a synthetic peptide
corresponding to the first 39 residues of mouse IFN-y, IFN-
y(l-39), neutralized IFN-y activity by blocking IFN-y
receptor binding (87). Additionally, the epitope specificity
of a mAb shown to neutralize mouse IFN-y by blocking receptor
binding was mapped preliminarily to residues 1-20 of the N-
terminus of the molecule (88), supporting the role of the N-


11
terminus as a functional domain. Others have demonstrated
that two mAb specific for the N-terminus of mouse IFN-y also
can neutralize IFN-y activity (84). In a more direct
experiment the synthetic peptide IFN-y(1-39) was able to
block directly IFN-y function and compete specifically with
IFN-y for it receptor (88). Thus, the role of the N-terminus
in receptor binding seems clear.
Polyclonal antiserum raised against the C-terminal
peptide of mouse IFN-y, IFN-y(95-133), blocks IFN-y function
and receptor binding (87). The peptide, IFN-y(95-133) did
not compete with IFN-y directly (88). Two C-terminal
specific mAb can also neutralize mouse IFN-y activity (84).
In contrast, studies have shown that mAb specific for the C-
terminus of human IFN-y are unable to neutralize IFN-y
activity and have concluded that the C-terminus is not
important (89). However, the epitope specificities of these
mAb have been directed to the last 15 residues of IFN-y and
may have missed an important functional site.
The Synthetic Peptide Approach
The synthetic peptide approach has generated much
information with regard to the location of functional domains
on a number of different proteins. This approach can be
divided into three related methods that utilize synthetic
peptides to probe for functional domains. The first method
involves the use of synthetic peptides to directly interact


12
with receptors or other molecules. Synthetic peptides
corresponding to portions of ligands have been shown to
compete directly with the larger intact molecule, thereby
identifying a region critical for receptor interaction. A
synthetic peptide corresponding to residues 400-411 of gamma
chain human fibrinogen was shown to bind to fibrinogen
receptor on platelets and block fibrinogen binding to its
receptor (90). A site of interaction on the superantigen
staphylococcal enterotoxin A (SEA) to class II molecules was
found to be contained within the first 45 residues of SEA
using direct peptide competition (91). These examples
illustrate that synthetic peptides can act as direct probes
for functional domains on proteins.
A second facet of the synthetic peptide approach is the
use of synthetic peptides to map the epitope specificity of
mAb that have been raised to an intact protein. Monoclonal
antibodies that have been shown to interfere with the
activity of a molecule are presumably binding to a region on
the molecule that is important for its activity. Synthetic
peptides can then be used to determine the epitope
specificity of the mAb and thereby identify an important
functional region. An example of this method is the
determination of a functional site on the interleukin-2 (IL-
2) molecule (92). Monoclonal antibodies raised against IL-2
were shown to inhibit its function and were mapped to the
between residues 8-54 of the molecule using synthetic
peptides.


13
A third part of the synthetic peptide approach involves
the use of synthetic peptides to produce site-specific
antibodies to regions of proteins. These antibodies, both
polyclonal and monoclonal, can interact with the native
protein and block its activity by interfering with an
important domain. This technique has been successful in a
number of different systems. In the study of IFN-y, antisera
raised to a synthetic peptide corresponding to the N-terminal
20 residues of human IFN-y neutralized IFN-y activity (86) .
Polyclonal antibodies produced to a peptide from the 15 N-
terminal residues of human tumor necrosis factor neutralized
its activity (93). Antibodies raised to synthetic peptides
of erythropoietin located a functional domain in the region
between residues 99 and 129 (94).
As described, all of these uses of the synthetic peptide
approach have been successful at locating functional sites on
proteins. The interpretation of the results obtained from
these studies is important. Studies where synthetic peptides
can directly interact with receptors are easily interpreted.
The peptide contains all or part of a functional domain of
the protein. The identification of a synthetic peptide with
these properties is not always possible. Some peptides do
not adopt sufficient conformation in solution to be able to
react with receptors. Our laboratory has found that longer
peptides have a greater likelihood of possessing stable
secondary structure and a greater chance of direct
interaction. When direct peptide competition is not found,


14
the use of antibodies as probes for functional domains can be
successful. The antibody molecule, by binding to a region
critical for activity, can interfere with the function of the
protein studied. One important drawback to this approach is
the possibility that the antibody may be inducing a
conformational change in the molecule that disrupts a domain
in a distant region. Another is the possibility that the
antibody may only bind to a subset of denatured molecules.
One way to minimize these potential problems is to use a
combination of the approaches discussed. This would increase
the likelihood of identifying a true functional domain.
Summary and Objective
At present, the state of knowledge concerning IFN-y is
one in which a large amount of information has been generated
about it but there remain questions concerning the number,
location, and characterization of functional domains. From
earlier work with molecular biology, antibodies, and
synthetic peptides there appears to be at least two distinct
functional domains of IFN-y. These domains are believed to
be located in the N-terminus and the C-terminus of the
molecule. However, little is known about the functional role
of the internal regions of the sequence. Studies with site
directed mutation of human IFN-y provide some suggestive
evidence that the internal region is critical to IFN-y
function, at least by being involved in the maintenance of


15
tertiary structure (95). Single amino acid substitutions in
the core of the molecule reduced receptor binding and
structure. Also, little has been done to further
characterize these potential domains with regard to precise
mapping of important regions.
It is the objective of this dissertation to further the
understanding of the structure/function properties of IFN-y
by attempting to identify other functional domains and by
further characterization of those that are known. This will
be accomplished through two main specific aims. First, the
N-terminal functional domain will be further characterized by
mapping the precise epitope specificity of an N-terminal
specific mAb that neutralizes IFN-y by blocking receptor
binding. Second, other regions of the molecule will be
explored for functional significance using long overlapping
synthetic peptides encompassing the entire sequence of IFN-y
and antiserum raised to these peptides.


CHAPTER 2
STRUCTURE OF AN EPITOPE IN A REGION OF THE IFN-y MOLECULE
THAT IS INVOLVED IN RECEPTOR INTERACTION
Introduction
The N-terminal region of IFN-y has been shown to contain
a binding site for the IFN-y receptor based on specific
blockage of IFN-y receptor binding by a synthetic peptide
corresponding to the first 39 residues of IFN-y and based on
blockage of IFN-y function by the N-terminal 39 residues as
well as the first 20 residues of IFN-y (88) A monoclonal
antibody has been produced that has been shown to neutralize
IFN-y function (88). This mAb neutralizes IFN-y activity by
blocking IFN-y binding to its receptor (88). This suggests
that this mAb binds to a functional site on the IFN-y
molecule that is involved in receptor interaction. The
epitope to which this mAb binds is partially mapped to the
first 20 residues of IFN-y using synthetic peptides as
competitors for binding of radiolabeled IFN-y to the mAb
(88). Interestingly, this region of IFN-y is predicted to
contain an amphipathic a-helix between positions three and
eleven. Amphipathic helices are thought to be involved in
immunodominance of antigens related to B-cell and T-cell
recognition (96,97).
16


17
The first specific aim of this dissertation is to
characterize the epitope specificity of this mAb in more
detail. Competition studies with peptide truncations
determined that residues 3,4, and 5 of IFN-y were required
for binding to the mAb. These residues are predicted to
participate in an amphipathic a-helix spanning residues 3-11
of IFN-y. The tyrosine at position 14 was also required as
its removal in C-terminal truncations caused the loss of
blocking ability. It can be concluded that the IFN-y epitope
for the neutralizing mAb involves residues 3-14, spanning 12
residues, and it appears that residues 3, 4, 5, and 14 are an
important part of the epitope and that this epitope can be
considered linear and discontinuous. The data presented here
provide further insight to the structure of a site that is
involved in receptor interaction.
Material .and Methods
Synthetic peptides. Peptides were synthesized with a
Biosearch 9500AT automated peptide synthesizer using FMOC
chemistry (98). Specific analogs were partially constructed
using a manual FMOC synthesis system (RaMPS, Dupont).
Peptides were assembled to a given point in the sequence with
the automated synthesizer. The peptide bearing resin was
then divided and individual analogs completed with the manual
system. Peptides were cleaved at 25 C from the resins using
trifluoroacetic acid/phenol/ethanedithiol at a volume ratio


18
of 95.0/4.5/0.5, respectively. The cleaved peptide was then
precipitated in ether and ethyl acetate and subsequently
dissolved in water and lyophilized. Reverse phase HPLC
analysis of crude peptides indicated one major peak in each
profile. Synthetic peptides were submitted to the Protein
Core Facility at the University of Florida for amino acid
analysis. Figures 2-1 through 2-4 are HPLC profiles and
amino acid analyses of the crude peptides used in these
studies.
Radioiodination of recombinant IFN-y. Recombinant
murine IFN-y was obtained from Schering-Plough Pharmaceutical
(Bloomfield, NJ) with a specific activity of 5 x 10^ units
per mg of protein. A unit of IFN-y activity is defined as
the concentration of IFN-y that reduces by 50% the plaque
formation of vesicular stomatitis virus on mouse L cells
(99). IFN-y was labeled with 125j by using chloramine T as
described (87). The specific activity of ^-25j_jpN-y was
generally 25-35 JiCi/ |lg protein (ICi = 37 GBq) .
MAb to IFN-y. MAb 5.102.12 was previously produced
(87) from the fusion of spleen cells from an Armenian hamster
immunized with IFN-y. Tissue culture supernatants containing
mAb were applied to a protein-A sepharose column. Antibody
was eluted with 0.58% acetic acid in 0.15 M NaCl. Eluted
fractions were immediately neutralized with one-tenth volume
of 1.5 M tris-HCl, pH 8.8, and dialyzed against phosphate


19
Amino acid
analysis
Asx
1.05
Thr
0.94
Ser
0.89
Glx
0.99
Gly
0.93
Val
0.70
lie
0.84
Leu
1.00
Tyr
0.97
Phe
0.95
His
0.94
Figure 2-1. HPLC profile and amino acid analysis of IFN-
y(l-20). Amino acid analysis values are the ratio of
expected number of residues divided by the actual number of
residues observed.


20
Figure 2-2. Purity data for synthetic peptides. A.
IFNy(2-20) HPLC profile. This peptide was synthetsized from
the same resin as IFNy(l-20) so no amino acid analysis was
done. B. IFNy(l-14) HPLC profile and amino acid analysis.


21
Figure 2-3. Purity information of synthetic peptides of
mouse IFNy. A. Amino acid analysis and HPLC profile of
IFNy(3-20) B. HPLC profile of IFNy(4-20) C. HPLC profile
IFNy(5-20). D. HPLC profile of IFNy(6-20). Amino acid
analysis was performed on IFNy(3-20) only because all other
peptides were synthesized from the same resin.


22
Figure 2-4. Purity information of synthetic peptides of
mouse IFNy. A. Amino acid analysis and HPLC profile of
IFNy(3-20) S3I4L5. B. Amino acid analysis and HPLC profile
of IFNy(3-20) S3L4V5. C. Amino acid analysis and HPLC
profile of IFNy(3-20) I3K4Y5. D. Amino acid analysis and
HPLC profile of IFNy(3-20) L3Y4K5.


23
buffered saline, pH 7.4. MAb 5.102.12 is an N-terminal
specific mAb raised against IFN-y and has been partially
characterized previously (87,88). This mAb has the ability
to neutralize the antiviral and macrophage priming activity
of IFN-y as well as block the specific binding of ^-25j_jpN-y
to its cell surface receptor. The N-terminal specificity of
the mAb was demonstrated by its binding to a ^25i-iabeled
synthetic peptide corresponding to the first 39 residues of
IFN-y, IFN-y(l-39) and by its ability to block the binding
of IFN-y by 5.102.12 in a competitive radioimmunoassay
(RIA)(87). The epitope was further localized to the first 20
residues of IFN-y, IFN-y(l-20) using a competitive RIA (88) .
Radioimmunoassay (RIA) and ELISA. Assays were performed
in duplicate at room temperature as described (100).
Briefly, mAb 5.102.12 was added to protein A (Sigma, P6650)
coated wells of 96 well PVC microtiter plates (Falcon, 3912).
Plates were washed of excess mAb after which 20 |ll of 125j_
IFN-y was added. An equal volume of competitor or medium was
added thirty minutes prior to the addition of labeled IFN-y.
After one hour, wells were washed, cut out and counted on a
gamma counter (LKB). Competition was assessed by comparing
cpm values to that of medium controls. Competitive ELISA was
carried out with the same protocol except biotinylated IFN-y
was substituted for the radiolabeled IFN-y. After the
mixture was allowed to react for one hour, the plates were
washed and alkaline phosphatase conjugated streptavidin
(Sigma) was added to the wells. After one hour the plates


24
were washed and substrate was added. Color development was
monitored on a microplate reader (BioRad).
Circular dichroism (CD). CD for selected peptides was
determined at room temperature using a JASCO 500C
spectropolarimeter. Scans were done with a 0.1 cm pathlength
cell at a sensitivity of 0.5-2.0 and a time constant of 8
seconds. The wavelength range measured was from 250 nm to
200 nm at a scan rate of 20 nm/ min. Scans were carried out
on peptides in 25% trifluoroethanol (TFE) in water at
concentrations of 0.1 to 0.5 mg/ml (101-103). The CD spectra
were expressed in terms of A£ related to the mean residue
ellipticity at a given wavelength ( [0]^) for each peptide.
The following formula was used to generate A£ (104):
^ X. ^ observed
l-6 U- cxl
A£=
3298
where [0]^ and [^ ] 0bservecl are exPressed in degrees
equals the mean residue concentration in moles/liter,
is the pathlength of the cell in cm.
/ c
and 1
Results -an.d-Ei.sgiis.sipn
Preliminary studies suggested that the N-terminal
residues of IFN-y(l-20) played an important role in the
epitope for mAb 5.102.12 (88). In order to ascertain the N-
terminal residue requirement for the epitope of this mAb in
more detail, I synthesized IFN-y(l-20) and its truncated
forms, IFN-y(2-20) IFN-y(3-20) IFN-y(4-20), IFN-y(5-20),
and IFN-y(6-20), and tested the peptides for their ability to


25
block specific binding of -*-25];_iFN_y to 5.102.12 in a
competitive RIA. The peptides IFN-y(l-20), IFN-y(2-20) and
IFN-y(3-20) all had similar ability to block IFN-y binding
(Figure 2-5). In addition, as the peptides were shortened
they correspondingly lost blocking ability incrementally
until they reached position six where IFN-y(6-20) lost
blocking function. The third, fourth, and fifth residues
appear to be the most important because the peptides lost
their blocking ability when these residues were deleted.
The observation that it took at least 1000 times more
peptide to block IFN-y binding to mAb illustrates the type of
reaction that is occurring in peptide competition studies of
this nature. The interaction of a synthetic peptide with a
monoclonal antibody raised against a native protein is
actually a cross reaction (105). The peptide is not the
actual antigen and probably adopts the conformation of the
intact protein for only a small percentage of the time. This
can account for the lower affinities commonly seen with
peptide-antibody interactions.
It had been previously reported that IFN-y(3-20) had a
reduced blocking activity compared to IFN-y(l-20)(88). The
greater activity of IFN-y(3-20) observed in the present
report can possibly be attributed to a change in synthesis
chemistry. The peptides in the previous report were
synthesized by t-Boc chemistry and cleaved by HF cleavage
procedures, whereas the peptides utilized in the present
study were synthesized by FMOC chemistry. The purity data


26
Peptide concentration (log M)
Figure 2-5. Competitive RIA with synthetic peptides of IFN-y
and mAb 5.102.12. The ability of synthetic peptides,
IFN-y(l-20) and its N-terminal truncations, to block
125j-jfn-Y (20nM) binding to mAb 5.102.12 was assessed.
Competition is expressed as percent of control binding to
wells that contain no peptide competitors. Control binding
was 33,816 100 cpm (cpm S.D.)in the absence of
competition. The figure is representative of three
independent experiments. Symbols: IFN-y(l-20) (!) ,
IFN-y(2-20) () IFN-y(3-20) (O) IFN-y(4-20) (A) IFN-y(5-
20) (A) IFN-y(6-20) ().


27
from the t-Boc synthesis of IFN-y(3-20) was unavailable. In
order to illustrate the differences between FMOC and t-Boc
synthesis chemistries, I have compared the purity of the
peptide IFN-y(l-20) synthesized by both methods. HPLC
profiles of IFN-y(1-20)FMOC and IFN-y(1-20)t-Boc are shown in
Figure 2-6. The FMOC peptide exists as one major species
while the t-Boc peptide is heterogeneous. The multiple peaks
seen in HPLC profiles of peptides can contain different
components. Some of these peaks are truncations and
deletions of the main sequence that are produced during the
synthesis. Some peaks are incompletely deprotected peptides
that still possess some of the side chain protecting groups
that are used in the synthesis. Finally, the remainder of
the peaks are peptides that have been chemically modified
during the cleavage in hydrogen fluoride which is used for
t-Boc cleavage and can be harsh on peptides through the
generation of carbonium ions (106). It is likely that the
difference seen in the blocking activity of the t-Boc IFN-
7(3-20) and the FMOC IFN-y(3-20) is due to modifications of
the t-Boc peptide. However, this is only speculation and
cannot be determined unequivocally without the original
peptide.
One way that residues three to five may function in the
epitope for mAb 5.102.12 is that they may actually be a part
of the epitope and serve as contact residues for mAb
5.102.12. Another possibility is that they may act to
stabilize adjacent contact regions of the epitope. The


28
Figure 2-6. HPLC profiles of t-Boc and FMOC peptides. A.
IFN-yd-20) synthesized by FMOC chemistry. B.IFN-yd-20)
synthesized by t-Boc chemistry.


29
predicted secondary structure of IFN-y(l-20) is presented in
Figure 2-7A, and is based on the method of Chou and Fasmen
(107,108) The region of 12-20 is proposed to form a loop
that may be found on the surface of the molecule and thus
have a high probability of forming an antigenic
determinant(s) (109). The remainder of the molecule is
predicted to be primarily a-helix. The N-terminal residues
three to five, which are depicted as initiating the helix
may act to stabilize the helix region thereby stabilizing the
main epitope. Interestingly, the predicted helix between
residues 3 and 12 is amphipathic in nature (96). Figure 2-7B
represents a helix wheel in which the residues found in the
predicted helix region are plotted in relation to their
positions in the helix. Hydrophilic and hydrophobic residues
are separated which is characteristic of an amphipathic
helix. The hydrophilic side of the helix presumably
interacts with the hydrophilic environment. Amphipathic
regions are thought to play a role in immunodominance in B-
cell and T-cell recognition (96,97). Amphipathic helices
have also been shown to be important in the functional sites
of some proteins (110) The amphipathic helix in the C-
terminus of IL-2 has been shown to be required for IL-2
function (111).
To gain further insight into the role of the N-terminal
residues of IFN-y(l-20) in the epitope, peptide analogs were
synthesized with either conservative or nonconservative amino
acid substitutions. The criteria for conservative and


30
Figure. 2-7. Structure of the N-terminal 20 residues of
IFN-y. A. Predicted secondary structure of IFN-Y(l-20) .
This model is derived from the predicted structure of
IFN-y(3) Alpha helical structure is depicted as coils,
however the number of turns is not intended to represent the
actual topography of the region. Wavy lines represent
regions that are not predicted to have stable structure (e.g.
residues 1-3) The (3-bend is represented as a trapezoid
shape (residues 16-20). B. Helix wheel depicting amphipathic
helix of IFN-y found between residues three and eleven.
Hydrophobic residues are circled.


31
nonconservative substitutions was based on side chain
structure, charge, and hydrophobicity (112) of amino acid
residues. Due to the incremental nature of the decrease in
binding of truncations, positions three, four, and five were
changed simultaneously. Table 2-1 presents the native
residues as well as analogs of IFN-y(3-20) The native
sequence for IFN-y(l-20) at positions three, four, and five
consists of threonine, valine, and isoleucine, respectively.
Two conservative and two nonconservative analogs were
constructed. The first conservative analog contained serine,
isoleucine, and leucine at these positions. The second
conservative analog had serine, leucine, and valine. These
substitutions are similar to the native sequence with respect
to charge and side chain structure and are designed to
maintain the charge in this region. One nonconservative
analog contained isoleucine, lysine, and tyrosine at
positions three, four, and five, while the other contained
leucine, tyrosine, and lysine. The charge and side chain
structure of these substitutions are different than the
native residues and are designed to disrupt the charge and
hydropathicity in this region. The data presented in Figure
2-8 illustrate the similar ability of the two conservative
analogs to block binding of ^25j_ipN_y to 5.102.12 relative
to IFN-y(3-20) In contrast, there was a significant
reduction in blocking activity by both nonconservative
analogs. Thus, the structure of residues three to five of


Table 2-1. Amino acid sequences of IFN-y(l-20) and its truncated analogs.
Type of substitution Sequence
IFN-y(1-20) HIS GLY THR VAL ILE GLU SER LEU GLU SER LEU ASN ASN TYR PHE ASN SER SER GLY ILE
Native
IFN-y(3-20)
SER3,LEU4, VAL5
Conservative
SER I LE LEU -- -- -- -- -- -- --
IFN-y(3-20)
SER3,LEU4'VAL5
Conservative
SER LEU VAL -
IFN-y(3-20)
ILE3,LYS4,TYR5
Nonconservative
I LE LYS TYR -- -- -- -- -- -- -- £
IFN-y(3-20)
leu3,tyr4,lys5
Nonconservative
LEU TYR LYS -- -- -- -- -- -- --


33
Figure. 2-8. Competitive RIA with peptide analogs of IFN-y
and mAb 5.102.12. The ability of IFN-y(l-20), IFN-y(3-20),
and their analogs to block 125j-iFN-y (8 nM) binding to mAb
5.102.12 was assessed. Competition is expressed as percent
of control binding to wells that contain no peptide
competitors. Control binding was 69471451 cpm (cpm S.D.).
The figure is representative of three independent
experiments. Symbols: IFN-y(l-20) () IFN-y(3-20) ( O ) ,
conservative IFN-y(3-20) SEr3, ile4' leu5 (A), conservative
IFN-y(3-20) SER3, LEU4' VALS (A) nonconservative IFN-y(3-
20) ILe3, lys4' TYR5 (), nonconservative IFN-y(3-20) LEU3,
TYR4, LYS5 (X), IFN-y(6-2 0) () .


34
IFN-y appears to play an important role in the epitope
specificity of mAb 5.102.12.
To verify the presence of a-helical structure in the
peptides, CD spectroscopy was employed (104,113). The CD
spectra of the peptides are obtained in 25% trifluoroethanol
(a secondary structure stabilizing agent) and show the
potential of each peptide to form a particular secondary
structure. As shown in Figure 2-9, the CD spectra of peptide
IFN-y(l-20) indicated mainly a-helix characterized by a
pronounced minimum at 220 nm. IFN-y(3-20) also had helical
structure that was less than that of IFN-y(l-20).
Conversely, the truncated peptide IFN-y(6-20), which lacked
function, lost most of its a-helical structure. IFN-y(3-20) ,
SER^, ILE^, LEU^, a conservative analog, had a-helical
structure that was slightly less than IFN-y(l-20) but greater
than the native truncation IFN-y(3-20). As indicated, this
conservative analog was as effective as IFN-y(3-20) in
blocking IFN-y binding to mAb 5.102.12. The nonconservative
analog, IFN-y(3-20) ILE^, LYS^, TYR^/ which lost blocking
activity, exhibited almost identical secondary structure to
the IFN-y(l-20) (primarily a-helix). Thus, the loss of
blocking activity of substituted analogs was not due to loss
of a-helical structure. Positions three, four, and five
apparently participate in the formation and/or stabilization
of the a-helix, based on the lack of helix found in the
truncation IFN-y(6-20). The interesting observation that the
nonconservative analog did not block function but still


35
Wavelength (nm)
Figure. 2-9. CD spectra of IFN-y(l-20), its analogs, and N-
terminal truncations. Spectra were obtained with peptides in
25% TFE as described in the Materials and Methods. Symbols:
IFN-y(1-20) ( ), IFN-y(3-20) ( ), IFN-y(6-20)
( ), Conservative analog IFN-y(3-20) SER3, ile4/ LEU$
( ), Nonconservative analog IFN-y(3-20) ILE3, LYS4^ TYR5
( )


36
retained stable secondary structure would suggest that the a-
helix alone is not critical and that residues three to five
are directly involved in the epitope of the N-terminal
peptide. The nonconservative substitutions are predicted to
participate in a-helix formation as effectively as the
conservative substitutions which is consistent with the
stable secondary structure of the analog containing these
residues (107). Additionally, the conservative analogs
examined maintain the amphipathic nature of the helix, while
the nonconservative analogs disrupt it through changes in
hydrophobicity and charge. Thus, the structural and
functional data implicate the third, fourth, and fifth
residues directly in the epitope of mAb 5.102.12, and that
the charge and hydropathicity of these residues is important.
Peptides were synthesized with C-terminal truncations in
order to determine the role of residues in the putative loop
region in the epitope specificity of mAb 5.102.12.
IFN-Y(1-14) and IFN-y(l-20) had similar blocking ability,
whereas IFN-y(l-13) possessed no activity, which is evidence
that tyrosine at position 14 is important for epitope
structure (figure 2-10). IFN-y(3-14) also had full blocking
activity. Thus, the minimum length requirement of the
epitope for mAb 5.102.12 involves residues 3 to 14.
I next examined the secondary structure of C-terminal
truncations of IFN-y(l-20). Both IFN-y(l-14) and IFN-y(l-13)
had an apparent reduced helical structure, but not complete
abrogation, with an apparent increase in random structure


37
Peptide concentration (log M)
Figure. 2-10. Competitive RIA with C-terminal truncations
of IFN-y(1-20) and mAb 5.102.12. The ability of IFN-y(l-20)
and its C-terminal truncations to block 125i-iFN-y (22 nM)
binding to mAb 5.102.12 was assessed. Competition is
expressed as percent of control binding to wells that contain
no peptide competitors. Control binding was 56,204 153 cpm
(cpm S.D.). The figure is representative of three
independent experiments. Symbols: IFN-y(l-20) (!) ,
IFN-yd-13) (O) IFN-y( 1-14) (A) IFN-y(3-14) () .


38
(minimum at 205-200 nm) (figure 2-11) The reduction in (X-
helix in these truncations may involve a partial loss of
helix in the C-terminal region of the peptide caused by the
removal of residues C-terminal to the helix (residues 3-11).
Since both peptides had similar reduction in helix and
IFN-yd-14) but not IFN-yd-13) blocked mAb binding, it
suggests that factors additional to a-helix may play a role
in the epitope.
All of the experiments described have used crude
peptides. Although, as I have shown, the purity of FMOC
peptides is higher than t-Boc, the differences in purity as
shown by HPLC profiles of the peptides used warrants further
examination. To attempt to address this issue, wo peptides,
IFN-y(1-20) and IFN-y(l-13) were purified by HPLC.
IFN-y(l-20) had a final purity of 90% and IFN-y(l-13) had a
final purity of 96% as determined by HPLC. Amino acid
analysis and HPLC profiles of the purified are shown in
figure 2-12. The results of a competitive ELISA (Figure 2-
13) show that crude and purified IFN-y(l-20) have identical
blocking profiles. Purified IFN-y(l-13) has no blocking
activity. Therefore, no impurity was responsible for
blocking activity. Additionally, the lack of purity was not
responsible for a lack of blocking activity.
To further explore this issue, I will attempt to explain
the nature of the impurities found in crude synthetic
peptides and relate this to my findings. Much of the
information that I present here comes from my own


39
Wavelength (ntn)
Figure 2-11. CD spectra of IFN-y(l-20) and its C-terminal
truncations. Spectra were obtained with peptides in 25% TFE
as described in the Materials and Methods. Symbols:
IFN-y(1-20) ( ) IFN-y(l-13) ( ),
IFN-y(l-14) ( ) .


40
Figure 2-12. Amino acid analysis and HPLC profiles on
purified synthetic peptides. Amino acid analysis values are
the ratio of expected number of residues divided by the
actual number of residues observed. A. IFN-y(l-13) crude.
B. IFN-yd-13) purified. C. IFN-y (1-20) crude. D.
IFN-y(l-20) purified.


41
o
u
o
U
o
120
100 -
Peptide concentration (log M)
Figure 2-13. Competitive ELISA of purified synthetic
peptides of IFN-y. ELISA was carried out under the
conditions described in materials and methods. Biotinylated
IFN-y concentration used was 25 nm. Symbols: IFN-y(l-20)
crude (), IFN-y(l-20) purified (o), IFN-y(l-13) purified
(A) .


42
observations and from discussion with peptide chemists at
Biosearch and Advanced Chemtech. Most of the impurities are
due to amino acid deletions and/or the incomplete removal of
side chain protecting groups. Amino acid deletions can be
identified by amino acid analysis. Incomplete deprotection
is commonly seen with the MTR group on arginine and the TRT
group on histidine. However, other protecting groups can
also be difficult to remove, depending on the sequence of the
peptide. Frequently these "impure" peptides, due to the
nature of the assay being used, exhibit the same activity as
complete sequence. This may be because some of the residues
affected may not be important for the activity of the
peptide.
It has been my observation that HPLC alone cannot give a
clear picture as to the purity of a synthetic peptide.
Protein sequencing is the only certain way of determining,
beyond question, the content of a peptide. As this is
impractical in routine situations, amino acid analysis must
be used to verify the presence of each amino acid residue.
When interpreting an amino acid analysis, the ratio of
observed to expected should be close to 1.0 +/- 10%, with
some exceptions. Serine, valine, methionine, isoleucine,
histidine, and tyrosine can be found in lower amounts due to
modifications during the hydrolysis reaction for the amino
acid analysis. Tryptophan and cysteine require special
hydrolysis. In my opinion, each peptide presented here has


43
an acceptable amino acid analysis and all of the residues are
present in reasonable quantities.
Differences in synthesis can be responsible for varied
purity of peptides. Each synthesis is unique to the sequence
being synthesized. Although two sequences are similar one
sequence may be more easily synthesized than the other.
Another observation that I have made concerns the
content of peaks seen in HPLC profiles. Due to the high
absorption seen with some protecting groups used in the
synthesis, a relatively large peak can contain a
disproportionately small amount (by weight) of material when
isolated and analyzed. The large peak observed can be
misleading.
A further indication of the suitability of crude
peptides in these mapping studies can be illustrated with the
following examples. Blocking studies presented in this
dissertation are interpreted in a qualitative manner. A
peptide either blocks or does not block IFN-y binding to mAb.
One exception to this involves the N-terminal truncations of
IFN-y(1-20) (IFN-y(3-20), iFN-y(4-20), lFN-y(5-20), and
IFN-y(6-20) ) These peptides lose blocking activity
incrementally as residues are left off. If one examines the
HPLC profiles of IFN-y(5-20) and IFN-y(6-20) (Figure 2-3 C,D)
they appear to be qualitatively similar. Also, these
peptides were synthesized from the same resin. However, when
ILE at position 5 is left off as in IFN-y(6-20) blocking
activity is reduced (Figure 2-5). This would suggest that


44
the lack of ILE at position 5 is responsible for this
difference in blocking activity, and not a difference in
purity. The difference seen in activity appears to be
greater than a difference in the HPLC profiles of the
peptides. The peptide IFN-y(1-14) has blocking activity
similar to IFN-y(l-20) as shown in figure 2-10. The amino
acid analysis appears to be reasonable based on the sequence
(Figure 2-2). It shows the presence of TYR in the peptide
while the amino acid analysis of IFN-y(l-13) does not (Figure
2-12). The HPLC profile contains two major peaks, each about
30-40% of the total. The content of the second peak is not
known but it is likely to be an incompletely deprotected
peptide. This impurity seems to not have an effect on
blocking activity. Also, if the peptide were three fold
greater in purity (>90% pure), one would expect little
difference in blocking activity. The profile for IFN-y(l-14)
would be shifted slightly to the left one half log and not be
very different than the profile of IFN-y(l-20) The lack of
TYR in IFN-y(l-13) seems to be what is responsible for a lack
of blocking activity.
The data presented in this chapter indicate that the
epitope specificity of the IFN-y neutralizing mAb 5.102.12
spans 12 amino acid residues, and is located between the
third and fourteenth residues of IFN-y with residues 3, 4, 5,
and 14 critical to the interaction of mAb 5.102.12 with IFN-
y. Epitopes are classified into four groups, linear,
conformational, continuous, and discontinuous (105).


45
Discontinuous and conformational epitopes are those that are
made up of more than one distant site on the surface of a
protein. The contact residues are separated by non-critical
residues (105). A classic conformational epitope that has
been studied is in the molecule lysozyme. X-ray
crystallographic studies of a monoclonal antibody binding to
an epitope on lysozyme have detailed a conformational
epitope. Linear and continuous epitopes are epitopes found
on short sequences not more than eight to ten residues in
length that involve a single stretch of residues (105,114-
118). Synthetic peptides are common continuous and linear
epitopes. This epitope for 5.102.12 does not appear to be
continuous in the classical sense based on its length, twelve
residues. It also does not appear to require a stable
conformation based on CD studies. Based on the requirement
of the residues 3,4,5,and 14, the linear peptide IFN-y(3-14)
appears to contain a discontinuous epitope. The role that
internal residues may play in this epitope is as yet
undetermined. Thus, the data suggest that the mAb binding
site on IFN-y exists as a linear discontinuous epitope. The
classification of linear discontinuous epitope has been used
in another report (119). In this case the terminology was
discontinuous linear epitope and was used to describe the
epitope specificities of several mAb to synthetic peptides.


CHAPTER 3
TOPOLOGY OF RECEPTOR BINDING DOMAINS OF MOUSE IFN-y THAT
INCLUDE THE N-AND C-TERMINI.
Introduction
IFN-y possesses a number of immunologic activities that
are critical to immune function. The identification of the
receptor binding domains should further enhance the
understanding of its mechanism of action. This information
would be useful for the design of agonists and antagonists of
IFN-y function. The location of these domains is unclear.
The N-terminus of IFN-y has been shown to bind to the
receptor as determined by competition studies where the first
39 residues of IFN-y, IFN-y(l-39), compete with IFN-y for its
receptor (88). Antibody studies confirm the role of the N-
terminus of IFN-y in receptor binding (87,88). The epitope
specificity of an IFN-yneutralizing monoclonal antibody was
mapped to the first 14 residues of IFN-y (Chapter 2). The
binding site on IFN-y for receptor probably involves regions
in addition to IFN-y(l-39), since this peptide binds to
receptor at l/1000th the affinity of IFN-y (88). The
functional significance of the remainder of the molecule has
not been determined. Additionally, the role of the C-
terminus in IFN-y function has been controversial. A
46


47
truncated form of recombinant human IFN-y missing the C-
terminal 23 residues was shown to possess full activity (82).
In contrast, the removal of 11 residues from the C-terminus
of IFN-y, with limited proteolysis, yielded IFN-y with
significantly reduced activity (81). Studies have shown that
polyclonal antibodies directed to a C-terminal synthetic
peptide, IFN-y(95-133) neutralized IFN-y activity (87).
Similarly, mAb that appear to be directed to the C-terminus
of IFN-y also neutralize IFN-y activity (84) .
The second specific aim of this dissertation is to
explore other regions of the molecule for functional
significance using long overlapping synthetic peptides
encompassing the entire sequence of IFN-y and antiserum
raised to these peptides. The data strongly suggests that
the C-terminus, in addition to the N-terminus, of mouse IFN-y
is important in receptor binding and function. Using
synthetic peptides, antisera raised against these peptides,
circular dichroism spectra for secondary structure, and
predictive algorithms for secondary and tertiary structure,
a three dimensional model of IFN-y has been constructed that
includes both the N and C-termini as receptor binding
domains.
Materials and Methods
Animals and immunizations. Female New Zealand White
rabbits (2-2.5kg) were immunized with synthetic peptides


48
conjugated with keyhole-limpet hemocyanin using
gluteraldehyde conjugation (120). One hundred micrograms of
conjugated peptide were injected with Complete Freund's
adjuvant in a total volume of 0.5 ml followed 30 days later
with 100 |i.g of conjugated peptide in Incomplete Freund's
adjuvant. Seven days later blood was drawn and serum was
collected. Subsequent boost injections occurred at 30 day
intervals followed by bleeds seven days later.
Synthetic peptides. Peptides were synthesized with a
Biosearch 9500AT automated peptide synthesizer using FMOC
chemistry (98). Peptides were cleaved at 25 C from the
resins using trifluoroacetic acid/phenol/ethanedithiol at a
volume ratio of 95.0/4.5/0.5. The cleaved peptides were then
precipitated in ether and ethyl acetate and subsequently
dissolved in water and lyophilized. The purity of peptides
was assessed by reverse phase HPLC and verified by amino acid
analysis. Amino acid analysis and HPLC profiles for the
peptides used for immunization are found in figure 3-1. The
peptides used in epitope mapping studies were purified using
a preparative reverse phase HPLC column (Vydac). The major
peak was collected, lyophilized, and checked for purity with
analytical HPLC and amino acid analysis. The amino acid
analysis and HPLC profiles for purified peptides are found in
figure 3-2.


49
Figure 3-1. Purity information of overlapping synthetic
peptides of mouse IFN-y. A. IFN-y(36-60) B. IFN-y(54-
91). C. IFN-y(78-107) D. IFN-y(95-133).


50
Figure 3-2. Purity information on purified synthetic
peptides used in C-terminal specific antibody mapping
studies. A. IFN-y(7 8-107) B. IFN-y(95-133) C.
IFN-y(108-133) D. IFN-y(120-133) E. IFN-y(108-119) .


51
ELISA. Synthetic peptides and IFN-y were dissolved in
0.1 M sodium carbonate buffer, pH 9.6, at a concentration of
12 |lg/ml and 50 |ll of solution were added to each well of a
96 well flat bottom tissue culture plate (Falcon). After
overnight incubation at room temperature, each plate was
dried with mild heat applied by a standard blow dryer.
Nonspecific binding sites were blocked with 200 |ll/well 5%
Carnation powdered milk dissolved in PBS for 2 hours. Plates
were then washed with 0.15 M NaCl/0.05% Tween 20 five times.
Dilutions of antisera were added in 50 |ll amounts and
incubated at room temperature for 2 hours. After washing,
horseradish peroxidase conjugated goat anti-rabbit IgG
antibodies (Cappel) were added to each well and incubated for
one hour. The plates were washed and 0-phenylenediamine
(Sigma) and H2O2 were added and color development was allowed
to occur for 15-30 minute after which 25 |ll of H2SO4 was
added to stop the reaction. Absorbance was determined at 492
nm in an ELISA plate reader (BioRad). Endpoints were defined
as the highest dilution of antisera with absorbance readings
that were twice those of the blank.
Circular dichroism (CD). CD for the peptides was
determined as described in chapter 2 except for the following
changes. Scans were done with a 0.01 cm pathlength cell at a
sensitivity of 1.0 and a time constant of 8 seconds. The
wavelength range measured was from 260 nm to 184 nm at a scan
rate of 5 nm/ min.


52
IFN-y neutralization assay. Varying concentrations of
IFN-y were incubated with dilutions of rabbit antisera for 30
min at 37C. Residual IFN-y activity was then measured as
described (99). Briefly, IFN-y samples in the presence or
absence of antibodies were incubated with mouse L cells for
16 to 18 hr at 37C after which inhibition of virus
replication was determined in a plaque reduction assay with
vesicular stomatitus virus. One unit of IFN-y was defined as
that which caused a 50% reduction in plaque formation. For
epitope mapping studies, synthetic peptides were incubated
with antibodies for 30 min at 37C prior to their addition to
IFN-y. The peptide inhibition of IFN-y neutralization by
anti-peptide antisera was compared to IFN-y alone and to
antibody plus IFN-y in the absence of peptide. For peptide
blocking studies, synthetic peptides were substituted for the
antibodies. Peptides were dissolved in HMEM containing 2%
FBS. No preincubation with IFN-y was performed.
Results and Discussion
To examine the role of the remainder of IFN-y, long
overlapping peptides were synthesized covering the entire 133
amino acids of IFN-y. The peptides synthesized were: IFN-
7(3 6-60), IFN-y(54-91) IFN-y(78-107) and IFN-y(95-133) and
are shown in Table 3-1. These peptides were tested for their
abilities to inhibit IFN-y antiviral activity. N-terminal
peptide, IFN-y(l-39), had the ability to directly inhibit


53
Peptide
Sequence
Hydropathy3
IFN-yd-39)
HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDG
-0.559
IFN-y(36-60)
QKDGDMKILQSQIISFYLRLFEVLK
-0.036
IFN-y(54-91)
RLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAF
-0.189
IFN-y(78-107)
TTFFSNSKAKKDAFMSIAKFEVNNPQVQRQ
-0.617
IFN-y(95-133)
AKFEVNNPQVQRQAFNELIRWHQLLPESSLRKRKRSRC
-0.792
a Average hydropathy values were calculated by talcing the sum of the
hydropathy values for each amino acid divided by the number of amino acids in
each sequence. Hydropathy values were taken from Kyte and Doolittle (112).


54
IFN-Y function as shown previously (88) while the remainder
of the peptides had no effect (Table 3-2). However, based on
competition studies, IFN-y(l-39) interacted with receptor
with 1/1000 the affinity of IFN-y (88) Thus, other regions
of the IFN-y molecule are likely to be involved in receptor
interaction. To determine the role of these other regions of
IFN-y, rabbit antibodies were produced to the overlapping
synthetic peptides and tested for their abilities to interact
with and inhibit IFN-yfunction. Antisera to IFN-y(l-39) and
IFN-y(95-133) had been produced previously (87). These
antibodies to IFN-y(l-39) and IFN-y(95-133) were shown to
neutralize IFN-y activity by blocking IFN-y receptor binding
(87). All of the peptides elicited antibodies reactive with
IFN-y as determined by ELISA (Table 3-3). Antibodies
directed against N-terminal peptide, IFN-y(l-39) also bound
and neutralized IFN-yas shown previously (87). Similarly,
antiserum to the C-terminus had the ability to neutralize
IFN-y antiviral activity. In contrast, antisera to the
internal three peptides had no effect although they bound
IFN-y. The differential neutralizing ability was probably
not due to the differences in titer. When the neutralization
titer is normalized on the basis of ELISA titer to IFN-y by
taking the ratio of neutralizing titer to ELISA titer, the
ratios were very different. If elevated titer alone was
responsible for neutralization then similar ratios would be
expected. Additionally, the ELISA titer to
anti-IFN-y(78-107) is similar to the titers of the


55
Table 3-2. Direct peptide blocking of mouse IFN-Y antiviral activity by
IFN-Y synthetic peptides.
Peptides
Peptide concentration3
IFN-y(l-39)
15 |iM
IFN-y(36-60)
>500 JIM
IFN-y(54-91)
>500 \m
IFN-Y(78-107)
>500 |IM
IFN~Y(95-133)
>500 |1M
aHighest peptide concentration that will block 10 units of IFN-Y
antiviral activity.


56
Table 3-3. IFN-y neutralization efficiency of antipeptide antisera.
Peptide
ELISA titer
Peptide
(E)a
IFN-y
Neutralization (N)
aqainst 10U IFN-y
Ratio13
E/N (xlOO)
lFN-yd-39)
ND
1/10,000
1/3000
30.0
IFN-y(36-60)
1/1000
1/1000
<1/3
<0.3
IFN-y(54-91)
1/1000
1/600
<1/3
<0.05
IFN-y(78-107)
1/100,000
1/20,000
<1/3
<0.015
IFN-y(95-133)
1/300,000
1/60,000
1/6000
10.0
aELISA performed as described in materials and methods. Six
hundred nanograms of each peptide or IFN-y was used in each well.
^Ratio calculated as ELISA titer to IFN-y divided by
neutralization titer multiplied by 100.
CND indicates not done.


57
N- and C-terminal antisera, but does not possess any
neutralizing activity. The results confirm the role of the
N-terminus as a receptor interaction site and suggest that
the C-terminus forms an additional binding domain.
The N-terminal binding domain has been characterized
previously using direct peptide competition and epitope
mapping of an N-terminal specific IFN-y neutralizing mAb (88,
Chapter 2). I was therefore interested in localizing the
epitope to which the C-terminal neutralizing antibodies bind
thereby further characterizing this site on the molecule.
The antiserum, anti-IFN-y(95-133), is polyclonal and
potentially contains antibodies to several epitopes, so there
is the possibility that only a subset of these antibodies is
responsible for the neutralizing activity seen in this
antiserum. Peptide truncations of IFN-y(95-133) were
synthesized and used to map its neutralizing epitope(s).
Figure 3-3 illustrates the peptides synthesized in these
experiments and their relationship to one another. To map
the neutralizing epitope (s) of anti-IFN-y(95-133) a
modification of the antibody neutralization assay was used in
which synthetic peptides were added to inhibit the IFN-y
neutralization by anti-IFN-y(95-133) The immunizing
peptide, IFN-y(95-133) was able to completely inhibit the
neutralizing activity of anti-IFN-y(95-133) (Table 3-4). A
truncation, IFN-y(108-133) was able to inhibit
neutralization about 30% at the same concentration.
IFN-y(78-107) as well as the shorter peptides IFN-y(120-133)


58
78 90 100 110 120 133
.... TTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC -COOH
78 107
95 133
108 133
120 133
108
119
Figure 3-3. C-terminal synthetic peptides of IFN-y used to
map the neutralizing epitope specificity of the neutralizing
antiserum produced against IFN-y(95-133) The sequence from
residues 78-133 is listed on top with the individual peptides
represented by lines under the sequence.


Table 3-4. Epitope mapping of the C-terminal specific IFN-y neutralizing antiserum3 and
identification of the neutralizing epitopes.
IFN-y titer (units)13
Peptide Cone.
(mM)
IFN-y(95-
-133) IFN-y(108-133)
IFN-y(78-107)
IFN-y(108-119)
IFN-y(120-133)
IFN-y(78-107)
IFN-y(108-119)
IFN-y(120-133)
0.1
300
100
30
3
30
300
0.03
300
100
30
3
30
300
0.01
300
100
30
3
30
300
0.003
300
100
30
3
30
300
0.001
300
100
30
3
30
300
0.0003
300
100
30
3
10
300
0.0001
100
60
30
3
6
300
0.00003
10
6
10
3
3
100
aAntiserum was
used at
a dilution of 1/1000.
^IFN-y titer in the absence of antiserum was 300 units. In the presence of antiserum the titer was <3
units.
cThe concentration of the mixture of peptides in the last column reflects the individual peptide
concentrations listed in the lefthand column.


60
and IFN-y(108-119) had a slight inhibitory ability when added
separately. However, when the longer sequence was
reconstructed with the combination of the shorter peptides,
lFN-y(78-107), iFN-y(108-119), and lFN-y(120-133), full
inhibitory activity was restored. This suggests that there
are multiple neutralizing epitopes involved in this region
that can function cooperatively and that the antiserum is
probably directed toward several linear sequences.
CD studies of human IFN-y have shown that the molecule
is about 66% a-helix with turn and random regions making up
the difference (80). Mouse and human IFN-y share about 40%
identical sequence homology (20). About 30% of the amino
acid changes are conservative substitutions. Both mouse and
human IFN-y possess very similar surface profiles as shown in
figure 3-4. The surface profile predicts which regions of a
protein may be found on the surface of the molecule. The
surface profile takes into account HPLC mobility, segmental
mobility and surface accessibility parameters for amino acid
residues calculated from model proteins and peptides (121) .
The overlapping profiles suggest that human and mouse IFN-y
are very similar at the structural level. Therefore, it is
reasonable that information obtained concerning human IFN-y
may be applicable to mouse IFN-y. Due to the overall
structural homology between human and mouse IFN-y mouse
IFN-y is likely to contain a similar amount of a-helix. I
was unable to obtain mouse IFN-y in sufficient quantities to
measure the CD directly; however, I did measure the CD of the


61
O 20 40 60 80 100 120
Residue Number
Figure 3-4. Surface profile of mouse IFN-y (dashed lines)
and human IFN-y (solid lines).


62
overlapping synthetic peptides that encompass the entire
sequence. CD of the peptides was measured in 25%TFE, a helix
stabilizing agent. TFE is commonly used in the CD
measurement of synthetic peptides in order to enhance
existing structure (11,101-103). Alpha helical structure was
observed in all of the peptides characterized by a minimum at
220 nm and a maximum at 190 nm (Figure 3-5). IFN-y(36-60)
was not sufficiently soluble in the concentration required
for CD measurement due to its hydrophobicity. Although the
CD of peptides may differ from that of the native molecule,
these results suggest the probability that the peptides form
a-helix and the likelihood that the native molecule is mainly
a-helical.
Based on the above results an overall topology of mouse
IFN-y can be pictured. Both the N-terminus and C-terminus
participate, in some way, in receptor interaction. The N-
terminus, based on direct peptide competition studies can
interact with receptor. Additionally, others have shown that
when the N-terminal 8-10 residues of human IFN-y are removed
by limited proteolysis, functional activity is reduced along
with a corresponding loss in a-helical structure (80). The
C-terminus, based on my antibody neutralization data, may
also be interacting with receptor or act to stabilize the
structure of the N-terminus. Two secondary structure
prediction algorithms predict that the molecule contains six
a-helices, no P-sheet, and the remainder turns and random
structure (107,108,122). The secondary structure prediction


63
Figure 3-5. CD spectra of overlapping synthetic peptides of
IFN-y. Spectra were obtained in 25%TFE as described in
Materials and Methods. The spectra for IFN-y(l-39) was
provided by H.I. Magazine and was generated under the same
conditions as the other peptides. Symbols: IFN-y(l-39)
( ), IFN-y(54-91) ( ), IFN-y(78-107) ( ),
IFN-y(95-133) < ) .


64
is shown in figure 3-6. These predictions are in agreement
with what is observed with CD studies of human IFN-y (80) and
of synthetic peptides of mouse IFN-y. All six of the a-
helices possess some degree of apolar periodicity that is
characteristic of a globular protein that can fold into a
common tertiary motif called a four-a-helix bundle (123-125).
The apolar periodicity forms a hydrophobic ridge on one side
of an a-helix that would allow it to interact with other
helices to form a four helix bundle.
Considering these results, a simple three dimensional
model of IFN-y was constructed (Figure 3-7) It has become
clear that many proteins, although they have different
primary sequences, fold into similar tertiary structure
motifs (126). Seemingly unrelated proteins have been found
to have virtually the same topology (126). A pattern of
structural motifs are repeated throughout proteins of known
structure. IFN-y and IL-2 share a number of structural
components, so the IFN-y tertiary structure was principally
patterned after the three dimensional structure of IL-2 which
has been derived by X-ray crystallography (127). Therefore,
the following similarities form the rationale for basing the
structure of IFN-y on the tertiary structure of IL-2. Both
proteins are of a similar length. Both proteins are
predicted to contain around 60% a-helix and no p-sheet as
determined by predictive algorithms (107,108,122), CD studies
of mouse IFN-y synthetic peptides and human IFN-y (80), and
by X-ray crystallographic studies for IL-2 (127).


65
1 3
1 1
35
20
Glycosilation
site
Figure 3-6. Predicted secondary structure of mouse IFN-y.
Wavy lines depict random structure, coils represent a-helix,
and lines represent turns and loops. There is a predicted
omega loop structure between residues 12-20. Hatched ovals
represent glycosylation sites.


66
COOH
Figure 3-7. Predicted tertiary structure of murine IFN-y.
The barrels depict a-helices and the letters indicate the
order of each helix along the sequence from N-terminus to C-
terminus.


67
Interleukin-2 has six helices, one of which contains a
proline kink, while IFN-y is predicted to contain six
helices. Interleukin-2 possesses an extended loop in the N-
terminus between the first and second helix and IFN-y is
predicted to have a similar loop (109). There is a predicted
amphipathic helix in the C-terminus of IFN-y (Figure 3-8) and
a known amphipathic helix in the C-terminus of IL-2 (111).
Interleukin-2 folds into a four-helix bundle motif (127).
This bundle motif, as well as most of the other structural
components of IL-2, were successfully predicted using similar
methods as those described here (128).
The predicted tertiary structure of IFN-y follows the
backbone of IL-2 closely, with two major exceptions.
Interleukin-2 has a disulfide bridge that links the second
and fourth turn. Interferon y has no disulfide bonds. Also,
in IL-2, the fifth helix is six residues in length and is
shorter than the others. This helix does not participate in
the four-helix-bundle and forms a bridge between the fourth
and sixth helices. The fourth helix in IFN-y is the shorter,
six residue helix. Therefore, the fourth and fifth helices
were transposed in the model of IFN-y. The fourth helix
forms a bridge between the third and fifth helices.
This model fits well with the existing data concerning the
structure and function relationship of IFN-y and a list of
this information can be found in Table 5. The key feature
involves the location of the functional domains. Evidence
suggests that the N- and C-terminus are important for


68
Figure 3-8. Helix wheel depicting amphipathic a-helix in the
C-terminus of mouse IFN-y between residues 106-120.
Hydrophobic residues are circled.


69
Table 3-5. Summary of information used to generate the 3-dimensional
model of mouse IFN-y.
N-terminal domain
N-terminal peptide, IFN-y(l-39), directly blocks IFN-y
binding and function
Reference
88
Polyclonal and monoclonal antibodies directed to the
N-terminus neutralize function through the blockage
of receptor binding.
87, 88
chapter 2
C-terminal domain
Site directed polyclonal antisera to the C-terminus
block IFN-y receptor binding and neutralize function.
4,this
work
Truncations of the C-terminus of human IFN-y abrogate
function.
81,83
Central region
Antibodies to the central region of the molecule have
no effect on IFN-y activity.
this work
Secondary structure prediction
Predicted location of turns: Residues- 16-20. 36-40.
60-64, 81-85, 100-104.
107,108,
122
The remainder of the the molecule is predicted to form
a-helices with some random structure. No P-sheet is
predicted.
107,108,
122
Circular dichroism
Human IFN-y consists of 66% a-helix. No P-sheet present.
80
Circular dichroism of overlapping synthetic peptides of
mouse IFN-y confirm the presence of mainly a-helical
structure and the absence of P-sheet.
this work
Tertiary structure prediction
Presence of apolar periodicity noted in all a-helices
which suggests the probability of a four-a-helix
bundle motif.
123-125
Functional data suggest the N and C-termini are
functional domains interacting with the receptor
which places them close together.
87,88,
chapter 2
Structural similarity to IL-2. Reasonable to base model
on backbone of IL-2 three dimensional structure.


70
receptor binding and are in close proximity. In this model
the N- and C-terminal helices are close together and both
could presumably interact with receptor. It has been
postulated that the functional unit of IFN-y exists as a
dimer (75-77) or tetramer (78) in solution. However, IFN-y
has been shown to have activity when isolated in its monomer
form (75). Thus, the functional form of IFN-y has yet to be
resolved. For simplicity, the model has been built based on
a single polypeptide chain. It is difficult to speculate,
based on existing data, which structural features might be
involved in the formation and maintenance of multimer IFN-y.
One additional observation involves the glycosylation sites
on the molecule. The glycosylation sites are included in
secondary structure prediction in figure 3-6. The sites
would fall in the N-terminal loop region and in the turn
between helix C and helix D on the tertiary structure
prediction (figure 3-7). These sites are exposed and
accessible to sugar residues in the model.
Although other orientations are possible, this model was
chosen as the most probable based on existing data.
Additionally, the overall structural features, the four-a-
helix bundle and the N- and C-termini exposed and together,
are the most important characteristics. This model provides
a working base that will be useful in the design of future
studies involving this important lymphokine.


CHAPTER 4
SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS
IFN-y is an important immunoregulatory lymphokine that
is involved in a diverse number of activities related to the
immune response. By understanding its mechanism of action
one may be able to modulate its activities in some way that
may be beneficial. Possibly, its many activities may be
dissected and the generation of agonists or antagonists that
differentially modulate IFN-y activity may be able to be
designed. To achieve these ambitious goals one must first
understand, at the molecular level, what structures are
involved in IFN-y function. There has been a great deal of
work carried out in this area but much information is
lacking. The work that has been described within this
dissertation focuses on the identification and
characterization of functional domains of mouse IFN-y through
the use of monoclonal and polyclonal antibodies and the
synthetic peptide approach.
A mAb neutralizes IFN-y by blocking IFN-y binding to
receptor, so a description of the epitope to which it binds
may also give insight to this IFN-y binding domain. The
epitope specificity of this mAb was mapped with synthetic
peptides to the N-terminus of the molecule and found to be
contained between residues 3-14 of IFN-y. This region is
predicted to contain an amphipathic a-helix between residues
71


72
3-11. The residues at positions three, four and five were
shown to be important when analogs with nonconservative
substitutions that abrogated the amphipathicity of the (X-
helix were found not to bind the mAb. A truncated peptide
that lacked tyrosine at position 14 also lacked binding
ability. Circular dichroism analysis confirmed the presence
of a-helix in the peptides in the presence of TFE but not in
water alone, suggesting that secondary structure is not
important for epitope structure, however secondary structure
is certainly present in the intact molecule. The epitope can
be described as a linear discontinuous epitope because of the
lack of secondary structure requirement and the fact that it
is longer than most continuous epitopes (8-10 residues). The
charge and hydropathy of the residues contained in the N-
terminal part of the amphipathic helix as well as the
tyrosine at position 14 are critical to the epitope. These
studies have characterized the site of interaction of a mAb
that binds to an important region on IFN-y.
It has been previously demonstrated that a peptide of
IFN-y that corresponds to the first 39 residues of the
molecule has the ability to block IFN-y function and receptor
binding (88). This peptide does not have agonist properties
and interacts with the receptor with l/1000th affinity as
IFN-y. Therefore, other regions of IFN-y must be important
in receptor binding. Studies have suggested that the C-
terminus may also be involved but have not been conclusive
(81-84,87). Additionally, the remainder of the molecule has


73
not been examined. Long overlapping peptides that correspond
to the entire sequence of IFN-y were synthesized. Only IFN-
y(l-39), as shown previously (88), had the ability to compete
for receptor. Antibodies raised to the peptides were able to
bind to IFN-y but only antibodies to the N-terminus and to
the C-terminus could neutralize IFN-y activity. These
neutralizing antibodies blocked IFN-y receptor binding. The
neutralizing epitopes of the C-terminal specific antiserum
are found along the entire region as evidenced by
peptide/antibody competition studies. Only peptides
corresponding to the entire C-terminus or combinations of
peptides that reconstruct the C-terminus with shorter
sequences could effectively compete with IFN-y for antiserum
binding to block IFN-y neutralization. Thus, both the N-
terminus and the C-terminus are involved in binding.
Data presented here support a two domain model for IFN-y
binding to its receptor. The N-terminus and C-terminus are
the only regions shown to be directly involved in IFN-y
receptor binding and since only one receptor protein has been
identified (17-21), the N-terminus and C-terminus are
probably relatively close together in the active IFN-y
molecule. Circular dichroism studies of human IFN-y show
that the molecule is 66% a-helix and no P-sheet. Circular
dichroism spectra of overlapping synthetic peptides of mouse
IFN-y confirm the ability of these peptides to form a-helical
conformation with the absence of P-sheet. Predictive
algorithms divide the molecule into six ahelices and five


74
turns and each of the helicies has a degree of apolar
periodicity. These characteristics allow for the potential
that the IFN-y forms a four-helix-bundle motif with the N-
terminus and C-terminus close together forming receptor
binding domains. The internal a-helices are hydrophobic and
buried within the structure. Ideally, x-ray crystallographic
data would be most helpful in understanding IFN-y. This
model is based on the similarities that exists between IFN-y
and IL-2. IL-2 is comprised of seven a-helices with a four
helix bundle motif. The N-terminus and C-terminus are also
in close proximity.
These studies have described in further detail and have
advanced the understanding of the structure/function
relationship of IFN-y. They describe a molecule that
interacts with its receptor with two domains, one in the N-
terminus and one in the C-terminus. The role of the N-
terminus is clear. Based on previous studies the N-terminus
participates directly in receptor binding (88). The role of
the C-terminus is, as yet, unclear. It may also participate
directly in binding or may act to stabilize another part of
the molecule. Further study is required to determine its
role.
Future studies should concentrate on the study of IFN-y
using molecular biology techniques. In the past, the
usefulness of this approach has been minimal due to the lack
of a rational basis for site-directed mutations. Random
mutations have yielded molecules with reduced activity but


75
have resulted in little understanding as to the reason for
the change in function. A more directed approach is required
where deletions and substitutions are targeted toward areas
shown to be involved in binding. The synthetic peptide and
antibody approach used in this and other studies have
identified regions of the IFN-y molecule for site-directed
mutations. With the information at hand we are in a position
to begin engineering the IFN-y molecule, attempting to design
agonists and antagonists that can modulate its function.
Another area of potential interest concerns the molecular
basis for species specificity of the IFN-y molecule. The
synthetic peptide approach would also be useful in
determining critical residues of IFN-y that are responsible
for species specificity. Chimeric peptides of human and
mouse IFN-y could be designed that contain sequences in
common to the two species as well as residues unique to each.
These peptides could then be assessed for their relative
abilities to block IFN-y binding to receptor. With this
information, recombinant molecules could then be constructed
to further study the basis for species specificity of IFN-y.


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128.Cohen, F.E., Kosen, P.A., Kuntz, I.D., Epstien, L.B.,
Ciardelli, T.L., and Smith, K.A., Structure-activity
studies of interleukin-2. Science 234:349. 1986.


BIOGRAPHICAL SKETCH
Michael Andrew Jarpe was born on July 19, 1962, to Jay
Stephen and Marion Blair Jarpe in St. Joseph, Michigan. He
has one sister, Jennifer, and four brothers, Stephen, Geoff,
Matthew, and Andrew. After moving to New Mexico with his
family in 1975, Michael grew up in a rural setting which had
a strong influence on his development. He graduated from Los
Lunas High School in 1980 and enrolled in New Mexico
Institute of Mining And Technology, a small science oriented
college, to obtain a degree in biology. There he met and
married his wife, Alyssa Joy Casper, on June 17, 1984. He
earned his B.S. in biology in December, 1984.
In August, 1985, Michael entered the Ph.D program in
immunology in the department of Pathology and Laboratory
Medicine at the University of Florida. He began his study
under Dr. Stephen W. Russell and subsequently moved to the
laboratory of Dr. Howard M. Johnson in August, 1987. After
receiving his degree in August, 1990, Michael will be
pursuing the study of IFN-y at the molecular biology level
with Dr. Johnson in a postdoctoral position.
89


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy
Howard M. Johnson, Chair
Graduate Research Professor
of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Docftof of Philoepplw. Q
¡i.
John R. Dankert
Assistant Professor of
Veterinary Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, pru scope and quality, as
a dissertation for the degree of Doqitqr^of^ Philosophy.
Paul A. Hargraj/e
Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Linds# M. Hutt Fletcher
Associate Professor of
Pathology and Laboratory-
Medicine


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Aiuv^C^F^L
Associate Professor of
Pathology and Laboratory
Medicine
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy
August, 1990
_ l. ^ V7 >
Dean; College of Medicine
Dean, Graduate School


Full Text
UNIVERSITY OF FLORIDA
I in in mu in .
3 1262 08554 3931


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STRUCTURE-FUNCTION STUDIES OF MURINE GAMMA INTERFERON: A
SYNTHETIC PEPTIDE AND ANTIBODY APPROACH
BY
MICHAEL ANDREW JARPE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990

ACKNOWLEDGEMENTS
I wish to acknowledge the following individuals who were
instrumental in helping me at some point in my life as a
student. First of all, thanks go to Howard for his patience
and scientific philosophy and for not getting sick of my face
before this was complete. Thanks belong to my committee
members, John Dankert, Paul Hargrave, Lindsey Hutt-Fletcher,
and Brough Peck, for their helpful guidance through this long
process called graduate school. I credit Allen G. Harmsen
for giving me my first taste of science. I thank my parents,
Jay and Marion, for bringing me up the way they did and my
bothers and sister for not killing me on long car trips.
Thank go to Myron, Carol, Sarah, Jeff, Mark, Rob, Jeanne,
Russell, Lori, Steve, Steve, and Doug, my comrades in arms,
who have helped me along the way by calming me down when
things got too serious or livening things up when they became
boring. And finally, I thank my wife, Alyssa, for without
her love and support (financial and otherwise) this whole
ordeal would not have been worth it.
ii

TABLE OF CONTENTS
ease
ACKNOWLEDGEMENTS ii
ABSTRACT iv
CHAPTERS
1. INTRODUCTION
Interferons: Historical Perspective 1
IFN-y Function 5
Structure/Function Studies of IFN-y 8
The Synthetic Peptide Approach 11
Summary and Objective 14
2. STRUCTURE OF AN EPITOPE IN A REGION OF THE IFN-y
MOLECULE THAT IS INVOLVED IN RECEPTOR
INTERACTION
Introduction 16
Material and Methods 17
Results and Discussion 24
3. TOPOLOGY OF RECEPTOR BINDING DOMAINS OF MOUSE IFN-y
THAT INCLUDE THE N-AND C-TERMINI.
Introduction 4 6
Materials and Methods 47
Results and Discussion 52
4. SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS 71
LIST OF REFERENCES 7 6
BIOGRAPHICAL SKETCH 89
iii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STRUCTURE/FUNCTION STUDIES OF MURINE GAMMA INTERFERON: A
SYNTHETIC PEPTIDE AND ANTIBODY APPROACH
By
Michael Andrew Jarpe
August, 1990
Chairman: Howard M. Johnson
Major Department: Pathology and Laboratory Medicine
Gamma Interferon (IFN-y) is an important
immunoregulatory lymphokine that affects a wide variety of
immune functions. I have undertaken studies to elucidate the
structural basis for IFN-y function. These
structure/function studies of IFN-y have yielded data on the
location of functional domains.
A monoclonal antibody (mAb) specific for the N-terminus
of mouse IFN-y was shown to neutralize IFN-y function by
blocking receptor binding. I mapped the epitope specificity
of this monoclonal antibody to residues 3 through 14.
Additionally, residues at positions 3, 4, and 5, are
particularly important in the structure of the epitope, since
peptides that lacked these residues had reduced ability to
interact with the mAb. Furthermore, peptide analogs that
iv

replaced these residues with nonconservative substitutions
lost antibody binding activity. Tyrosine in position 14 was
also found to be critical, since peptides lacking it also
lost binding activity. The epitope specificity for this mAb
encompasses the 12 residues between 3 and 14 and is linear
and discontinuous. Because this mAb blocks IFN-y receptor
binding, this epitope may be involved in receptor
interaction.
Antibodies raised against five overlapping synthetic
peptides that encompass the entire sequence of mouse IFN-y
were tested for their ability to inhibit IFN-y function.
Only antisera raised against the N-terminal and the C-
terminal peptides could neutralize IFN-y by blocking receptor
binding. Further mapping studies showed that the C-terminal
specific antiserum was directed to several neutralizing
epitopes within the region. The antibody neutralization data
suggest that IFN-y contains two binding domains, one in the
N-terminus and one in the C-terminus. A three dimensional
model of IFN-y has been constructed utilizing both functional
data, predictive algorithms for secondary and tertiary
structure, and comparison to the known structure of IL-2.
The molecule is predicted to form six a-helices divided by
five turns and to form a four-helix bundle motif. The N-
terminus and C-terminus form two receptor binding domains and
are found close together. This model provides a working base
for future studies.
v

CHAPTER 1
INTRODUCTION
Interferons: Historical Perspective
Interferon (IFN) was first discovered in 1957 by Isaacs
and Lindenmann (1) as a substance that had the ability to
protect host cells against viral infection. The term
interferon has subsequently come to describe a class of
proteins that fall into three distinct groups designated as
IFN-a, IFN-P, and IFN-y. IFN-a and IFN-P are classically
produced in cells induced by viruses or polyribonucleotides
(2). IFN-y is classically produced by T-lymphocytes and
natural killer (NK) cells in response to antigen or mitogen
stimulation (2).
Genes for all three classes of IFNs have been cloned and
sequenced. IFN-a and IFN~P have about 29% sequence homology
while IFN-y has little homology to IFN-a or IFN~p. IFN-a and
IFN~P have been shown to exist in most vertebrates; however,
IFN-y has been shown to exist only in mammals (3). IFN-a is
actually a family of 23 genes each without introns and 15 of
these genes encode for full length proteins of between 165
and 172 amino acid residues (4). There are three IFN~P
genes, one without introns coding for a protein of 166 amino
acid residues and two with four introns each (5-10). The
1

2
genes containing introns are called IFN-J32 or IL-6 (5) . IFN-
(32 has been shown to be a B-cell differentiation factor
(originally called BSF-2) that can induce B-cell
proliferation and enhance antibody production (6,8).
Although IFN-p2 activity can be neutralized by antiserum to
IFN-{3, IFN-f}2 bears little homology to the other IFN-f$
molecules and shows little antiviral activity, therefore its
classification as an IFN is debatable (11).
IFN-y has been cloned in human (12,13), mouse (14), rat
(15), and bovine (16) and is encoded by a gene with three
introns and it appears that there is only one IFN-ygene
present in the genome. A strong species specificity exists
with IFN-y (11). Table 1-1 illustrates the amino acid
sequence homology between the sequences of the four known
IFN-y proteins from human, cattle, rat and mouse. The
sequence analysis was performed on a microVAX using sequence
data from the GenBank database and the program GAP. GAP
aligns sequences and produces a best fit by inserting gaps
within the sequences. The program reports the percentage of
residues that are identical between sequences and also
reports the percentage similarity. Similarity is defined as
the percentage of identical residues plus the percentage of
residues that are conservative substitutions. In most of the
comparisons the percentage identity is low. For example,
when human IFN-y and mouse IFN-y are compared, there is a 40%
identity (Table I). However, the percentage similarity is
67%. Sixty-seven percent similarity indicates there are many

3
conservative amino acid substitutions. This same trend
appears in all of the comparisons. The lack of homology may
account for the species specificity. Based on the degree of
similarity, it is likely that the proteins are structurally
similar. This suggests that information concerning the
location of functional domains in one species may be
applicable to other species.
A receptor for IFN-y has been identified, cloned and
sequenced in both human and mouse (17-21) and shown to exist
on chromosome six in the human (22). The deduced amino acid
sequence yields a protein of 451 and 472 amino acids for
mouse and human, respectively (17-21). There is about 55%
overall homology between the mouse and human proteins (18-
21). A predicted transmembrane domain divides the protein in
half and both domains bear no appreciable homology to any
known receptors (17-21). The external domain comprises the
binding domain and when expressed on the surface of a cell
exhibits identical binding characteristics as the native
receptor which suggest that there is only one binding protein
for IFN-y (17-21). The expressed protein has no function
when IFN-y binds to it (17-21). However, if the short arm of
human chromosome 22 is present in hamster/human hybrid cells
in addition to the receptor, function is restored, suggesting
that there is some other factor required for functional
activity in addition to binding of IFN-y (23) . The
requirements of the receptor for binding and the requirements

4
Table 1-1. Amino acid sequence homology between known IFN-y
proteins from different species.
Species
Human
Bovine
Rat
Bovine
62(76)
-
-
Rat
37(60)
45(68)
-
Mouse
40(67)
43(67)
86(95)
Values are percent
amino acid homology.
Values in parentheses
are
percent homology plus percent conservative substitutions.

5
of other as yet undiscovered factors for receptor function
need further study.
IFN-y Function
IFN-y is a glycoprotein of approximately 20,000 daltons
that possesses many diverse activities. IFN-y was
classically described as a product of white blood cells
stimulated with phytohaemagglutinin, a T-cell mitogen, that
possessed antiviral activity (24). Later, this product was
shown to be different from the type I interferons (IFN-a and
IFN-fJ) in its instability at pH 2 (24) . IFN-y was also found
to be antigenically different from type I IFNs (25). These
characteristics, in part, classify it as type II IFN or
immune IFN. The observation that IFN had immunomodulatory
activities was initially made with IFN-a and IFN-(} when it
was shown that they had a suppressive effect on antibody
production (26-31). IFN was also shown to regulate delayed
type hypersensitivity (32-36), cellular immunity (32-36), and
natural killer cytotoxic activity (37-40). IFN-y was shown
to possess immunomodulatory activities, some in common to and
others unique from IFN-a and IFN-p (41). In fact, the
immunoregulatory potency of IFN-y is much greater than that
of IFN-a and IFN-p (42) .
IFN-y has the ability to modulate antibody production by
B lymphocytes both positively and negatively (43-49). IFN-y
was shown to suppress in vitro antibody response, in mice,

6
before clonal expansion of B-cells had occurred (44,45,47).
Conversely, IFN-y had a positive effect when added to B-
lymphocytes that had already undergone clonal expansion by
inducing antibody secretion and terminal differentiation
(43, 46, 48,49) .
The increased expression of a variety of cell surface
molecules can be achieved with IFN-y. IFN-y can increase the
expression of interleukin-2 receptors on lymphocytes (50).
IFN-y can increase surface expression of Fc receptors on
macrophages which are involved in antibody binding and
antibody dependent cell cytotoxicity (51,52). Class-I major
histocompatibilty antigen expression is increased by IFN-y
(53,54). Class-I expression is required for T-cell
recognition for the killing of virally infected cells.
Class-II molecule expression, which is critical for antigen
presentation and the induction of an immune response, is also
increased by IFN-y (55,56).
Another immunomodulatory function of IFN-y, and possibly
one that has received the most attention, is the activation
of a variety of cytotoxic cells that have the ability to kill
tumor cells. Both NK cell activity and cytotoxic T-cell
activity are increased by IFN-y (37-40,57). The tumoricidal
activity of macrophages has been shown to be augmented by
factor(s) liberated from antigen or mitogen stimulated
lymphocytes and has been termed macrophage activating factor
or MAF (58-62). The process of activation actually occurs in
a series of steps, beginning with priming of macrophages by

7
MAF then subsequent triggering by a small amount of
lipopolysaccharide (LPS) (60,61) . Macrophages so treated
have a greatly enhanced ability to kill tumor cells in vitro.
MAF activity has been shown to be mainly, if not exclusively,
due to IFN-y (59,60,63). Recombinant or purified natural
IFN-y has potent MAF activity, and IFN-y neutralizing
antibodies can remove MAF activity completely from MAF
containing cultures (60, 63). Both IFN-a and IFN-|3 have MAF
activity (58) , although they are much less potent when
compared with IFN-y (64).
The in vivo properties of IFN-y have been studied
primarily in the context of its potential anti-cancer
properties. Studies have shown, at least in experimental
animal systems, that IFN-y has a direct role in host tumor
defense (65,66). In a mouse tumor model where tumors can be
implanted that either progress or spontaneously regress when
the dose of tumor cells is varied, higher levels of IFN-y
were observed in regressing tumors (65). Additionally, when
an IFN-y neutralizing monoclonal antibody (mAb) was injected
intraperitoneally into mice that possessed regressing tumors,
the tumors were converted to progressing tumors. A non¬
neutralizing mAb that bound IFN-y but did not neutralize its
function had no effect on tumor growth. Others have shown
similar results using direct tumor injection of IFN-y
neutralizing mAbs in similar tumor models (66). These
finding suggest that IFN-y may be functioning in host tumor
defense under normal circumstances, but when administered in

8
clinical trials, IFN-y seems to be ineffective in treating
tumors (67-74). The reason for its ineffectivity is unknown.
Structure/Function Studies of IFN-y
When the genes for human and mouse IFN-y were cloned in
the early 1980s and the primary sequence was determined, it
was thought that an understanding of the mechanism for IFN-y
function would soon follow. To the contrary, the
structure/function relationship of IFN-y has proven to be
more complicated than first thought. Studies designed to
explore this area have involved the use of a variety of
techniques and can be divided into approaches using molecular
biology, antibodies, synthetic peptides, and combinations
thereof. Some of these studies have yielded conflicting
results.
Some studies have shown that IFN-y may exist as
homodimer (75-77) or larger multimer (78); however, the exact
role of these multimer structures is unclear. It has been
shown that IFN-y isolated in its monomer form has activity
(75). Recombinant IFN-y expressed in E. coli lacks
glycosylation but retains full activity (12,14) which shows
that glycosylation is not required for IFN-y activity. It
has been postulated that glycosylation of IFN-y provides
protection of the mature protein against proteolysis (75).
Initially, the amino (N) terminus of IFN-y was thought to be
the amino acid sequence CYS-TYR-CYS which left the

9
possibility of disulfide bond formation (12,14). This
possibility was rejected when N-terminal sequencing of human
natural IFN-y showed that the N-terminus was pyroglutamic
acid (75). Therefore, no disulfide bonding occurs in IFN-y.
Structure/function studies have been carried out with
IFN-y using truncated molecules generated through mutation of
cDNA clones or through limited proteolysis of recombinant
IFN-y (79-83). Human and mouse IFN-y lacking the N-terminal
eight or nine amino acid residues have been shown to have
significantly reduced functional activity and reduced
receptor binding activity (79-80). This abrogation of
activity in human IFN-y was shown to correlate to a
significant loss in a-helical structure (80). Recombinant
human IFN-y lacking portions of its C-terminus had either a
significant reduction in activity (81,83) or no change in
activity (82). Human recombinant IFN-y treated with
proteases that removed 11 (81) or 13 (83) residues from the
C-terminus showed a significant decrease in IFN-y function
and receptor binding activity. Conversely, a mutant
recombinant IFN-y from E. coli that lacked the 23 C-terminal
residues retained full activity (82). The reason for this
discrepancy remains unclear and may have something to do with
proteolytic attack altering more than just the C-terminus,
although in each proteolytic experiment N-terminal sequencing
confirmed that the N-terminus was not altered (81,83).
The use of antibodies, both polyclonal and monoclonal,
to probe for functional sites along the IFN-y molecule has

10
proven useful in the study of the structure/function
relationship of IFN-y. Most of these studies hint at a two
or multiple functional domain model of IFN-y; however,
controversy surrounds the issue of where these domains lie.
Some evidence exists that suggests that there are different
functional domains for different IFN-y functions such as
antiviral activity and macrophage priming activity (84);
however, this has yet to be confirmed. One study using 21
neutralizing mAb to human IFN-y supports a multi-domain model
(85). These mAb were divided into two groups using antibody-
antibody competition assays with each group having a
different epitope specificity, suggesting more than one
functional domain. The location of these epitopes is unknown
as they have not been mapped. Other studies have either
localized mAb epitopes or raised site directed antibodies to
synthetic peptides of the IFN-y molecule. Antibodies raised
against a synthetic peptide corresponding to the first 20
residues of human IFN-y (the first three residues were CYS-
TYR-CYS) neutralized IFN-y activity, suggesting that the N-
terminal region of IFN-y was involved in function (86). A
polyclonal antiserum raised against a synthetic peptide
corresponding to the first 39 residues of mouse IFN-y, IFN-
y(l-39), neutralized IFN-y activity by blocking IFN-y
receptor binding (87). Additionally, the epitope specificity
of a mAb shown to neutralize mouse IFN-y by blocking receptor
binding was mapped preliminarily to residues 1-20 of the N-
terminus of the molecule (88), supporting the role of the N-

11
terminus as a functional domain. Others have demonstrated
that two mAb specific for the N-terminus of mouse IFN-y also
can neutralize IFN-y activity (84). In a more direct
experiment the synthetic peptide IFN-y(1-39) was able to
block directly IFN-y function and compete specifically with
IFN-y for it receptor (88). Thus, the role of the N-terminus
in receptor binding seems clear.
Polyclonal antiserum raised against the C-terminal
peptide of mouse IFN-y, IFN-y(95-133), blocks IFN-y function
and receptor binding (87). The peptide, IFN-y(95-133) , did
not compete with IFN-y directly (88). Two C-terminal
specific mAb can also neutralize mouse IFN-y activity (84).
In contrast, studies have shown that mAb specific for the C-
terminus of human IFN-y are unable to neutralize IFN-y
activity and have concluded that the C-terminus is not
important (89). However, the epitope specificities of these
mAb have been directed to the last 15 residues of IFN-y and
may have missed an important functional site.
The Synthetic Peptide Approach
The synthetic peptide approach has generated much
information with regard to the location of functional domains
on a number of different proteins. This approach can be
divided into three related methods that utilize synthetic
peptides to probe for functional domains. The first method
involves the use of synthetic peptides to directly interact

12
with receptors or other molecules. Synthetic peptides
corresponding to portions of ligands have been shown to
compete directly with the larger intact molecule, thereby
identifying a region critical for receptor interaction. A
synthetic peptide corresponding to residues 400-411 of gamma
chain human fibrinogen was shown to bind to fibrinogen
receptor on platelets and block fibrinogen binding to its
receptor (90). A site of interaction on the superantigen
staphylococcal enterotoxin A (SEA) to class II molecules was
found to be contained within the first 45 residues of SEA
using direct peptide competition (91). These examples
illustrate that synthetic peptides can act as direct probes
for functional domains on proteins.
A second facet of the synthetic peptide approach is the
use of synthetic peptides to map the epitope specificity of
mAb that have been raised to an intact protein. Monoclonal
antibodies that have been shown to interfere with the
activity of a molecule are presumably binding to a region on
the molecule that is important for its activity. Synthetic
peptides can then be used to determine the epitope
specificity of the mAb and thereby identify an important
functional region. An example of this method is the
determination of a functional site on the interleukin-2 (IL-
2) molecule (92). Monoclonal antibodies raised against IL-2
were shown to inhibit its function and were mapped to the
between residues 8-54 of the molecule using synthetic
peptides.

13
A third part of the synthetic peptide approach involves
the use of synthetic peptides to produce site-specific
antibodies to regions of proteins. These antibodies, both
polyclonal and monoclonal, can interact with the native
protein and block its activity by interfering with an
important domain. This technique has been successful in a
number of different systems. In the study of IFN-y, antisera
raised to a synthetic peptide corresponding to the N-terminal
20 residues of human IFN-y neutralized IFN-y activity (86) .
Polyclonal antibodies produced to a peptide from the 15 N-
terminal residues of human tumor necrosis factor neutralized
its activity (93). Antibodies raised to synthetic peptides
of erythropoietin located a functional domain in the region
between residues 99 and 129 (94).
As described, all of these uses of the synthetic peptide
approach have been successful at locating functional sites on
proteins. The interpretation of the results obtained from
these studies is important. Studies where synthetic peptides
can directly interact with receptors are easily interpreted.
The peptide contains all or part of a functional domain of
the protein. The identification of a synthetic peptide with
these properties is not always possible. Some peptides do
not adopt sufficient conformation in solution to be able to
react with receptors. Our laboratory has found that longer
peptides have a greater likelihood of possessing stable
secondary structure and a greater chance of direct
interaction. When direct peptide competition is not found,

14
the use of antibodies as probes for functional domains can be
successful. The antibody molecule, by binding to a region
critical for activity, can interfere with the function of the
protein studied. One important drawback to this approach is
the possibility that the antibody may be inducing a
conformational change in the molecule that disrupts a domain
in a distant region. Another is the possibility that the
antibody may only bind to a subset of denatured molecules.
One way to minimize these potential problems is to use a
combination of the approaches discussed. This would increase
the likelihood of identifying a true functional domain.
Summary and Objective
At present, the state of knowledge concerning IFN-y is
one in which a large amount of information has been generated
about it but there remain questions concerning the number,
location, and characterization of functional domains. From
earlier work with molecular biology, antibodies, and
synthetic peptides there appears to be at least two distinct
functional domains of IFN-y. These domains are believed to
be located in the N-terminus and the C-terminus of the
molecule. However, little is known about the functional role
of the internal regions of the sequence. Studies with site
directed mutation of human IFN-y provide some suggestive
evidence that the internal region is critical to IFN-y
function, at least by being involved in the maintenance of

15
tertiary structure (95). Single amino acid substitutions in
the core of the molecule reduced receptor binding and
structure. Also, little has been done to further
characterize these potential domains with regard to precise
mapping of important regions.
It is the objective of this dissertation to further the
understanding of the structure/function properties of IFN-y
by attempting to identify other functional domains and by
further characterization of those that are known. This will
be accomplished through two main specific aims. First, the
N-terminal functional domain will be further characterized by
mapping the precise epitope specificity of an N-terminal
specific mAb that neutralizes IFN-y by blocking receptor
binding. Second, other regions of the molecule will be
explored for functional significance using long overlapping
synthetic peptides encompassing the entire sequence of IFN-y
and antiserum raised to these peptides.

CHAPTER 2
STRUCTURE OF AN EPITOPE IN A REGION OF THE IFN-y MOLECULE
THAT IS INVOLVED IN RECEPTOR INTERACTION
Introduction
The N-terminal region of IFN-y has been shown to contain
a binding site for the IFN-y receptor based on specific
blockage of IFN-y receptor binding by a synthetic peptide
corresponding to the first 39 residues of IFN-y and based on
blockage of IFN-y function by the N-terminal 39 residues as
well as the first 20 residues of IFN-y (88) . A monoclonal
antibody has been produced that has been shown to neutralize
IFN-y function (88). This mAb neutralizes IFN-y activity by
blocking IFN-y binding to its receptor (88). This suggests
that this mAb binds to a functional site on the IFN-y
molecule that is involved in receptor interaction. The
epitope to which this mAb binds is partially mapped to the
first 20 residues of IFN-y using synthetic peptides as
competitors for binding of radiolabeled IFN-y to the mAb
(88). Interestingly, this region of IFN-y is predicted to
contain an amphipathic a-helix between positions three and
eleven. Amphipathic helices are thought to be involved in
immunodominance of antigens related to B-cell and T-cell
recognition (96,97).
16

17
The first specific aim of this dissertation is to
characterize the epitope specificity of this mAb in more
detail. Competition studies with peptide truncations
determined that residues 3,4, and 5 of IFN-y were required
for binding to the mAb. These residues are predicted to
participate in an amphipathic a-helix spanning residues 3-11
of IFN-y. The tyrosine at position 14 was also required as
its removal in C-terminal truncations caused the loss of
blocking ability. It can be concluded that the IFN-y epitope
for the neutralizing mAb involves residues 3-14, spanning 12
residues, and it appears that residues 3, 4, 5, and 14 are an
important part of the epitope and that this epitope can be
considered linear and discontinuous. The data presented here
provide further insight to the structure of a site that is
involved in receptor interaction.
Material .and Methods
Synthetic peptides. Peptides were synthesized with a
Biosearch 9500AT automated peptide synthesizer using FMOC
chemistry (98). Specific analogs were partially constructed
using a manual FMOC synthesis system (RaMPS, Dupont).
Peptides were assembled to a given point in the sequence with
the automated synthesizer. The peptide bearing resin was
then divided and individual analogs completed with the manual
system. Peptides were cleaved at 25° C from the resins using
trifluoroacetic acid/phenol/ethanedithiol at a volume ratio

18
of 95.0/4.5/0.5, respectively. The cleaved peptide was then
precipitated in ether and ethyl acetate and subsequently
dissolved in water and lyophilized. Reverse phase HPLC
analysis of crude peptides indicated one major peak in each
profile. Synthetic peptides were submitted to the Protein
Core Facility at the University of Florida for amino acid
analysis. Figures 2-1 through 2-4 are HPLC profiles and
amino acid analyses of the crude peptides used in these
studies.
Radioiodination of recombinant IFN-y. Recombinant
murine IFN-y was obtained from Schering-Plough Pharmaceutical
(Bloomfield, NJ) with a specific activity of 5 x 10^ units
per mg of protein. A unit of IFN-y activity is defined as
the concentration of IFN-y that reduces by 50% the plaque
formation of vesicular stomatitis virus on mouse L cells
(99). IFN-y was labeled with 125j by using chloramine T as
described (87). The specific activity of ^-25j_jpN-y was
generally 25-35 JiCi/ |lg protein (ICi = 37 GBq) .
MAb to IFN-y. MAb 5.102.12 was previously produced
(87) from the fusion of spleen cells from an Armenian hamster
immunized with IFN-y. Tissue culture supernatants containing
mAb were applied to a protein-A sepharose column. Antibody
was eluted with 0.58% acetic acid in 0.15 M NaCl. Eluted
fractions were immediately neutralized with one-tenth volume
of 1.5 M tris-HCl, pH 8.8, and dialyzed against phosphate

19
Amino acid
analysis
Asx
1.05
Thr
0.94
Ser
0.89
Glx
0.99
1 Gly
0.93
Val
0.70
lie
0.84
Leu
1.00
Tyr
0.97
Phe
0.95
His
0.94
Figure 2-1. HPLC profile and amino acid analysis of IFN-
y(l-20). Amino acid analysis values are the ratio of
expected number of residues divided by the actual number of
residues observed.

20
Figure 2-2. Purity data for synthetic peptides. A.
IFNy(2-20) HPLC profile. This peptide was synthetsized from
the same resin as IFNy(l-20) so no amino acid analysis was
done. B. IFNy(l-14) HPLC profile and amino acid analysis.

21
Figure 2-3. Purity information of synthetic peptides of
mouse IFNy. A. Amino acid analysis and HPLC profile of
IFNy(3-20) . B. HPLC profile of IFNy(4-20) . C. HPLC profile
IFNy(5-20). D. HPLC profile of IFNy(6-20). Amino acid
analysis was performed on IFNy(3-20) only because all other
peptides were synthesized from the same resin.

22
Figure 2-4. Purity information of synthetic peptides of
mouse IFNy. A. Amino acid analysis and HPLC profile of
IFNy(3-20) S3I4L5. B. Amino acid analysis and HPLC profile
of IFNy(3-20) S3L4V5. C. Amino acid analysis and HPLC
profile of IFNy(3-20) I3K4Y5. D. Amino acid analysis and
HPLC profile of IFNy(3-20) L3Y4K5.

23
buffered saline, pH 7.4. MAb 5.102.12 is an N-terminal
specific mAb raised against IFN-y and has been partially
characterized previously (87,88). This mAb has the ability
to neutralize the antiviral and macrophage priming activity
of IFN-y as well as block the specific binding of ^-25j_jpN-y
to its cell surface receptor. The N-terminal specificity of
the mAb was demonstrated by its binding to a ^5i-iabeled
synthetic peptide corresponding to the first 39 residues of
IFN-y, IFN-y(l-39) , and by its ability to block the binding
of IFN-y by 5.102.12 in a competitive radioimmunoassay
(RIA)(87). The epitope was further localized to the first 20
residues of IFN-y, IFN-y(l-20) , using a competitive RIA (88) .
Radioimmunoassay (RIA) and ELISA. Assays were performed
in duplicate at room temperature as described (100).
Briefly, mAb 5.102.12 was added to protein A (Sigma, P6650)
coated wells of 96 well PVC microtiter plates (Falcon, 3912).
Plates were washed of excess mAb after which 20 |ll of 125j_
IFN-y was added. An equal volume of competitor or medium was
added thirty minutes prior to the addition of labeled IFN-y.
After one hour, wells were washed, cut out and counted on a
gamma counter (LKB). Competition was assessed by comparing
cpm values to that of medium controls. Competitive ELISA was
carried out with the same protocol except biotinylated IFN-y
was substituted for the radiolabeled IFN-y. After the
mixture was allowed to react for one hour, the plates were
washed and alkaline phosphatase conjugated streptavidin
(Sigma) was added to the wells. After one hour the plates

24
were washed and substrate was added. Color development was
monitored on a microplate reader (BioRad).
Circular dichroism (CD). CD for selected peptides was
determined at room temperature using a JASCO 500C
spectropolarimeter. Scans were done with a 0.1 cm pathlength
cell at a sensitivity of 0.5-2.0 and a time constant of 8
seconds. The wavelength range measured was from 250 nm to
200 nm at a scan rate of 20 nm/ min. Scans were carried out
on peptides in 25% trifluoroethanol (TFE) in water at
concentrations of 0.1 to 0.5 mg/ml (101-103). The CD spectra
were expressed in terms of A£ related to the mean residue
ellipticity at a given wavelength ( [0]^) for each peptide.
The following formula was used to generate A£ (104):
^ ^ X _[® ] observed
L0-lx_ cxl
A£=
3298
where [0]^ and [^ ] 0bservecl are exPressed in degrees
equals the mean residue concentration in moles/liter,
is the pathlength of the cell in cm.
/ c
and 1
Results -an.d-.Ei.sgiis.sipn
Preliminary studies suggested that the N-terminal
residues of IFN-y(l-20) played an important role in the
epitope for mAb 5.102.12 (88). In order to ascertain the N-
terminal residue requirement for the epitope of this mAb in
more detail, I synthesized IFN-y(l-20) and its truncated
forms, IFN-y(2-20), IFN-y(3-20) , IFN-y(4-20), IFN-y(5-20),
and IFN-y(6-20), and tested the peptides for their ability to

25
block specific binding of -*-25];_iFN_y to 5.102.12 in a
competitive RIA. The peptides IFN-y(l-20), IFN-y(2-20) , and
IFN-y(3-20) all had similar ability to block IFN-y binding
(Figure 2-5). In addition, as the peptides were shortened
they correspondingly lost blocking ability incrementally
until they reached position six where IFN-y(6-20) lost
blocking function. The third, fourth, and fifth residues
appear to be the most important because the peptides lost
their blocking ability when these residues were deleted.
The observation that it took at least 1000 times more
peptide to block IFN-y binding to mAb illustrates the type of
reaction that is occurring in peptide competition studies of
this nature. The interaction of a synthetic peptide with a
monoclonal antibody raised against a native protein is
actually a cross reaction (105). The peptide is not the
actual antigen and probably adopts the conformation of the
intact protein for only a small percentage of the time. This
can account for the lower affinities commonly seen with
peptide-antibody interactions.
It had been previously reported that IFN-y(3-20) had a
reduced blocking activity compared to IFN-y(l-20) (88) . The
greater activity of IFN-y(3-20) observed in the present
report can possibly be attributed to a change in synthesis
chemistry. The peptides in the previous report were
synthesized by t-Boc chemistry and cleaved by HF cleavage
procedures, whereas the peptides utilized in the present
study were synthesized by FMOC chemistry. The purity data

26
Peptide concentration (log M)
Figure 2-5. Competitive RIA with synthetic peptides of IFN-y
and mAb 5.102.12. The ability of synthetic peptides,
IFN-y(l-20) and its N-terminal truncations, to block
125j-jfn-Y (20nM) binding to mAb 5.102.12 was assessed.
Competition is expressed as percent of control binding to
wells that contain no peptide competitors. Control binding
was 33,816 ±100 cpm (cpm ± S.D.)in the absence of
competition. The figure is representative of three
independent experiments. Symbols: IFN-y(l-20) (!) ,
IFN-y(2-20) (â–¡) , IFN-y(3-20) (O) , IFN-y(4-20) (A) , IFN-y(5-
20) (A) , IFN-y(6-20) (•).

27
from the t-Boc synthesis of IFN-y(3-20) was unavailable. In
order to illustrate the differences between FMOC and t-Boc
synthesis chemistries, I have compared the purity of the
peptide IFN-y(l-20) synthesized by both methods. HPLC
profiles of IFN-y(1-20)FMOC and IFN-y(1-20)t-Boc are shown in
Figure 2-6. The FMOC peptide exists as one major species
while the t-Boc peptide is heterogeneous. The multiple peaks
seen in HPLC profiles of peptides can contain different
components. Some of these peaks are truncations and
deletions of the main sequence that are produced during the
synthesis. Some peaks are incompletely deprotected peptides
that still possess some of the side chain protecting groups
that are used in the synthesis. Finally, the remainder of
the peaks are peptides that have been chemically modified
during the cleavage in hydrogen fluoride which is used for
t-Boc cleavage and can be harsh on peptides through the
generation of carbonium ions (106). It is likely that the
difference seen in the blocking activity of the t-Boc IFN-
7(3-20) and the FMOC IFN-y(3-20) is due to modifications of
the t-Boc peptide. However, this is only speculation and
cannot be determined unequivocally without the original
peptide.
One way that residues three to five may function in the
epitope for mAb 5.102.12 is that they may actually be a part
of the epitope and serve as contact residues for mAb
5.102.12. Another possibility is that they may act to
stabilize adjacent contact regions of the epitope. The

28
Figure 2-6. HPLC profiles of t-Boc and FMOC peptides. A.
IFN-yd-20) synthesized by FMOC chemistry. B.IFN-yd-20)
synthesized by t-Boc chemistry.

29
predicted secondary structure of IFN-y(l-20) is presented in
Figure 2-7A, and is based on the method of Chou and Fasmen
(107,108) . The region of 12-20 is proposed to form a loop
that may be found on the surface of the molecule and thus
have a high probability of forming an antigenic
determinant(s) (109). The remainder of the molecule is
predicted to be primarily a-helix. The N-terminal residues
three to five, which are depicted as initiating the helix
may act to stabilize the helix region thereby stabilizing the
main epitope. Interestingly, the predicted helix between
residues 3 and 12 is amphipathic in nature (96). Figure 2-7B
represents a helix wheel in which the residues found in the
predicted helix region are plotted in relation to their
positions in the helix. Hydrophilic and hydrophobic residues
are separated which is characteristic of an amphipathic
helix. The hydrophilic side of the helix presumably
interacts with the hydrophilic environment. Amphipathic
regions are thought to play a role in immunodominance in B-
cell and T-cell recognition (96,97). Amphipathic helices
have also been shown to be important in the functional sites
of some proteins (110) . The amphipathic helix in the C-
terminus of IL-2 has been shown to be required for IL-2
function (111).
To gain further insight into the role of the N-terminal
residues of IFN-y(l-20) in the epitope, peptide analogs were
synthesized with either conservative or nonconservative amino
acid substitutions. The criteria for conservative and

30
Figure. 2-7. Structure of the N-terminal 20 residues of
IFN-y. A. Predicted secondary structure of IFN-y(l-20) .
This model is derived from the predicted structure of
IFN-y(3) . Alpha helical structure is depicted as coils,
however the number of turns is not intended to represent the
actual topography of the region. Wavy lines represent
regions that are not predicted to have stable structure (e.g.
residues 1-3) . The (3-bend is represented as a trapezoid
shape (residues 16-20). B. Helix wheel depicting amphipathic
helix of IFN-y found between residues three and eleven.
Hydrophobic residues are circled.

31
nonconservative substitutions was based on side chain
structure, charge, and hydrophobicity (112) of amino acid
residues. Due to the incremental nature of the decrease in
binding of truncations, positions three, four, and five were
changed simultaneously. Table 2-1 presents the native
residues as well as analogs of IFN-y(3-20) . The native
sequence for IFN-y(l-20) at positions three, four, and five
consists of threonine, valine, and isoleucine, respectively.
Two conservative and two nonconservative analogs were
constructed. The first conservative analog contained serine,
isoleucine, and leucine at these positions. The second
conservative analog had serine, leucine, and valine. These
substitutions are similar to the native sequence with respect
to charge and side chain structure and are designed to
maintain the charge in this region. One nonconservative
analog contained isoleucine, lysine, and tyrosine at
positions three, four, and five, while the other contained
leucine, tyrosine, and lysine. The charge and side chain
structure of these substitutions are different than the
native residues and are designed to disrupt the charge and
hydropathicity in this region. The data presented in Figure
2-8 illustrate the similar ability of the two conservative
analogs to block binding of ^25j_ipN_y to 5.102.12 relative
to IFN-y(3-20). In contrast, there was a significant
reduction in blocking activity by both nonconservative
analogs. Thus, the structure of residues three to five of

Table 2-1. Amino acid sequences of IFN-y(l-20) and its truncated analogs.
Type of substitution Sequence
IFN-y(1-20) HIS GLY THR VAL ILE GLU SER LEU GLU SER LEU ASN ASN TYR PHE ASN SER SER GLY ILE
Native
IFN-y(3-20)
SER3,LEU4, VAL5
Conservative
SER I LE LEU - -- -- -- -- -- -- --
IFN-y(3-20)
SER3,LEU4'VAL5
Conservative
SER LEU VAL -
IFN-y(3-20)
ILE3,LYS4,TYR5
Nonconservative
I LE LYS TYR - -- -- -- -- -- -- -- £
IFN-y(3-20)
leu3,tyr4,lys5
Nonconservative
LEU TYR LYS - -- -- -- -- -- -- --

33
Figure. 2-8. Competitive RIA with peptide analogs of IFN-y
and mAb 5.102.12. The ability of IFN-y(l-20), IFN-y(3-20),
and their analogs to block 125j-iFN-y (8 nM) binding to mAb
5.102.12 was assessed. Competition is expressed as percent
of control binding to wells that contain no peptide
competitors. Control binding was 69471451 cpm (cpm ± S.D.).
The figure is representative of three independent
experiments. Symbols: IFN-y(l-20) (â– ) , IFN-y(3-20) ( O ) ,
conservative IFN-y(3-20) SEr3, ile4' leu5 (A), conservative
IFN-y(3-20) SER3, LEU4' VAL$ (A) , nonconservative IFN-y(3-
20) ILe3, lys4' TYR5 (â–¡), nonconservative IFN-y(3-20) LEU3,
TYR4, LYS5 (X), IFN-y(6-2 0) (•) .

34
IFN-y appears to play an important role in the epitope
specificity of mAb 5.102.12.
To verify the presence of a-helical structure in the
peptides, CD spectroscopy was employed (104,113). The CD
spectra of the peptides are obtained in 25% trifluoroethanol
(a secondary structure stabilizing agent) and show the
potential of each peptide to form a particular secondary
structure. As shown in Figure 2-9, the CD spectra of peptide
IFN-y(l-20) indicated mainly a-helix characterized by a
pronounced minimum at 220 nm. IFN-y(3-20) also had helical
structure that was less than that of IFN-y(l-20) .
Conversely, the truncated peptide IFN-y(6-20), which lacked
function, lost most of its a-helical structure. IFN-y(3-20) ,
SER^, ILE^, LEU^, a conservative analog, had a-helical
structure that was slightly less than IFN-y(l-20) but greater
than the native truncation IFN-y(3-20). As indicated, this
conservative analog was as effective as IFN-y(3-20) in
blocking IFN-y binding to mAb 5.102.12. The nonconservative
analog, IFN-y(3-20) ILE^, LYS^, TYR^/ which lost blocking
activity, exhibited almost identical secondary structure to
the IFN-y(l-20) (primarily a-helix). Thus, the loss of
blocking activity of substituted analogs was not due to loss
of a-helical structure. Positions three, four, and five
apparently participate in the formation and/or stabilization
of the a-helix, based on the lack of helix found in the
truncation IFN-y(6-20). The interesting observation that the
nonconservative analog did not block function but still

35
Wavelength (nm)
Figure. 2-9. CD spectra of IFN-y(l-20), its analogs, and N-
terminal truncations. Spectra were obtained with peptides in
25% TFE as described in the Materials and Methods. Symbols:
IFN-y(1-20) ( ), IFN-y(3-20) ( ), IFN-y(6-20)
( ), Conservative analog IFN-y(3-20) SER3, ile4/ LEUS
( ), Nonconservative analog IFN-y(3-20) ILE3, LYS4^ TYR5
( ) •

36
retained stable secondary structure would suggest that the a-
helix alone is not critical and that residues three to five
are directly involved in the epitope of the N-terminal
peptide. The nonconservative substitutions are predicted to
participate in a-helix formation as effectively as the
conservative substitutions which is consistent with the
stable secondary structure of the analog containing these
residues (107). Additionally, the conservative analogs
examined maintain the amphipathic nature of the helix, while
the nonconservative analogs disrupt it through changes in
hydrophobicity and charge. Thus, the structural and
functional data implicate the third, fourth, and fifth
residues directly in the epitope of mAb 5.102.12, and that
the charge and hydropathicity of these residues is important.
Peptides were synthesized with C-terminal truncations in
order to determine the role of residues in the putative loop
region in the epitope specificity of mAb 5.102.12.
IFN-y(l-14) and IFN-y(l-20) had similar blocking ability,
whereas IFN-y(l-13) possessed no activity, which is evidence
that tyrosine at position 14 is important for epitope
structure (figure 2-10). IFN-y(3-14) also had full blocking
activity. Thus, the minimum length requirement of the
epitope for mAb 5.102.12 involves residues 3 to 14.
I next examined the secondary structure of C-terminal
truncations of IFN-y(l-20). Both IFN-y(l-14) and IFN-y(l-13)
had an apparent reduced helical structure, but not complete
abrogation, with an apparent increase in random structure

37
Peptide concentration (log M)
Figure. 2-10. Competitive RIA with C-terminal truncations
of IFN-y(1-20) and mAb 5.102.12. The ability of IFN-y(l-20)
and its C-terminal truncations to block 125i-iFN-y (22 nM)
binding to mAb 5.102.12 was assessed. Competition is
expressed as percent of control binding to wells that contain
no peptide competitors. Control binding was 56,204 ±153 cpm
(cpm ± S.D.). The figure is representative of three
independent experiments. Symbols: IFN-y(l-20) (!) ,
IFN-yd-13) (O) , IFN-y( 1-14) (A) , IFN-y(3-14) (â–¡) .

38
(minimum at 205-200 nm) (figure 2-11) . The reduction in (X-
helix in these truncations may involve a partial loss of
helix in the C-terminal region of the peptide caused by the
removal of residues C-terminal to the helix (residues 3-11).
Since both peptides had similar reduction in helix and
IFN-yd-14) but not IFN-yd-13) blocked mAb binding, it
suggests that factors additional to a-helix may play a role
in the epitope.
All of the experiments described have used crude
peptides. Although, as I have shown, the purity of FMOC
peptides is higher than t-Boc, the differences in purity as
shown by HPLC profiles of the peptides used warrants further
examination. To attempt to address this issue, wo peptides,
IFN-y(1-20) and IFN-y(l-13) were purified by HPLC.
IFN-y(l-20) had a final purity of 90% and IFN-y(l-13) had a
final purity of 96% as determined by HPLC. Amino acid
analysis and HPLC profiles of the purified are shown in
figure 2-12. The results of a competitive ELISA (Figure 2-
13) show that crude and purified IFN-y(l-20) have identical
blocking profiles. Purified IFN-y(l-13) has no blocking
activity. Therefore, no impurity was responsible for
blocking activity. Additionally, the lack of purity was not
responsible for a lack of blocking activity.
To further explore this issue, I will attempt to explain
the nature of the impurities found in crude synthetic
peptides and relate this to my findings. Much of the
information that I present here comes from my own

39
Wavelength (ntn)
Figure 2-11. CD spectra of IFN-y(l-20) and its C-terminal
truncations. Spectra were obtained with peptides in 25% TFE
as described in the Materials and Methods. Symbols:
IFN-y(1-20) ( ) , IFN-y(l-13) ( ),
IFN-y(l-14) ( ) .

40
Figure 2-12. Amino acid analysis and HPLC profiles on
purified synthetic peptides. Amino acid analysis values are
the ratio of expected number of residues divided by the
actual number of residues observed. A. IFN-y(l-13) crude.
B. IFN-yd-13) purified. C. IFN-y (1-20) crude. D.
IFN-y(l-20) purified.

41
o
u
o
U
o
120
100 -
Peptide concentration (log M)
Figure 2-13. Competitive ELISA of purified synthetic
peptides of IFN-y. ELISA was carried out under the
conditions described in materials and methods. Biotinylated
IFN-y concentration used was 25 nm. Symbols: IFN-y(l-20)
crude (•), IFN-y(l-20) purified (o), IFN-y(l-13) purified
(A) .

42
observations and from discussion with peptide chemists at
Biosearch and Advanced Chemtech. Most of the impurities are
due to amino acid deletions and/or the incomplete removal of
side chain protecting groups. Amino acid deletions can be
identified by amino acid analysis. Incomplete deprotection
is commonly seen with the MTR group on arginine and the TRT
group on histidine. However, other protecting groups can
also be difficult to remove, depending on the sequence of the
peptide. Frequently these "impure" peptides, due to the
nature of the assay being used, exhibit the same activity as
complete sequence. This may be because some of the residues
affected may not be important for the activity of the
peptide.
It has been my observation that HPLC alone cannot give a
clear picture as to the purity of a synthetic peptide.
Protein sequencing is the only certain way of determining,
beyond question, the content of a peptide. As this is
impractical in routine situations, amino acid analysis must
be used to verify the presence of each amino acid residue.
When interpreting an amino acid analysis, the ratio of
observed to expected should be close to 1.0 +/- 10%, with
some exceptions. Serine, valine, methionine, isoleucine,
histidine, and tyrosine can be found in lower amounts due to
modifications during the hydrolysis reaction for the amino
acid analysis. Tryptophan and cysteine require special
hydrolysis. In my opinion, each peptide presented here has

43
an acceptable amino acid analysis and all of the residues are
present in reasonable quantities.
Differences in synthesis can be responsible for varied
purity of peptides. Each synthesis is unique to the sequence
being synthesized. Although two sequences are similar one
sequence may be more easily synthesized than the other.
Another observation that I have made concerns the
content of peaks seen in HPLC profiles. Due to the high
absorption seen with some protecting groups used in the
synthesis, a relatively large peak can contain a
disproportionately small amount (by weight) of material when
isolated and analyzed. The large peak observed can be
misleading.
A further indication of the suitability of crude
peptides in these mapping studies can be illustrated with the
following examples. Blocking studies presented in this
dissertation are interpreted in a qualitative manner. A
peptide either blocks or does not block IFN-y binding to mAb.
One exception to this involves the N-terminal truncations of
IFN-y(1-20) (IFN-y(3-20), iFN-y(4-20), lFN-y(5-20), and
IFN-y(6-20) ) . These peptides lose blocking activity
incrementally as residues are left off. If one examines the
HPLC profiles of IFN-y(5-20) and IFN-y(6-20) (Figure 2-3 C,D)
they appear to be qualitatively similar. Also, these
peptides were synthesized from the same resin. However, when
ILE at position 5 is left off as in IFN-y(6-20) blocking
activity is reduced (Figure 2-5). This would suggest that

44
the lack of ILE at position 5 is responsible for this
difference in blocking activity, and not a difference in
purity. The difference seen in activity appears to be
greater than a difference in the HPLC profiles of the
peptides. The peptide IFN-y(1-14) has blocking activity
similar to IFN-y(l-20) as shown in figure 2-10. The amino
acid analysis appears to be reasonable based on the sequence
(Figure 2-2). It shows the presence of TYR in the peptide
while the amino acid analysis of IFN-y(l-13) does not (Figure
2-12). The HPLC profile contains two major peaks, each about
30-40% of the total. The content of the second peak is not
known but it is likely to be an incompletely deprotected
peptide. This impurity seems to not have an effect on
blocking activity. Also, if the peptide were three fold
greater in purity (>90% pure), one would expect little
difference in blocking activity. The profile for IFN-y(l-14)
would be shifted slightly to the left one half log and not be
very different than the profile of IFN-y(l-20) . The lack of
TYR in IFN-y(l-13) seems to be what is responsible for a lack
of blocking activity.
The data presented in this chapter indicate that the
epitope specificity of the IFN-y neutralizing mAb 5.102.12
spans 12 amino acid residues, and is located between the
third and fourteenth residues of IFN-y with residues 3, 4, 5,
and 14 critical to the interaction of mAb 5.102.12 with IFN-
y. Epitopes are classified into four groups, linear,
conformational, continuous, and discontinuous (105).

45
Discontinuous and conformational epitopes are those that are
made up of more than one distant site on the surface of a
protein. The contact residues are separated by non-critical
residues (105). A classic conformational epitope that has
been studied is in the molecule lysozyme. X-ray
crystallographic studies of a monoclonal antibody binding to
an epitope on lysozyme have detailed a conformational
epitope. Linear and continuous epitopes are epitopes found
on short sequences not more than eight to ten residues in
length that involve a single stretch of residues (105,114-
118). Synthetic peptides are common continuous and linear
epitopes. This epitope for 5.102.12 does not appear to be
continuous in the classical sense based on its length, twelve
residues. It also does not appear to require a stable
conformation based on CD studies. Based on the requirement
of the residues 3,4,5,and 14, the linear peptide IFN-y(3-14)
appears to contain a discontinuous epitope. The role that
internal residues may play in this epitope is as yet
undetermined. Thus, the data suggest that the mAb binding
site on IFN-y exists as a linear discontinuous epitope. The
classification of linear discontinuous epitope has been used
in another report (119). In this case the terminology was
discontinuous linear epitope and was used to describe the
epitope specificities of several mAb to synthetic peptides.

CHAPTER 3
TOPOLOGY OF RECEPTOR BINDING DOMAINS OF MOUSE IFN-y THAT
INCLUDE THE N-AND C-TERMINI.
Introduction
IFN-y possesses a number of immunologic activities that
are critical to immune function. The identification of the
receptor binding domains should further enhance the
understanding of its mechanism of action. This information
would be useful for the design of agonists and antagonists of
IFN-y function. The location of these domains is unclear.
The N-terminus of IFN-y has been shown to bind to the
receptor as determined by competition studies where the first
39 residues of IFN-y, IFN-y(l-39), compete with IFN-y for its
receptor (88). Antibody studies confirm the role of the N-
terminus of IFN-y in receptor binding (87,88). The epitope
specificity of an IFN-yneutralizing monoclonal antibody was
mapped to the first 14 residues of IFN-y (Chapter 2). The
binding site on IFN-y for receptor probably involves regions
in addition to IFN-y(l-39), since this peptide binds to
receptor at l/1000th the affinity of IFN-y (88). The
functional significance of the remainder of the molecule has
not been determined. Additionally, the role of the C-
terminus in IFN-y function has been controversial. A
46

47
truncated form of recombinant human IFN-y missing the C-
terminal 23 residues was shown to possess full activity (82).
In contrast, the removal of 11 residues from the C-terminus
of IFN-y, with limited proteolysis, yielded IFN-y with
significantly reduced activity (81). Studies have shown that
polyclonal antibodies directed to a C-terminal synthetic
peptide, IFN-y(95-133) , neutralized IFN-y activity (87).
Similarly, mAb that appear to be directed to the C-terminus
of IFN-y also neutralize IFN-y activity (84) .
The second specific aim of this dissertation is to
explore other regions of the molecule for functional
significance using long overlapping synthetic peptides
encompassing the entire sequence of IFN-y and antiserum
raised to these peptides. The data strongly suggests that
the C-terminus, in addition to the N-terminus, of mouse IFN-y
is important in receptor binding and function. Using
synthetic peptides, antisera raised against these peptides,
circular dichroism spectra for secondary structure, and
predictive algorithms for secondary and tertiary structure,
a three dimensional model of IFN-y has been constructed that
includes both the N and C-termini as receptor binding
domains.
Materials and Methods
Animals and immunizations. Female New Zealand White
rabbits (2-2.5kg) were immunized with synthetic peptides

48
conjugated with keyhole-limpet hemocyanin using
gluteraldehyde conjugation (120). One hundred micrograms of
conjugated peptide were injected with Complete Freund's
adjuvant in a total volume of 0.5 ml followed 30 days later
with 100 |lg of conjugated peptide in Incomplete Freund's
adjuvant. Seven days later blood was drawn and serum was
collected. Subsequent boost injections occurred at 30 day
intervals followed by bleeds seven days later.
Synthetic peptides. Peptides were synthesized with a
Biosearch 9500AT automated peptide synthesizer using FMOC
chemistry (98). Peptides were cleaved at 25° C from the
resins using trifluoroacetic acid/phenol/ethanedithiol at a
volume ratio of 95.0/4.5/0.5. The cleaved peptides were then
precipitated in ether and ethyl acetate and subsequently
dissolved in water and lyophilized. The purity of peptides
was assessed by reverse phase HPLC and verified by amino acid
analysis. Amino acid analysis and HPLC profiles for the
peptides used for immunization are found in figure 3-1. The
peptides used in epitope mapping studies were purified using
a preparative reverse phase HPLC column (Vydac). The major
peak was collected, lyophilized, and checked for purity with
analytical HPLC and amino acid analysis. The amino acid
analysis and HPLC profiles for purified peptides are found in
figure 3-2.

49
Figure 3-1. Purity information of overlapping synthetic
peptides of mouse IFN-y. A. IFN-y(36-60) . B. IFN-y(54-
91). C. IFN-y(78-107) . D. IFN-y(95-133).

50
Figure 3-2. Purity information on purified synthetic
peptides used in C-terminal specific antibody mapping
studies. A. IFN-y(7 8-107) . B. IFN-y(95-133) . C.
IFN-y(108-133) . D. IFN-y(120-133) . E. IFN-y(108-119) .

51
ELISA. Synthetic peptides and IFN-y were dissolved in
0.1 M sodium carbonate buffer, pH 9.6, at a concentration of
12 |lg/ml and 50 |ll of solution were added to each well of a
96 well flat bottom tissue culture plate (Falcon). After
overnight incubation at room temperature, each plate was
dried with mild heat applied by a standard blow dryer.
Nonspecific binding sites were blocked with 200 |ll/well 5%
Carnation powdered milk dissolved in PBS for 2 hours. Plates
were then washed with 0.15 M NaCl/0.05% Tween 20 five times.
Dilutions of antisera were added in 50 |ll amounts and
incubated at room temperature for 2 hours. After washing,
horseradish peroxidase conjugated goat anti-rabbit IgG
antibodies (Cappel) were added to each well and incubated for
one hour. The plates were washed and 0-phenylenediamine
(Sigma) and H2O2 were added and color development was allowed
to occur for 15-30 minute after which 25 |ll of H2SO4 was
added to stop the reaction. Absorbance was determined at 492
nm in an ELISA plate reader (BioRad). Endpoints were defined
as the highest dilution of antisera with absorbance readings
that were twice those of the blank.
Circular dichroism (CD). CD for the peptides was
determined as described in chapter 2 except for the following
changes. Scans were done with a 0.01 cm pathlength cell at a
sensitivity of 1.0 and a time constant of 8 seconds. The
wavelength range measured was from 260 nm to 184 nm at a scan
rate of 5 nm/ min.

52
IFN-y neutralization assay. Varying concentrations of
IFN-y were incubated with dilutions of rabbit antisera for 30
min at 37°C. Residual IFN-y activity was then measured as
described (99). Briefly, IFN-y samples in the presence or
absence of antibodies were incubated with mouse L cells for
16 to 18 hr at 37°C after which inhibition of virus
replication was determined in a plaque reduction assay with
vesicular stomatitus virus. One unit of IFN-y was defined as
that which caused a 50% reduction in plaque formation. For
epitope mapping studies, synthetic peptides were incubated
with antibodies for 30 min at 37°C prior to their addition to
IFN-y. The peptide inhibition of IFN-y neutralization by
anti-peptide antisera was compared to IFN-y alone and to
antibody plus IFN-y in the absence of peptide. For peptide
blocking studies, synthetic peptides were substituted for the
antibodies. Peptides were dissolved in HMEM containing 2%
FBS. No preincubation with IFN-y was performed.
Results and Discussion
To examine the role of the remainder of IFN-y, long
overlapping peptides were synthesized covering the entire 133
amino acids of IFN-y. The peptides synthesized were: IFN-
7(3 6-60), IFN-y(54-91) , IFN-y(78-107) , and IFN-y(95-133) and
are shown in Table 3-1. These peptides were tested for their
abilities to inhibit IFN-y antiviral activity. N-terminal
peptide, IFN-y(l-39), had the ability to directly inhibit

53
Peptide
Sequence
Hydropathy3
IFN-yd-39)
HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDG
-0.559
IFN-y(36-60)
QKDGDMKILQSQIISFYLRLFEVLK
-0.036
IFN-y(54-91)
RLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAF
-0.189
IFN-y(78-107)
TTFFSNSKAKKDAFMSIAKFEVNNPQVQRQ
-0.617
IFN-y(95-133)
AKFEVNNPQVQRQAFNELIRWHQLLPESSLRKRKRSRC
-0.792
a Average hydropathy values were calculated by talcing the sum of the
hydropathy values for each amino acid divided by the number of amino acids in
each sequence. Hydropathy values were taken from Kyte and Doolittle (112).

54
IFN-Y function as shown previously (88) while the remainder
of the peptides had no effect (Table 3-2). However, based on
competition studies, IFN-y(l-39) interacted with receptor
with 1/1000 the affinity of IFN-y (88) . Thus, other regions
of the IFN-y molecule are likely to be involved in receptor
interaction. To determine the role of these other regions of
IFN-y, rabbit antibodies were produced to the overlapping
synthetic peptides and tested for their abilities to interact
with and inhibit IFN-yfunction. Antisera to IFN-y(l-39) and
IFN-y(95-133) had been produced previously (87). These
antibodies to IFN-y(l-39) and IFN-y(95-133) were shown to
neutralize IFN-y activity by blocking IFN-y receptor binding
(87). All of the peptides elicited antibodies reactive with
IFN-y as determined by ELISA (Table 3-3). Antibodies
directed against N-terminal peptide, IFN-y(l-39) , also bound
and neutralized IFN-yas shown previously (87). Similarly,
antiserum to the C-terminus had the ability to neutralize
IFN-y antiviral activity. In contrast, antisera to the
internal three peptides had no effect although they bound
IFN-y. The differential neutralizing ability was probably
not due to the differences in titer. When the neutralization
titer is normalized on the basis of ELISA titer to IFN-y by
taking the ratio of neutralizing titer to ELISA titer, the
ratios were very different. If elevated titer alone was
responsible for neutralization then similar ratios would be
expected. Additionally, the ELISA titer to
anti-IFN-y(78-107) is similar to the titers of the

55
Table 3-2. Direct peptide blocking of mouse IFN-Y antiviral activity by
IFN-Y synthetic peptides.
Peptides
Peptide concentration3
IFN-y(l-39)
15 |iM
IFN-y(36-60)
>500 JIM
IFN-y(54-91)
>500 \m
IFN-Y(78-107)
>500 |IM
IFN~Y(95-133)
>500 |1M
aHighest peptide concentration that will block 10 units of IFN-Y
antiviral activity.

56
Table 3-3. IFN-y neutralization efficiency of antipeptide antisera.
Peptide
ELISA titer
Peptide
(E)a
IFN-y
Neutralization (N)
aqainst 10U IFN-y
Ratio13
E/N (xlOO)
lFN-yd-39)
ND
1/10,000
1/3000
30.0
IFN-y(36-60)
1/1000
1/1000
<1/3
<0.3
IFN-y(54-91)
1/1000
1/600
<1/3
<0.05
IFN-y(78-107)
1/100,000
1/20,000
<1/3
<0.015
IFN-y(95-133)
1/300,000
1/60,000
1/6000
10.0
aELISA performed as described in materials and methods. Six
hundred nanograms of each peptide or IFN-y was used in each well.
^Ratio calculated as ELISA titer to IFN-y divided by
neutralization titer multiplied by 100.
CND indicates not done.

57
N- and C-terminal antisera, but does not possess any
neutralizing activity. The results confirm the role of the
N-terminus as a receptor interaction site and suggest that
the C-terminus forms an additional binding domain.
The N-terminal binding domain has been characterized
previously using direct peptide competition and epitope
mapping of an N-terminal specific IFN-y neutralizing mAb (88,
Chapter 2). I was therefore interested in localizing the
epitope to which the C-terminal neutralizing antibodies bind
thereby further characterizing this site on the molecule.
The antiserum, anti-IFN-y(95-133), is polyclonal and
potentially contains antibodies to several epitopes, so there
is the possibility that only a subset of these antibodies is
responsible for the neutralizing activity seen in this
antiserum. Peptide truncations of IFN-y(95-133) were
synthesized and used to map its neutralizing epitope(s).
Figure 3-3 illustrates the peptides synthesized in these
experiments and their relationship to one another. To map
the neutralizing epitope(s) of anti-IFN-y(95-133) , a
modification of the antibody neutralization assay was used in
which synthetic peptides were added to inhibit the IFN-y
neutralization by anti-IFN-y(95-133) . The immunizing
peptide, IFN-y(95-133) , was able to completely inhibit the
neutralizing activity of anti-IFN-y(95-133) (Table 3-4). A
truncation, IFN-y(108-133) , was able to inhibit
neutralization about 30% at the same concentration.
IFN-y(78-107) as well as the shorter peptides IFN-y(120-133)

58
78 90 100 110 120 133
.... TTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC -COOH
78 107
95 133
108 133
120 133
108
119
Figure 3-3. C-terminal synthetic peptides of IFN-y used to
map the neutralizing epitope specificity of the neutralizing
antiserum produced against IFN-y(95-133) . The sequence from
residues 78-133 is listed on top with the individual peptides
represented by lines under the sequence.

Table 3-4. Epitope mapping of the C-terminal specific IFN-y neutralizing antiserum3 and
identification of the neutralizing epitopes.
IFN-y titer (units)b
Peptide Cone.
(mM)
IFN-y(95-
-133) IFN-y(108-133)
IFN-y(78-107)
IFN-y(108-119)
IFN-y(120-133)
IFN-y(78-107)
IFN-y(108-119)
IFN-y(120-133)
0.1
300
100
30
3
30
300
0.03
300
100
30
3
30
300
0.01
300
100
30
3
30
300
0.003
300
100
30
3
30
300
0.001
300
100
30
3
30
300
0.0003
300
100
30
3
10
300
0.0001
100
60
30
3
6
300
0.00003
10
6
10
3
3
100
aAntiserum was
used at
a dilution of 1/1000.
^IFN-y titer in the absence of antiserum was 300 units. In the presence of antiserum the titer was <3
units.
cThe concentration of the mixture of peptides in the last column reflects the individual peptide
concentrations listed in the lefthand column.

60
and IFN-y(108-119) had a slight inhibitory ability when added
separately. However, when the longer sequence was
reconstructed with the combination of the shorter peptides,
lFN-y(78-107), lFN-y(108-119), and lFN-y(120-133), full
inhibitory activity was restored. This suggests that there
are multiple neutralizing epitopes involved in this region
that can function cooperatively and that the antiserum is
probably directed toward several linear sequences.
CD studies of human IFN-y have shown that the molecule
is about 66% a-helix with turn and random regions making up
the difference (80). Mouse and human IFN-y share about 40%
identical sequence homology (20). About 30% of the amino
acid changes are conservative substitutions. Both mouse and
human IFN-y possess very similar surface profiles as shown in
figure 3-4. The surface profile predicts which regions of a
protein may be found on the surface of the molecule. The
surface profile takes into account HPLC mobility, segmental
mobility and surface accessibility parameters for amino acid
residues calculated from model proteins and peptides (121) .
The overlapping profiles suggest that human and mouse IFN-y
are very similar at the structural level. Therefore, it is
reasonable that information obtained concerning human IFN-y
may be applicable to mouse IFN-y. Due to the overall
structural homology between human and mouse IFN-y , mouse
IFN-y is likely to contain a similar amount of a-helix. I
was unable to obtain mouse IFN-y in sufficient quantities to
measure the CD directly; however, I did measure the CD of the

61
O 20 40 60 80 100 120
Residue Number
Figure 3-4. Surface profile of mouse IFN-y (dashed lines)
and human IFN-y (solid lines).

62
overlapping synthetic peptides that encompass the entire
sequence. CD of the peptides was measured in 25%TFE, a helix
stabilizing agent. TFE is commonly used in the CD
measurement of synthetic peptides in order to enhance
existing structure (11,101-103). Alpha helical structure was
observed in all of the peptides characterized by a minimum at
220 nm and a maximum at 190 nm (Figure 3-5). IFN-y(36-60)
was not sufficiently soluble in the concentration required
for CD measurement due to its hydrophobicity. Although the
CD of peptides may differ from that of the native molecule,
these results suggest the probability that the peptides form
a-helix and the likelihood that the native molecule is mainly
a-helical.
Based on the above results an overall topology of mouse
IFN-y can be pictured. Both the N-terminus and C-terminus
participate, in some way, in receptor interaction. The N-
terminus, based on direct peptide competition studies can
interact with receptor. Additionally, others have shown that
when the N-terminal 8-10 residues of human IFN-y are removed
by limited proteolysis, functional activity is reduced along
with a corresponding loss in a-helical structure (80). The
C-terminus, based on my antibody neutralization data, may
also be interacting with receptor or act to stabilize the
structure of the N-terminus. Two secondary structure
prediction algorithms predict that the molecule contains six
a-helices, no P-sheet, and the remainder turns and random
structure (107,108,122). The secondary structure prediction

63
Figure 3-5. CD spectra of overlapping synthetic peptides of
IFN-y. Spectra were obtained in 25%TFE as described in
Materials and Methods. The spectra for IFN-y(l-39) was
provided by H.I. Magazine and was generated under the same
conditions as the other peptides. Symbols: IFN-y(l-39)
( ), IFN-y(54-91) ( ), IFN-y(78-107) ( ),
IFN-y(95-133) < ) .

64
is shown in figure 3-6. These predictions are in agreement
with what is observed with CD studies of human IFN-y (80) and
of synthetic peptides of mouse IFN-y. All six of the a-
helices possess some degree of apolar periodicity that is
characteristic of a globular protein that can fold into a
common tertiary motif called a four-a-helix bundle (123-125).
The apolar periodicity forms a hydrophobic ridge on one side
of an a-helix that would allow it to interact with other
helices to form a four helix bundle.
Considering these results, a simple three dimensional
model of IFN-y was constructed (Figure 3-7) . It has become
clear that many proteins, although they have different
primary sequences, fold into similar tertiary structure
motifs (126). Seemingly unrelated proteins have been found
to have virtually the same topology (126). A pattern of
structural motifs are repeated throughout proteins of known
structure. IFN-y and IL-2 share a number of structural
components, so the IFN-y tertiary structure was principally
patterned after the three dimensional structure of IL-2 which
has been derived by X-ray crystallography (127). Therefore,
the following similarities form the rationale for basing the
structure of IFN-y on the tertiary structure of IL-2. Both
proteins are of a similar length. Both proteins are
predicted to contain around 60% a-helix and no p-sheet as
determined by predictive algorithms (107,108,122), CD studies
of mouse IFN-y synthetic peptides and human IFN-y (80), and
by X-ray crystallographic studies for IL-2 (127).

65
1 3
1 1
^MIJISÍJIa^n
35
20
Glycosilation
site
Figure 3-6. Predicted secondary structure of mouse IFN-y.
Wavy lines depict random structure, coils represent a-helix,
and lines represent turns and loops. There is a predicted
omega loop structure between residues 12-20. Hatched ovals
represent glycosylation sites.

66
COOH
Figure 3-7. Predicted tertiary structure of murine IFN-y.
The barrels depict a-helices and the letters indicate the
order of each helix along the sequence from N-terminus to C-
terminus.

67
Interleukin-2 has six helices, one of which contains a
proline kink, while IFN-y is predicted to contain six
helices. Interleukin-2 possesses an extended loop in the N-
terminus between the first and second helix and IFN-y is
predicted to have a similar loop (109). There is a predicted
amphipathic helix in the C-terminus of IFN-y (Figure 3-8) and
a known amphipathic helix in the C-terminus of IL-2 (111).
Interleukin-2 folds into a four-helix bundle motif (127).
This bundle motif, as well as most of the other structural
components of IL-2, were successfully predicted using similar
methods as those described here (128).
The predicted tertiary structure of IFN-y follows the
backbone of IL-2 closely, with two major exceptions.
Interleukin-2 has a disulfide bridge that links the second
and fourth turn. Interferon y has no disulfide bonds. Also,
in IL-2, the fifth helix is six residues in length and is
shorter than the others. This helix does not participate in
the four-helix-bundle and forms a bridge between the fourth
and sixth helices. The fourth helix in IFN-y is the shorter,
six residue helix. Therefore, the fourth and fifth helices
were transposed in the model of IFN-y. The fourth helix
forms a bridge between the third and fifth helices.
This model fits well with the existing data concerning the
structure and function relationship of IFN-y and a list of
this information can be found in Table 5. The key feature
involves the location of the functional domains. Evidence
suggests that the N- and C-terminus are important for

68
Figure 3-8. Helix wheel depicting amphipathic a-helix in the
C-terminus of mouse IFN-y between residues 106-120.
Hydrophobic residues are circled.

69
Table 3-5. Summary of information used to generate the 3-dimensional
model of mouse IFN-y.
N-terminal domain
N-terminal peptide, IFN-y(l-39), directly blocks IFN-y
binding and function
Reference
88
Polyclonal and monoclonal antibodies directed to the
N-terminus neutralize function through the blockage
of receptor binding.
87, 88
chapter 2
C-terminal domain
Site directed polyclonal antisera to the C-terminus
block IFN-y receptor binding and neutralize function.
4,this
work
Truncations of the C-terminus of human IFN-y abrogate
function.
81,83
Central region
Antibodies to the central region of the molecule have
no effect on IFN-y activity.
this work
Secondary structure prediction
Predicted location of turns: Residues- 16-20. 36-40.
60-64, 81-85, 100-104.
107,108,
122
The remainder of the the molecule is predicted to form
a-helices with some random structure. No P-sheet is
predicted.
107,108,
122
Circular dichroism
Human IFN-y consists of 66% a-helix. No P-sheet present.
80
Circular dichroism of overlapping synthetic peptides of
mouse IFN-y confirm the presence of mainly a-helical
structure and the absence of P-sheet.
this work
Tertiary structure prediction
Presence of apolar periodicity noted in all a-helices
which suggests the probability of a four-a-helix
bundle motif.
123-125
Functional data suggest the N and C-termini are
functional domains interacting with the receptor
which places them close together.
87,88,
chapter 2
Structural similarity to IL-2. Reasonable to base model
on backbone of IL-2 three dimensional structure.

70
receptor binding and are in close proximity. In this model
the N- and C-terminal helices are close together and both
could presumably interact with receptor. It has been
postulated that the functional unit of IFN-y exists as a
dimer (75-77) or tetramer (78) in solution. However, IFN-y
has been shown to have activity when isolated in its monomer
form (75). Thus, the functional form of IFN-y has yet to be
resolved. For simplicity, the model has been built based on
a single polypeptide chain. It is difficult to speculate,
based on existing data, which structural features might be
involved in the formation and maintenance of multimer IFN-y.
One additional observation involves the glycosylation sites
on the molecule. The glycosylation sites are included in
secondary structure prediction in figure 3-6. The sites
would fall in the N-terminal loop region and in the turn
between helix C and helix D on the tertiary structure
prediction (figure 3-7). These sites are exposed and
accessible to sugar residues in the model.
Although other orientations are possible, this model was
chosen as the most probable based on existing data.
Additionally, the overall structural features, the four-a-
helix bundle and the N- and C-termini exposed and together,
are the most important characteristics. This model provides
a working base that will be useful in the design of future
studies involving this important lymphokine.

CHAPTER 4
SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS
IFN-y is an important immunoregulatory lymphokine that
is involved in a diverse number of activities related to the
immune response. By understanding its mechanism of action
one may be able to modulate its activities in some way that
may be beneficial. Possibly, its many activities may be
dissected and the generation of agonists or antagonists that
differentially modulate IFN-y activity may be able to be
designed. To achieve these ambitious goals one must first
understand, at the molecular level, what structures are
involved in IFN-y function. There has been a great deal of
work carried out in this area but much information is
lacking. The work that has been described within this
dissertation focuses on the identification and
characterization of functional domains of mouse IFN-y through
the use of monoclonal and polyclonal antibodies and the
synthetic peptide approach.
A mAb neutralizes IFN-y by blocking IFN-y binding to
receptor, so a description of the epitope to which it binds
may also give insight to this IFN-y binding domain. The
epitope specificity of this mAb was mapped with synthetic
peptides to the N-terminus of the molecule and found to be
contained between residues 3-14 of IFN-y. This region is
predicted to contain an amphipathic a-helix between residues
71

72
3-11. The residues at positions three, four and five were
shown to be important when analogs with nonconservative
substitutions that abrogated the amphipathicity of the OC-
helix were found not to bind the mAb. A truncated peptide
that lacked tyrosine at position 14 also lacked binding
ability. Circular dichroism analysis confirmed the presence
of a-helix in the peptides in the presence of TFE but not in
water alone, suggesting that secondary structure is not
important for epitope structure, however secondary structure
is certainly present in the intact molecule. The epitope can
be described as a linear discontinuous epitope because of the
lack of secondary structure requirement and the fact that it
is longer than most continuous epitopes (8-10 residues). The
charge and hydropathy of the residues contained in the N-
terminal part of the amphipathic helix as well as the
tyrosine at position 14 are critical to the epitope. These
studies have characterized the site of interaction of a mAb
that binds to an important region on IFN-y.
It has been previously demonstrated that a peptide of
IFN-y that corresponds to the first 39 residues of the
molecule has the ability to block IFN-y function and receptor
binding (88). This peptide does not have agonist properties
and interacts with the receptor with l/1000th affinity as
IFN-y. Therefore, other regions of IFN-y must be important
in receptor binding. Studies have suggested that the C-
terminus may also be involved but have not been conclusive
(81-84,87). Additionally, the remainder of the molecule has

73
not been examined. Long overlapping peptides that correspond
to the entire sequence of IFN-y were synthesized. Only IFN-
y(l-39), as shown previously (88), had the ability to compete
for receptor. Antibodies raised to the peptides were able to
bind to IFN-y but only antibodies to the N-terminus and to
the C-terminus could neutralize IFN-y activity. These
neutralizing antibodies blocked IFN-y receptor binding. The
neutralizing epitopes of the C-terminal specific antiserum
are found along the entire region as evidenced by
peptide/antibody competition studies. Only peptides
corresponding to the entire C-terminus or combinations of
peptides that reconstruct the C-terminus with shorter
sequences could effectively compete with IFN-y for antiserum
binding to block IFN-y neutralization. Thus, both the N-
terminus and the C-terminus are involved in binding.
Data presented here support a two domain model for IFN-y
binding to its receptor. The N-terminus and C-terminus are
the only regions shown to be directly involved in IFN-y
receptor binding and since only one receptor protein has been
identified (17-21), the N-terminus and C-terminus are
probably relatively close together in the active IFN-y
molecule. Circular dichroism studies of human IFN-y show
that the molecule is 66% a-helix and no P-sheet. Circular
dichroism spectra of overlapping synthetic peptides of mouse
IFN-y confirm the ability of these peptides to form a-helical
conformation with the absence of P-sheet. Predictive
algorithms divide the molecule into six a—helices and five

74
turns and each of the helicies has a degree of apolar
periodicity. These characteristics allow for the potential
that the IFN-y forms a four-helix-bundle motif with the N-
terminus and C-terminus close together forming receptor
binding domains. The internal a-helices are hydrophobic and
buried within the structure. Ideally, x-ray crystallographic
data would be most helpful in understanding IFN-y. This
model is based on the similarities that exists between IFN-y
and IL-2. IL-2 is comprised of seven a-helices with a four
helix bundle motif. The N-terminus and C-terminus are also
in close proximity.
These studies have described in further detail and have
advanced the understanding of the structure/function
relationship of IFN-y. They describe a molecule that
interacts with its receptor with two domains, one in the N-
terminus and one in the C-terminus. The role of the N-
terminus is clear. Based on previous studies the N-terminus
participates directly in receptor binding (88). The role of
the C-terminus is, as yet, unclear. It may also participate
directly in binding or may act to stabilize another part of
the molecule. Further study is required to determine its
role.
Future studies should concentrate on the study of IFN-y
using molecular biology techniques. In the past, the
usefulness of this approach has been minimal due to the lack
of a rational basis for site-directed mutations. Random
mutations have yielded molecules with reduced activity but

75
have resulted in little understanding as to the reason for
the change in function. A more directed approach is required
where deletions and substitutions are targeted toward areas
shown to be involved in binding. The synthetic peptide and
antibody approach used in this and other studies have
identified regions of the IFN-y molecule for site-directed
mutations. With the information at hand we are in a position
to begin engineering the IFN-y molecule, attempting to design
agonists and antagonists that can modulate its function.
Another area of potential interest concerns the molecular
basis for species specificity of the IFN-y molecule. The
synthetic peptide approach would also be useful in
determining critical residues of IFN-y that are responsible
for species specificity. Chimeric peptides of human and
mouse IFN-y could be designed that contain sequences in
common to the two species as well as residues unique to each.
These peptides could then be assessed for their relative
abilities to block IFN-y binding to receptor. With this
information, recombinant molecules could then be constructed
to further study the basis for species specificity of IFN-y.

LIST OF REFERENCES
1. Isaacs, A. & Lindenmann, J., Virus interference. I. the
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BIOGRAPHICAL SKETCH
Michael Andrew Jarpe was born on July 19, 1962, to Jay
Stephen and Marion Blair Jarpe in St. Joseph, Michigan. He
has one sister, Jennifer, and four brothers, Stephen, Geoff,
Matthew, and Andrew. After moving to New Mexico with his
family in 1975, Michael grew up in a rural setting which had
a strong influence on his development. He graduated from Los
Lunas High School in 1980 and enrolled in New Mexico
Institute of Mining And Technology, a small science oriented
college, to obtain a degree in biology. There he met and
married his wife, Alyssa Joy Casper, on June 17, 1984. He
earned his B.S. in biology in December, 1984.
In August, 1985, Michael entered the Ph.D program in
immunology in the department of Pathology and Laboratory
Medicine at the University of Florida. He began his study
under Dr. Stephen W. Russell and subsequently moved to the
laboratory of Dr. Howard M. Johnson in August, 1987. After
receiving his degree in August, 1990, Michael will be
pursuing the study of IFN-y at the molecular biology level
with Dr. Johnson in a postdoctoral position.
89

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy
Howard M. Johnson, Chair
Graduate Research Professor
of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Docftof of Philoeoplw. Q
6i.
John R. Dankert
Assistant Professor of
Veterinary Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, iru scope and quality, as
a dissertation for the degree of Doqitqr^of^ P^losophy.
ms-
Paul A. Hargraj/e
Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Lindsé# M. Hutt - Fletcher
Associate Professor of
Pathology and Laboratory-
Medicine

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Atu^F^L
Associate Professor of
Pathology and Laboratory
Medicine
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy
August, 1990
_ c „ ^ V7 >
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA
I in in mu in» _ .
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