Structure-function studies of murine gamma interferon

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Structure-function studies of murine gamma interferon a synthetic peptide and antibody approach
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Synthetic peptide and antibody approach
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Jarpe, Michael Andrew, 1962-
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Interferon Type II -- physiology   ( mesh )
Antibodies, Monoclonal -- physiology   ( mesh )
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Molecular Sequence Data   ( mesh )
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Amino Acid Sequence   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 76-88.
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by Michael Andrew Jarpe.
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Typescript.
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Vita.

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Full Text

















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.















TABLE OF CONTENTS


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. ............................. 46
Materials and Methods...................... 47
Results and Discussion..................... 52

4. SUMMARY, CONCLUSIONS, AND FUTURE DIRECTIONS ........... 71

LIST OF REFERENCES ........................................ 76

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









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.














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-J, 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-0 have about 29% sequence homology

while IFN-y has little homology to IFN-a or IFN-0. IFN-a and

IFN-0 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








genes containing introns are called IFN-02 or IL-6 (5). IFN-

(2 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-02 activity can be neutralized by antiserum to

IFN-P, IFN-P2 bears little homology to the other IFN-P

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-7 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









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

















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.









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-P) 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-P 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,









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

factors) 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









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-P 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









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









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









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(1-39), 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-









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









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.









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,









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









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).









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 250 C from the resins using

trifluoroacetic acid/phenol/ethanedithiol at a volume ratio









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 105 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 1251 by using chloramine T as

described (87). The specific activity of 125I-IFN-y was

generally 25-35 gCi/gg 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 NaC1. Eluted

fractions were immediately neutralized with one-tenth volume

of 1.5 M tris-HCl, pH 8.8, and dialyzed against phosphate
















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.









Amino acid analysis


A.1


Asx
Thr
Ser
Glx
Gly
Val
le
Lcu
Tyr
His


JJUK


0.98
0.83
0.94
1.05
0.87
0.68
0.55
1.00
1.07
040


Figure 2-2. Purity data for synthetic peptides. A.
IFNy(2-20) HPLC profile. This peptide was synthetsized from
the same resin as IFNy(1-20) so no amino acid analysis was
done. B. IFNy(1-14) HPLC profile and amino acid analysis.


040








A. Amino acid analysis
Asx 1.21
Thr 0.83
Ser 0.68
Glx 0.88
Gly 1.08
Val 1.14
Be 1.10
Leu 1.00
Tyr 1.05
S Phe 1.10


-J


B.


U


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.


~


JIj











































Figure 2-4. Purity information of synthetic peptides of
mouse IFNy. A. Amino acid analysis and HPLC profile of
IFNy(3-20) S314L5. 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.









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 1251-IFN-y

to its cell surface receptor. The N-terminal specificity of

the mAb was demonstrated by its binding to a 125I-labeled

synthetic peptide corresponding to the first 39 residues of

IFN-Y, IFN-y(1-39), 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(1-20), 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 L1 of 125I-

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








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 AE related to the mean residue
ellipticity at a given wavelength ( [e] ) for each peptide.

The following formula was used to generate AS (104):
[_e [ ] observed
A'= 32i98-- [] -- cxl
where [9] and observedd are expressed in degrees, c

equals the mean residue concentration in moles/liter, and 1

is the pathlength of the cell in cm.


Results and Discussion


Preliminary studies suggested that the N-terminal

residues of IFN-y(1-20) 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(1-20) 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








block specific binding of 125I-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











120



100






20-
















-6 -5 -4 -3

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(1-20) and its N-terminal truncations, to block
125I-IFN-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(1-20) (),
IFN-y(2-20) (0), IFN-y(3-20) (0), IFN-y(4-20) (A), IFN-y(5-
20) (A), IFN-y(6-20) (0).









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(1-20) synthesized by both methods. HPLC

profiles of IFN-y(1-20)FMOC and IFN-y(l-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-

y(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



















A.


Figure 2-6.
IFN-y(1-20)
synthesized


U


a


HPLC profiles of t-Boc and FMOC peptides. A.
synthesized by FMOC chemistry. B.IFN-y(1-20)
by t-Boc chemistry.








predicted secondary structure of IFN-y(1-20) 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(1-20) in the epitope, peptide analogs were

synthesized with either conservative or nonconservative amino

acid substitutions. The criteria for conservative and
















A. B. TH
SER 10 3
7 SER
GLU6
1 3 12 16



4 G

GLU9
20

8
5




Figure. 2-7. Structure of the N-terminal 20 residues of
IFN-y. A. Predicted secondary structure of IFN-y(1-20).
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 P-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.








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(1-20) 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 125I-IFN-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











































































C)




S 4 >




In )U
44 W
Hm U


Ul)
104

C))



in


M O U
HAU


0


c
WO
HZ

H Z


4,

>4.J



O 0
E-4C

DC
WzO
tAZ









111 1Y


100 -




80
so-



60 -




40




20-



0- ------ ------ --- --
-6 -5 -4 -3

Peptide concentration (log M)



Figure. 2-8. Competitive RIA with peptide analogs of IFN-y
and mAb 5.102.12. The ability of IFN-y(1-20), IFN-y(3-20),
and their analogs to block 125I-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 6947451 cpm (cpm S.D.).
The figure is representative of three independent
experiments. Symbols: IFN-y(1-20) (), IFN-y(3-20)( 0 )
conservative IFN-y(3-20) SER3, ILE4, LEU5 (A), conservative
IFN-y(3-20) SER3, LEU4, VAL5 (A) nonconservative IFN-y(3-
20) ILE3, LYS4, TYR5 (0), nonconservative IFN-y(3-20) LEU3,
TYR4, LYS5 (x), IFN-y(6-20) (@).








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(1-20) 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(1-20).

Conversely, the truncated peptide IFN-y(6-20), which lacked

function, lost most of its a-helical structure. IFN-y(3-20),

SER3, ILE4, LEU5, a conservative analog, had a-helical

structure that was slightly less than IFN-y(1-20) 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) ILE3, LYS4, TYR5' which lost blocking

activity, exhibited almost identical secondary structure to

the IFN-y(1-20) (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



























4-,











-6 -- -- -- -- -- -- -
190 200 210 220 230 240 250 260


Wavelength (nm)


Figure. 2-9. CD spectra of IFN-y(1-20), 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) (2 ), IFN-y(3-20) (, IFN-y(6-20)
( .........), Conservative analog IFN-y(3-20) SER3, ILE4, LEU5
( )- Nonconservative analog IFN-y(3-20) ILE3, LYS4, TYR5
( )








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(1-13) 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(1-20). Both IFN-y(1-14) and IFN-y(1-13)

had an apparent reduced helical structure, but not complete

abrogation, with an apparent increase in random structure












120




100








60-








20





-6 -5 -4 -3

Peptide concentration (log M)

Figure. 2-10. Competitive RIA with C-terminal truncations
of IFN-y(I-20) and mAb 5.102.12. The ability of IFN-y(1-20)
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(1-20) (0),
IFN-Y(1-13) (0) IFN-y(1-14) (A) IFN-y(3-14) (0) .








(minimum at 205-200 nm) (figure 2-11). The reduction in a-

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-y(1-14) but not IFN-y(1-13) 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(1-13) were purified by HPLC.

IFN-y(1-20) had a final purity of 90% and IFN-y(1-13) 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(1-20) 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














2












-2-2





-4





-6-
190 200 210 220 230 240 250 260


Wavelength (nm)


Figure 2-11. CD spectra of IFN-y(1-20) 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(1-13) (.........),
IFN-Y(1-14) (---) .













































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(1-13) crude.
B. IFN-y(l-13) purified. C. IFN-y(1-20) crude. D.
IFN-y(1-20) purified.
















120



100



80



60



40



20




1 0-7 10-6 10-5 10-4 10-3


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(1-20)
crude (0), IFN-y(1-20) purified (o), IFN-y(1-13) purified
(A).









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









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), IFN-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









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(I-14) has blocking activity

similar to IFN-y(1-20) 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(1-13) 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(1-14)

would be shifted slightly to the left one half log and not be

very different than the profile of IFN-y(1-20). The lack of

TYR in IFN-y(1-13) 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).









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(1-39), 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(1-39), since this peptide binds to

receptor at 1/1000th 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









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









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 gg 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 250 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.












































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).






































Asx
Glx
Ala
Val
Ile
Leu
Phe
His
Arg


1.05
1.01
0.95
0.70
0.96
1.02
0.99
0.98
0.98


Figure 3-2. Purity information on purified synthetic
peptides used in C-terminal specific antibody mapping
studies. A. IFN-y(78-107). B. IFN-y(95-133). C.
IFN-y(108-133). D. IFN-y(120-133) E. IFN-y(108-119).


Amino acid analysis B. Amino acid analysis
Asx 0.77 Asx 1.11
Thr 0.73 Ser 0.99
Ser 0.89 Glx 1.04
Glx 1.34 Pro 0.89
Pro 1.09 Ala 1.00
Ala 1.00 Val 0.90
Val 1.10 \ e 1.05
Met 0.43 Leu 1.00
Ile 1.03 Phe 1.03
Phe 0.90 His 1.34
His 0.96 _Lys 1.14
Arg2.40 Arg 1.11

Amino acid analysis D. Amino acid analysis
Asx 1.15 Ser 0.95
Ser 0.89 Glx 1.00
Pro 1.00 Pro 0.80
Gly 0.79 Gly 1.00
Ala 0.98 Lys 1.08
Val 0.62 Arg 1.08
Ile 0.93
Leu 1.01
Phe 0.95
His 1.50
Lys 1.04
Arg 1.05








ELISA. Synthetic peptides and IFN-y were dissolved in

0.1 M sodium carbonate buffer, pH 9.6, at a concentration of

12 gg/ml and 50 pL 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 Il/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 p. 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 H202 were added and color development was allowed

to occur for 15-30 minute after which 25 gl 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.









IFN-Y neutralization assay. Varying concentrations of

IFN-y were incubated with dilutions of rabbit antisera for 30

min at 370C. 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 370C 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 370C 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-

Y(36-60), 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(1-39), had the ability to directly inhibit











Table 3-1. Hydropathicity of IFN-yoverlapping synthetic peptides.
Peptide Sequence Hydropathya

IFN-Y(1-39) 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) AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC -0.792

a Average hydropathy values were calculated by taking 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).









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(1-39) 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(1-39) and

IFN-Y(95-133) had been produced previously (87). These

antibodies to IFN-y(1-39) 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(1-39), 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

















Table 3-2. Direct peptide blocking of mouse IFN-y antiviral activity by
IFN-y synthetic peptides.
Peptides Peptide concentrationa

IFN-Ty(-39) 15 LM

IFN-Y(36-60) >500 pM

IFN-y(54-91) >500 iM

IFN-y(78-107) >500 iM

IFN-y(95-133) >500 JM
aHighest peptide concentration that will block 10 units of IFN-y
antiviral activity.














Table 3-3. IFN-Y neutralization efficiency of antipeptide antisera.

ELISA titer (E)a Neutralization (N) Ratiob

Peptide Peptide IFN-y against 10U IFN-Y E/N (x100)

IFN-y(1-39) 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.
bRatio calculated as ELISA titer to IFN-y divided by
neutralization titer multiplied by 100.
CND indicates not done.









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)

















.... TFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC -COOH


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.


I

































10
11













C
N




H1
0






44 )




c I
0 4
0 a>

M O






c c
3


0














-4
0
I0 44
) 0
U C


o qo




C C
04 a

c i -i



04










0
-i
.0


0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
m %I) m C c) c) m r-I





















































o o 0 0 0 D 0












o 0 0 0 0 0 0
o o 0 0 0 0 0
o o 000 0 '












> 0


0
0
0
1-
-4


0


.H
4A
-1


V.




4)



0




0
H
4)
4


-4
-H









-4








0

4.)
a)

0
-4














0



















1 -4
-9
c1




0
4)
u9





0
rI-
0 V
4)
I-I



0 r






Q4 0
.tI









o o

o o
0 0)







0 3














C! C









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,

IFN-y(78-107), IFN-y(108-119), and IFN-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










10o














0


0 20 40 60 80 100 120


Residue Number


Figure 3-4. Surface profile of mouse IFN-y
and human IFN-y (solid lines).


(dashed lines)









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













i i
itL




: w \i

a*



a a

-10
5 ** /


-15 -- I -i* I
180 200 220 240 260


Wavelength (nm)



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(1-39) was
provided by H.I. Magazine and was generated under the same
conditions as the other peptides. Symbols: IFN-y(1-39)
(-----.), IFN-Y(54-91) (-), IFN-y(78-107) (.........),
IFN-y(95-133) (.....) .









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).












35


75
82 5(



85


133
120


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.


Glycosilation
site


F


1 3 11

V^AQIQ--IOOP'
























COOH



A
D


NH2


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.









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





























GLN |
119 GLN ARG
108 115


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.









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(1-39), directly blocks IFN-y
binding and function
Polyclonal and monoclonal antibodies directed to the
N-terminus neutralize function through the blockage
of receptor binding.
C-terminal domain

Site directed polyclonal antisera to the C-terminus
block IFN-y receptor binding and neutralize function.

Truncations of the C-terminus of human IFN-y abrogate
function.


Reference
88


87,88
chapter 2


4,this
work

81,83


Central region


Antibodies to the central region of the molecule have
no effect on IFN-y activity.

Secondary structure prediction

Predicted location of turns: Residues- 16-20, 36-40,
60-64, 81-85, 100-104.
The remainder of the the molecule is predicted to form
a-helices with some random structure. No P-sheet is
predicted.
Circular dichroism

Human IFN-y consists of 66% a-helix. No P-sheet present.

Circular dichroism of overlapping synthetic peptides of
mouse IFN-y confirm the presence of mainly a-helical
structure and the absence of P-sheet.
Tertiary structure prediction

Presence of apolar periodicity noted in all a-helices
which suggests the probability of a four-a-helix
bundle motif.

Functional data suggest the N and C-termini are
functional domains interacting with the receptor
which places them close together.


this work


107,108,
122
107,108,
122


this work


123-125


87,88,

chapter 2


Structural similarity to IL-2. Reasonable to base model
on backbone of IL-2 three dimensional structure.









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









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 a-

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 1/1000th 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








not been examined. Long overlapping peptides that correspond

to the entire sequence of IFN-y were synthesized. Only IFN-

y(1-39), 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 3-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 3-sheet. Predictive

algorithms divide the molecule into six a-helices and five









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








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
interferon. Proc. R. Soc. London Ser. B. 141:258, 1957.

2. Johnson, H.M., Mechanism of interferon gamma production
and assessment of immunoregulatory properties.
Lymphokines 11:33, 1985.

3. Wilson, V., Jeffrys, A.J., Barrie, P.A., Boseley, P.G.,
Slocombe, P.M., Easton, A., and Burke, D.C., A comparison
of vertebrate interferon gene families detected by
hybridization with human interferon DNA. J. Mol. Biol.
615:457, 1983.

4. Henco, K., Brosius, J., Fujisana, A., Fujisana, J-I.,
Haynes, J.R., Hochstadt, J., Koviac, T., Pasek, M.,
Schambock, A., Schmid, J., Todokoro, K., Walchli, M.,
Nagata, S., and Weissmann, C., Structural relationship of
human interferon alpha genes and pseudogenes. J. Mol.
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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.








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 Philosoph.


Howard M. Johnon, 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 Doo of hil. op .


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, scope and quality, as
a dissertation for the degree of tr of Philosophy.


Pail A. Hargra 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.


Lindsky 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.



./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 P a
Dean, College of Medicine


Dean, Graduate School






























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