Title: Binding, kinetics, and cellular processing of ovine interferon tau and the type I interferon receptor /
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Title: Binding, kinetics, and cellular processing of ovine interferon tau and the type I interferon receptor /
Physical Description: xv, 174 leaves : ill. (some col.) ; 29 cm.
Language: English
Creator: Siler, Kendra Indhira
Publication Date: 2001
Copyright Date: 2001
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Subject: Microbiology and Cell Science thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF   ( lcsh )
Genre: bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )
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Summary: ABSTRACT: Although ovine interferon tau (IFNtau) was originally identified as a pregnancy recognition hormone and IFNtau genes have not been identified in humans, this type I IFN exerts plieotropic biological activities without species-specificity. Ovine IFNtau elicits function by binding the type I IFN receptor, which is shared by all type I IFNs. However, IFNtau is less toxic than FDA approved type I IFNs, yet comparable functionally. This study serves as a comprehensive analysis of the structurally and functionally significant interactions and cellular processing of IFNtau and its receptor. This research may contribute significantly to the design of hybrid type I IFN therapeutic agents. Biochemical aspects of ovine IFNtau, especially the efficient removal of ovine IFNtau from cells and the reduced rate of insertion of receptor into the cell membrane, may be factors in the reduction of the toxic effects that are characteristic of type I IFNs. For every four human IFNalphaA internalized one is degraded; the ratio of IFNtau internalization to degradation is 1:1. In addition, receptor is inserted into the cell membrane 70% more slowly in response to IFNtau than human IFNalphaA. Therefore, accumulation of human IFNalphaA in cells may contribute to toxicity. The display of these biochemical characteristics of ovine IFNtau by hybrid type I IFN molecules may be indicative of therapeutic potential. Specific binding regions of ovine IFNtau and IFNAR2 were elucidated to provide primary structure information for hybrid IFN design. Ovine IFNtau residues 1-38 and 77-138 interact with the N-terminus of IFNAR2 at residues 1-67.
Summary: KEYWORDS: interferon, receptor kinetics, steady state, fluorescent microscopy, receptor binding, ovine interferon tau
Thesis: Thesis (Ph. D.)--University of Florida, 2001.
Bibliography: Includes bibliographical references (leaves 158-172).
Additional Physical Form: Also available on the World Wide Web; Adobe Acrobat Reader required to view and print PDF file.
Statement of Responsibility: by Kendra Indhira Siler.
General Note: Printout.
General Note: Vita.
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Bibliographic ID: UF00100804
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 49232460
alephbibnum - 002763019
notis - ANP1039

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BINDING, KINETICS, AND CELLULAR PROCESSING OF OVINE
INTERFERON TAU AND THE TYPE I INTERFERON RECEPTOR








BY

KENDRA INDHIRA SILER


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


2001



























COPYRIGHT 2001

BY

KENDRA INDHIRA SILER



























To the loving memory of my abuelita, Gwendolyn Matthews, who
taught me patience, perseverance, compassion, and how to read.












ACKNOWLEDGMENTS

I would like to recognize the chairman of my committee, Dr.

Phillip M. Achey; and the members of my committee, Dr. Daniel C.

Sharp, Dr. Edward M. Hoffmann, and Dr. James F. Preston for their

positive contributions to my research and education. I am grateful to

Dr. Rachel Shireman, Dr. Gerard Shaw, Dr. William W. Thatcher, Dr.

Ben Dunn, Dr. Paul Chun, Dr. Martez M. Green, and Dr. Michael B.

Porter for assisting with experimental design, data analysis and

research presentation. I also greatly appreciate the technical advice

and services provided by Alfred Chung, Tim Vaught, Frank Michel,

George Rawls, Jose Clemente and Sam Frank. I was so fortunate to

have the opportunity to interact with Dr. Daniel C. Sharp, Dr. Rachel

Shireman, and Dr. B.D. Sivazlian. They are all excellent role models

and mentors because they do not limit what they teach to science;

they truly care about the well being of others. Dr. Samuel R. Farrah

deserves my deepest gratitude for his kindness; he treated me as if I

were one of his own students. I appreciate the time and laughter

shared with my fellow graduate students, especially those in Dr. Daniel

C. Sharp's laboratory, Dr. Samuel R. Farrah's laboratory, Dr. Linda

Bloom's laboratory, and Dr. Ben Dunn's laboratory. I am grateful for









the positive attitude, support, work ethic, and expertise of the integral

employees of the Microbiology and Cell Science Department, especially

Cathy Cassidy, Linda Parsons, and Barbara Perry. I will always cherish

the kindness and love that can only be provided by true friends. My

family, especially James H. Siler, Kumari Siler, and James K. Siler, will

always be adored for their love, moral support, and for teaching me

what is important in life.














TABLE OF CONTENTS


page

ACKNOW LEDG M ENTS ................................................................. iv

LIST O F TABLES ........................ ............ ........ ...... ..................... ix

LIST O F FIG U RES .............................. ......................................... x

1 BACKGROUND AND SIGNIFICANCE ............................................. 1
Structural and Biochemical Characteristics of Ovine IFN .................1
Biological Properties of Ovine IFNc ................................................ 2
Functional Regions on the Ovine IFNt Molecule............................... 6
Structural Characteristics of the Type I IFN Receptor ...................... 8
Binding and Biochemical Features of the Type I IFNs to the
Type I IFN Receptor ..................................................................... 9
Interactions between the Type I IFN Receptor and Intracellular
Signalling M olecules.......................... ................................. ... 10
Signal Transduction and Biological Response to Ovine IFNc ........... 11
Cellular Internalization and Processing ....................................... 12
Equilibrium Binding of the Type I IFNs to the Type IFN Receptor.... 14
Steady-state Models and Kinetics of the Type I IFNs and the Type I
IFN Receptor in W hole Cells...................................................... 17

2 MATERIALS AND METHODS .................................................... 24
C e ll Lines .................................................................................. 24
Recom binant Ovine IFNc .......................................................... 24
Peptide Synthesis and Purification............................................. 24
Biotin Labeling of IFNt Peptides and Ovine IFNc for
Synthetic Peptide Studies......................................................... 25
Radioiodination of Ovine IFNt for Synthetic Peptide Studies..........26
D direct Binding Assays............................................................... 27
Competitive Binding Assays on Viable Cells.................................28
Solid-phase Com petition Assays................................................ 28
Vesicular Stomatis Virus (VSV) Propagation ...............................29
Antiviral Cytopathic Effect Inhibition Assay .................................30
Localization of IFNAR2 in MDBK Cells Using Immunofluorescence ..30









Localization of Ovine IFNT with respect to the Lysosomes
in MDBK Cells Using Immunofluorescence..................................31
Iodogen Radioiodination of Ovine IFNT and Human IFNaA for
Equilibrium Binding and Kinetic Assays...................................... 33
Cellular Binding and Internalization Kinetic Assays .....................34
Equilibrium Binding Assay ........................................................ 36
Steady State Binding Assays ..................................................... 37
Unoccupied Receptor Turnover Binding Assays............................39

3 DETERMINATION OF STRUCTURAL AND FUNCTIONAL REGIONS OF
OVINE IFNt AND THE TYPE I IFN RECEPTOR .................................41
Direct Binding of Biotinylated Ovine IFNt to IFNAR2 Extracellular
Dom ain Peptides ...................... .............. .... .......... ................ 41
Extracellular Domain Receptor Peptides Block Ovine 125I-IFNC
Binding to Viable M DBK Cells.................................................... 44
Differential Binding Affinities of IFNAR2(1-38) and
IFNAR2(34-67) for Ovine 125I-IFN ............................................... 47
Structurally Important Extracellular IFNAR2 Peptides Block Antiviral
Activity of O vine IFNt............................................................... 49
Human IFNuD Antagonizes Biotinylated Ovine IFNc Binding to
IFNA R2(34-67)............................. .................................... .... 50
Direct Binding of Biotinylated Ovine IFNt to IFNAR2 Intracellular
D om ain Peptides ........ ...................... .................... ................ 50
An Intracellular Peptide Inhibits Ovine 125I-IFN- Binding
to IFNAR2 on Viable and Intact MDBK Cells.................................54
An Intracellular Peptide Inhibits Ovine IFNt Antiviral Activity ........59
Direct Binding of Biotin-conjugated Ovine IFNt Peptides to
Extracellular IFNAR2 Peptides.................................................... 59
Direct Binding of Biotin-conjugated Ovine IFNt Peptides to
Intracellular IFNAR2 Peptides ................................................... 60
Ovine IFNc Peptides Compete with Ovine 125I-IFN- for
IFNA R2(1-38) .............................. .................................... .. .. 63
Ovine IFNc Peptides Compete with Ovine 125I-IFN- for
IFNA R2(34-67)............................. .................................... .... 73
Ovine IFNc and Ovine IFNc Peptides Specifically Recognize
IFNA R2(287-315) .................................................................... 80
Saturation Binding Studies with 125I-Ovine IFNt Further Reveal
Specific Recognition of IFNAR2(287-315)...................................91
Determination of Binding Regions on Ovine IFNt for the
Type I IFN Receptor ................................................................. 92
D iscussion................................................................................. 92









4 TOPOGRAPHICAL FATE OF OVINE IFNt AND THE TYPE I IFN
RECEPTOR AS DETERMINED BY INDIRECT FLUORESCENT
M ICRO SCO PY ...................... ................. ............... ... ... .............. 98
Ovine IFNt Colocalizes with the Lysosomal Associated Matrix
Protein 1 .................................................................................. 98
Ovine IFNc is Degraded in the Lysosomes..................................112
IFNAR2 is Internalized in MDBK Cells Following Ovine IFNc
S tim u la tio n ................................................................ ............. 1 1 2
D iscussion............................................................................... 113

5 BINDING, INTERNALIZATION, AND PROCESSING OF OVINE IFNt,
HUMAN IFNaA, AND THE TYPE I IFN RECEPTOR BY MDBK CELLS: A
COMPARATIVE BIOCHEMICAL ANALYSIS .................................... 116
Equilibrium Binding of Ovine IFNt and Human IFNucA to the Type I
IFN Receptor on M DBK Cells................................................... 116
Dissociation Rate Constant (kd) of Ovine IFNc and Human IFNUcA
from the Type I IFN Receptor on MDBK Cells ........................... 123
Association Rate Constants (ka) of Ovine IFNc and Human IFNUcA
from the Type I IFN Receptor on MDBK Cells ........................... 124
Endocytotic Rate Constant (ke) of Human IFNucA is Significantly
Faster than that of Ovine IFNt into MDBK Cells .........................127
Surface Binding and Internalization Patterns of Human IFNucA and
Ovine IFNc in M DBK Cells ....................................................... 128
Rate Constants of Hydrolysis (kh) of Ovine IFNt and Human IFNUcA in
M D B K C e lls ................................................................ ............. 1 3 3
Steady State Binding Constants (Kss) of Ovine IFNt and Human
IFNu.A for the Type I IFN Receptor ............................................134
Reinsertion of Type I IFN Receptors into the Plasma Membrane
Occurs at a Faster Rate in MDBK Cells Stimulated with Human IFNUcA
than Ovine IFNt.................... ................ ..................... ......... 135
Human IFNucA and Ovine IFNc Accelerate the Internalization of
Ligand-Occupied Type I IFN Receptors into MDBK Cells............. 140
D iscussion............................................................................... 147

6 DISCUSSIO N ................. .................. ................. ... ...... ....... 153

LIST O F REFERENCES ............................................................... 158

BIOGRAPHICAL SKETCH............................................................ 173














LIST OF TABLES


Table Page

3-1. Sequences and secondary structure predictions of the synthetic
extracellular domain peptides of IFNAR2..............................42

3-2. Sequences and secondary structure predictions of the synthetic
intracellular domain peptides of IFNAR2. ..............................53

3-3. Sequences and secondary structure of the synthetic ovine IFN-
peptides of IFNA R2 ............................................................ 64














LIST OF FIGURES


Figure Page

3-1. Direct binding of biotinylated ovine IFNt to overlapping synthetic
peptides of the extracellular domain of IFNAR2 .....................43

3-2. Dose-response of unlabeled ovine IFN- and extracellular IFNAR2
peptide competitors on ovine IFNt binding to the type I IFN
receptor on viable MDBK cells............................................. 45

3-3. Dose-response of extracellular IFNAR2 peptide competitors,
IFNAR2(1-38) and IFNAR2(34-67) on ovine 125I-IFN- binding
to the type I IFN receptor on MDBK cells.............................46

3-4. Extracellular IFNAR2 peptide, IFNAR2(186-217), does not effect
ovine 125I-IFNc binding to the type I IFN receptor on MDBK
ce lls ................................................................................... 4 8

3-5. Extracellular IFNAR2 peptides inhibition of ovine IFN- antiviral
active ity ........................................................................... . . 5 1

3-6. Dose-dependent inhibition of biotinylated ovine IFNt binding to
IFNAR2(34-67) by unlabeled ovine IFN- and human IFNcD .....52

3-7. Direct binding of biotinylated ovine IFNt to overlapping synthetic
peptides of the intracellular domain of IFNAR2.....................55

3-8. Dose-response of unlabeled ovine IFN- and extracellular IFNAR2
peptide competitors on ovine IFNt binding to the type I IFN
receptor on M DBK cells ...................................................... 56

3-9. Dose-response of intracellular IFNAR2 peptide competitor,
IFNAR2(287-315) on ovine 125I-IFN- binding to the type I IFN
receptor on M DBK cells ...................................................... 57









3-10. Intracellular IFNAR2 peptide inhibition of ovine IFNt antiviral
active ity ........................................................................... . . 58

3-11. Binding of biotinylated ovine IFNt, and ovine IFNt peptides,
IFNt(1-38), IFNT(118-138), and IFNT(153-168) to overlapping
synthetic peptides corresponding to the extracellular domain
of IFNA R2........................................................................... 6 1

3-12. Binding of biotinylated ovine IFNt, and ovine IFNt peptides,
IFNt(1-38), IFNT(118-138), and IFNT(153-168) to overlapping
synthetic peptides corresponding to the intracellular domain
of IFNA R2........................................................................... 62

3-13. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(1-38) in
the presence of unlabeled ovine IFNt peptides. .....................65

3-14. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(1-38) in
the presence of unlabeled ovine IFN .................................. 71

3-15. Dose-response of extracellular IFNAR2 peptide competitors,
IFNAR2(1-38) and IFNAR2(186-217), on ovine 125I-IFNt
binding to the type I IFN receptor on MDBK cells..................72

3-16. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(34-67) in
the presence of unlabeled ovine IFNt peptides .....................74

3-17. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(34-67) in
the presence of unlabeled ovine IFN .................................. 81

3-18. Dose-response of extracellular IFNAR2 peptide competitors,
IFNAR2(34-67) and IFNAR2(186-217), on ovine 125I-IFNt
binding to IFNAR2(34-67) .................................................. 82

3-19. Dose-dependent binding of biotinylated ovine IFNt peptides,
IFNt(1-38), IFNT(118-138), and IFNt(153-168) to
IFNAR2(287-315) in the presence of unlabeled ovine IFNt
peptides. ...................................................................... . ... 83

3-20. Binding of biotinylated ovine IFNt to IFNAR2(287-315) in the
presence of unlabeled ovine IFN .. ........................................ 86

3-21. Dose-dependent binding of biotinylated ovine IFNt to
IFNAR2(287-315) in the presence of IFNAR2(287-315)...........87









3-22. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(287-315)
in the presence of unlabeled ovine IFNT...............................88

3-23. Dose-response of intracellular IFNAR2 peptide competitor,
IFNAR2(287-315), on ovine 125I-IFNt binding to
IFNA R2(287-315) .............................................................. 89

3-24. Dose-response of unlabeled ovine IFNT and extracellular IFNAR2
peptide competitors on ovine IFNT binding to the type I IFN
receptor on M DBK cells. ...................................................... 90

4-1. Translocation of ovine IFN- in respect to LAMP-1, a lysosomal
protein, at 32 m minutes ....................................................... 99

4-2. Time-dependent translocation of ovine IFNT in respect to
LAMP-1, a lysosomal protein, following ovine IFNT treatment
as shown by indirect immunofluorescence ............................103

4-3. The indirect immunofluorescence of LAMP-1 ............................104

4-4. MDBK cells treated only with secondary antibodies against
primary antibodies specific for (A) ovine IFNT and
(B) LA M P-1 ....................... ................................................. 105

4-5. Concentration of degraded ovine 125I-IFNT from 100 |tM final
concentration of chloroquine-treated MDBK cells ..................106

4-6. Time-dependent translocation of IFNAR2 following ovine IFNT
treatment as shown by indirect immunofluorescence .............110

4-7. MDBK cells treated only with secondary antibodies that were
against primary antibodies specific for IFNAR2.......................111

5-1. The equilibrium surface binding of human IFNuA to MDBK cells as
determined by (A) Scatchard analysis and (B) nonlinear
regression analysis ............................................................. 117

5-2. The equilibrium surface binding of ovine IFNT to MDBK cells as
determined by (A) Scatchard analysis and (B) nonlinear
regression analysis .............................................................. 119

5-3. The dissociation rate constants of human IFNuA and the type I
IFN receptor on M DBK cells.. ............................................... 125









5-4. The dissociation rate constants of ovine IFNT and the type I IFN
receptor on M DBK cells ....................................................... 126

5-5. The rate constants of endocytosis and hydrolysis of human IFNUA
and the type I IFN receptor on MDBK cells............................129

5-6. The rate constants of endocytosis and hydrolysis of ovine IFNT
and the type I IFN receptor on MDBK cells............................130

5-7. The surface binding and internalization pattern of human IFNUA
and the type I IFN receptor in relation to MDBK cells.............131

5-8. The surface binding and internalization pattern of ovine IFNT and
the type I IFN receptor in relation to MDBK cells ...................132

5-9. The steady state binding of human IFNaA to MDBK cells and the
rate of insertion of type I IFN receptors into MDBK cell
membranes following human IFNuA stimulation as determined
by (A) Scatchard analysis and (B) nonlinear regression
a na ly sis........................................................................... . 136

5-10. The steady state binding of 125I-ovine IFNT and the rate of
insertion of type I IFN receptors into MDBK cell membranes
following ovine IFNT stimulation as determined by
(A) Scatchard analysis and (B) nonlinear regression analysis...138

5-11. Total receptor concentration following human IFNuA stimulation
at a final concentration of 22 pM as determined by
(A) Scatchard analysis and (B) nonlinear regression analysis...142

5-12. Total receptor concentration following ovine IFNT stimulation at
a final concentration of 56 pM as determined by (A) Scatchard
analysis and (B) nonlinear regression analysis ......................144














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


Binding, Kinetics, and Cellular Processing of Ovine Interferon Tau and
the Type I Interferon Receptor

By

Kendra Indhira Siler

August, 2001


Chairman: Phillip M. Achey
Major Department: Microbiology and Cell Science

Although ovine interferon tau (IFNt) was originally identified as a

pregnancy recognition hormone and IFN- genes have not been

identified in humans, this type I IFN exerts plieotropic biological

activities without species-specificity. Ovine IFNc elicits function by

binding the type I IFN receptor, which is shared by all type I IFNs.

However, IFNc is less toxic than FDA approved type I IFNs, yet

comparable functionally. This study serves as a comprehensive

analysis of the structurally and functionally significant interactions and

cellular processing of IFNt and its receptor. This research may

contribute significantly to the design of hybrid type I IFN therapeutic

agents.









Biochemical aspects of ovine IFNt, especially the efficient

removal of ovine IFN- from cells and the reduced rate of insertion of

receptor into the cell membrane, may be factors in the reduction of

the toxic effects that are characteristic of type I IFNs. For every four

human IFNuA internalized one is degraded; the ratio of IFNC

internalization to degradation is 1:1. In addition, receptor is inserted

into the cell membrane 70% more slowly in response to IFN- than

human IFNuA. Therefore, accumulation of human IFNuA in cells may

contribute to toxicity. The display of these biochemical characteristics

of ovine IFN- by hybrid type I IFN molecules may be indicative of

therapeutic potential.

Specific binding regions of ovine IFNt and IFNAR2 were

elucidated to provide primary structure information for hybrid IFN

design. Ovine IFNt residues 1-38 and 77-138 interact with the N-

terminus of IFNAR2 at residues 1-67.













CHAPTER 1
BACKGROUND AND SIGNIFICANCE

Type I interferons (IFNs) are structurally and functionally related

cytokines that exert strong antiviral, antiproliferative, antitumor, and

immunomodulatory activities. At least fourteen IFNa subtypes, IFN3,

IFNo, and the newest member IFNt, originally identified as a

pregnancy recognition hormone for sheep, make up the type I IFN

family [1, 2]. Because of successful basic research and clinical trials,

IFNu has been approved by the FDA for treating hairy cell leukemia,

Kaposi's sarcoma, and chronic hepatitis B and C [1, 2, 3]. Interferonp

has received FDA approval for the treatment of relapsing-remitting

multiple sclerosis [1]. However, undesirable side effects at high

doses, such as severe flu-like symptoms and bone marrow suppression

limit more extensive and effective use of the type I IFNs [1, 4, 5].


Structural and Biochemical Characteristics of Ovine IFNc

Ovine IFNc shares 30 to 70% amino acid sequence identity with

type I IFNs of various species and subtypes [6]. The primary

sequence of ovine IFNt is 30% identical to that of ovine IFNp and 45 to

55% identical when compared to the amino acid sequences of human,

mouse, and pig IFNa subtypes. Primary structure identity increases to









70% when ovine IFNT is aligned with bovine IFNco. Also, like bovine

IFNco, the mature form of ovine IFNT is 172 amino acids in length [7].

The protein's precursor is 195 amino acids long; therefore, 23 amino

acids make up the cleaved signal sequence [2]. The molecular weight

is approximately 19 kD, equivalent to that of all type I IFNs. The

isoelectric point (pI) of various natural and recombinant ovine IFN-

molecules ranges between 5.3 and 5.8 [1, 2]. The pI values of IFNca

subtypes average 5.5. All of the type I IFNs are acid stable to pH 2-3

[8].

Ovine IFN- has the characteristic tertiary structure of the type I

IFN family, a four-helix bundle with a tightly associated fifth helix [9,

10]. Cellular response to all type I IFNs depends on ligand recognition

and interaction with the type I IFN receptor [7, 11-13]. Ovine IFNt

interacts with this receptor to exert function [8]. No other receptors

have been shown to specifically interact with ovine IFNt. Cytokines

that share receptor subunits, like the type I IFNs, elicit both redundant

and unique biological responses [14].


Biological Properties of Ovine IFN-

Like all type I IFNs, ovine IFNt has been shown to inhibit viral

replication. Specifically, ovine IFN- inhibits vesicular stomatis virus

(VSV), human papilloma virus (HPV), human and feline

immunodeficiency lentiviruses and ovine progressive pneumonia virus









(OPPV), a concern to sheep ranchers [8]. Therefore, the antiviral

activity is not species-specific. In addition, antiviral protection is

provided to many cell types including various epithelial cells and

fibroblasts [2, 8, 12]. Unlike other type I IFNs, however, neither viral

infections nor double-stranded RNA stimulates the production of ovine

IFNT by trophoblasts [15].

Type I IFNs invoke antiviral activity by multiple mechanisms.

The upregulation of 2'-5' oligoadenylate synthetase, ds-RNA-

dependent protein kinase (PKR) activation, and Mx protein synthesis

provides several different cell types with a comprehensive defense

against a host of viruses [16-21]. The 2'-5' oligoadenylate synthetase

production of 2', 5' oligoadenylates is stimulated by viral dsRNA [22].

The oligoadenylates bind and activate monomers of 2-5A-dependent

RNase L, which then dimerize and cleave ssRNA to block viral

replication. The activation of PKR ultimately causes the inhibition of

translation [20]. Double-stranded RNA binds to PKR, a serine-

threonine kinase, to initiate a kinase cascade that results in the

inactivation of guanine diphosphate bound eIF2 and eIF2B. Without

these active complexes, all translation, and therefore, viral translation,

stops. Both PKR and 2-5A-dependent RNase L have been implicated in

apoptosis, which also provides viral protection [23, 24]. Mx proteins

inhibit viral replication at levels of transcription and translation, at









various points in the viral life cycle. These proteins are GTPases of the

dynamin family [25]. Type I IFNs, including ovine IFNT, exert

antiretroviral activity by blocking reverse transcriptase [26, 27].

The plieotropic nature of type I IFNs is apparent by the broad

scope of immunomodulatory properties they possess. The cytotoxic

effects of natural killer cells are amplified by type IFNs [28-30]. Type

I IFN stimulation enhances CD8+ T cell responsiveness by increasing

the expression of major histocompatibity complex I (MHC I) on cell

membranes [31]. Production and development of T helper 1 cells is

stimulated [32]. In addition, the expression of cell adhesion molecules

on the cell membrane is enhanced [33]. These immunomodulatory

activities are in part responsible for the antiviral, antiproliferative and

antitumor capabilities of type I IFNs. Type I IFNs also exert

antiproliferative activities by arresting the cell cycle in cell lines such

as Madin-Darby kidney cells, mouse L929 fibroblasts, and human

WISH fibroblasts [8, 34]. Cell cycle arrest and apoptosis are also

believed to be mechanisms by which type I IFNs exert their antitumor

activities. Type I IFNs have been shown to down-regulate various

cyclins of the cell cycle, inhibit c-myc expression, and inhibit

phosphorylation of Rb [34, 35]. These cyclins and tumor suppressors

have all been determined to play roles as causative agents and factors

in various cancers. Various B-cell neoplasms, including hairy cell









leukemia and cryoglobulinemia, were sent into remission by IFNa

treatment in some patients [36]. Human breast, kidney, and

hematopoietic tumor cell growth has been inhibited by ovine IFNT in

vitro [8].

Despite structural and biochemical similarities with other type I

IFNs, IFNT is the only known type I IFN that serves as a pregnancy

survival hormone [7]. Signalling from embryo to mother was found to

occur simultaneously with the secretion of ovine IFNT in pregnant ewes

between days 13 and 21 of pregnancy. At day 16, high quantities,

approximately 200 itg, of ovine IFNT are secreted within 30 hours by a

sheep concepts. Ovine IFNT is produced specifically by the

trophectoderm, which is reflected by its original names, ovine

trophoblast protein 1 (oTP-1), trophoblastin, and type I trophoblast

interferon [37, 38].

The secretion of ovine IFN- by trophoblasts results in down-

regulation of oxytocin receptors in pregnant ewes [7]. Oxytocin

induces the pulsing secretions of prostaglandin F2a (PGF2a) of cycling

ewes and PGF2a, in turn, causes the corpus luteum to regress at the

end of a non-pregnant ewe's cycle [38]. If the PGF2a pulses are

blocked, the corpus luteum and therefore, pregnancy can be

maintained. High concentrations of ovine IFNt in sheep result in the

inhibition of the expression of the oxytocin receptor [37].









The reproductive function of ovine IFNT is considered to be luteostatic

because it prevents the regression of the corpus luteum [7, 37].

Another marked difference between ovine IFNT and other type I

IFNs is the duration and quantity of protein release [38]. Ovine IFNT

secretion occurs for several continuous days in ruminants, whereas the

release of IFNa in various species occurs only for a few hours in

response to an immunological challenge. As mentioned earlier,

approximately 200 itg, of ovine IFNt will be secreted within 30 hours

by a 16-day old sheep concepts. This amount is approximately 300

times the quantities of IFNa released. Animal models have been

shown to tolerate high quantities of ovine IFNt; however, even in short

durations, high doses of other type I IFNs result in apparent adverse

effects (i.e. fever, flu-like symptoms etc.) [1]. Interestingly, ovine

IFNt retains the beneficial antiviral and cell inhibitory properties in

tissue culture and animal models without the associated toxicity of the

other type I IFNs [38, 39].


Functional Regions on the Ovine IFNt Molecule

In previous studies, the function of ovine IFNt could be blocked

by peptides, but receptor binding could not be blocked [39].

Functional competition assays using ovine IFNt peptides, identified as

peptides oIFNt(1-37), oIFNt(62-92), oIFNT(119-150), and









oIFNt(139-172), as effective inhibitors of ovine IFNT induced antiviral

activity. Antiproliferative activity of ovine IFNT was blocked by

oIFNT(119-150), oIFNt(139-172), and to a lesser degree

oIFNt(90-122). Whole polyclonal antibodies to the peptides blocked

ovine IFNT biological activity with similar results to the competition

assays using the ovine IFNT peptides. All ovine IFNT peptides, except

oIFNt(1-37), blocked human and bovine IFNa antiviral activity on

MDBK cells, suggesting that the N-terminus of ovine IFNT differentially

recognizes the type I IFN receptor. Consistent with these studies,

x-ray crystallography identified significant structural differences

between the N-termini of human IFNa2 and ovine IFNT, primarily

involving residues 1-8 and 22-25 [9]. The crystal structure also

showed that the functional peptides corresponded to regions that lie

on the same face or side of the ovine IFNT molecule. Multiple binding

sites may be necessary for monomers of ovine IFNT to bind to receptor

with high affinity.

Previous studies using IFNa variants revealed that three regions

within residues 10-35, 78-107, and 123-166 were necessary for

growth inhibitory activity, antiviral activity, and receptor binding [39].

Another study using molecular variants of IFNa showed that truncated

human IFNu2 consisting of amino acids 1-110, but not the fragment of

human IFNu2 consisting of residues 111-153, blocked antiviral activity









of the intact molecule [40]. These studies combined suggest that N-

terminal regions of human IFNu2 are important for receptor

recognition and function.


Structural Characteristics of the Type I IFN Receptor

Type I IFNs initialize their biological properties by interacting

with the type I IFN receptor complex, a plasma membrane-bound

receptor [41]. This receptor is made up of at least two subunits,

IFNAR1 and IFNAR2. IFNAR1 and IFNAR2 were previously referred to

as the a and P subunits of the type I IFN receptor. IFNAR2 is

expressed in cells in two alternative variant forms, IFNAR2b and

IFNAR2c [42, 43]. Truncated IFNAR2 variant, IFNAR2b, was referred

to as the P short subunit in older literature [42]. It is only 331 amino

acids long and includes an extracellular, transmembrane, and

truncated intracellular domain [44]. Although it does not allow cellular

response to type I IFN stimulation, IFNAR2b is expressed in low levels

in type I IFN responsive cell lines [42]. If over-expressed, it competes

with the long IFNAR2c subunit for type I IFN binding and blocks its

biological effects [42, 43]. IFNAR2c is the 515 amino acid membrane-

bound form of IFNAR2 that upon ligand binding can initiate a cellular

response [43, 44]. This 90 kD protein is generally identified as

IFNAR2 unless otherwise specified. The ratio of expression of

IFNAR2c/IFNAR2b in U-266 cells is 10-20/1 [42]. IFNAR2a is a









recombinant, soluble variant of IFNAR2c that is expressed without the

hydrophobic transmembrane domain that is not typically present in

cells [42, 45]. The a subunit of the receptor, IFNAR1, is 557 amino

acids long and has a molecular weight of 110 kD [46, 47]. The

extracellular domain of IFNAR1 is 457 amino acids long; however, only

100 residues of IFNAR1 construct the intracellular domain [48].


Binding and Biochemical Features of the Type I IFNs to the Type I IFN
Receptor

Despite the substantial extracellular domain, IFNAR1 only serves

as an accessory protein with respect to type I IFN binding [49].

Binding studies have shown that IFNAR2 alone binds type I IFNs with

low/intermediate binding affinity, while IFNAR1 alone fails to bind type

I IFNs [42, 49]. Human IFNa bound to IFNAR2 expressed on the

membranes of mouse L929 fibroblasts with a dissociation equilibrium

constant (Kd) of 0.5 to 1 nM, whereas binding to IFNAR1 was

undetectable. When the receptor chains are completed, ligand is

bound with high affinity binding [42, 43, 44, 50-52]. Coexpression of

IFNAR1 and IFNAR2 on mouse L929 fibroblasts result in high affinity

receptor binding on the order of 10-100 pM [43]. On Madin-Darby

kidney cells, the dissociation constant (Kd) for ovine IFNT binding to

the type I IFN receptor on MDBK cells was 3.9 x 10-10 M [53]. In

competition assays, excess ovine IFNt did not block receptor binding









or cell death induced by IFNaA [53]. It followed that the cytotoxicity

associated with IFNaA was a result of type I IFN receptor saturation,

whereas antiviral activity only required a small fractional occupancy of

the receptors. In part, the higher binding affinity of IFNuA appeared

to account for the differences in toxicities.


Interactions between the Type I IFN Receptor and Intracellular
Signalling Molecules

Both receptor subunits contribute to the signaling required for

cellular function [12, 54, 55]. IFNAR1 has a cytoplasmic binding site

for the Janus kinase, Tyk2, and the signal transducer and activator of

transcription 1 (STAT1) transcription factor [56-58]. Tyk2

constitutively associates with IFNAR1 and is believed to contribute

structurally to IFNAR1 [57, 59, 60]. The JAKs and STATs are pre-

associated with the receptor complex in unactivated, unphosphorylated

forms [60, 61]. The STATs bind to the receptor subunits by their SH2

domains. However, without STAT2 completed to IFNAR2, STAT1 will

not bind IFNAR1 [42, 62]. In addition, unactivated STAT1 and STAT2

will complex in the cytoplasm without type I IFN stimulation; the

biological significance remains to be determined [63]. In cells that do

not express Tyk2, little IFNAR1 is expressed on the cell membrane

[60]. Possibly Tyk2 is structurally important for IFNAR1. IFNAR2 is

responsible for Janus kinase 1 (JAK 1) binding and interacts with both









STAT 1 and STAT2 [42]. JAK1 and STAT2 interaction with IFNAR2 is

continuous; however, the structural relevance of these proteins with

respect to IFNAR2 is unknown.


Signal Transduction and Biological Response to Ovine IFN-

Type I IFN receptor binding results in signal transduction,

primarily via the JAKs/STATs pathway [14, 64]. The type I IFN

recognition and initial binding to the type I IFN receptor brings the

receptor complex into close proximity, activating JAK1 and Tyk2 [57,

65, 66]. JAK1 and Tyk2 phosphorylate the receptor complex [14, 66].

Specifically, phosphorylation of tyrosine 466 of IFNAR1 results in a

conformational change that creates a docking site for STAT2 [58, 67].

The substitution of a phenylalanine for the tyrosine at 466 abrogates

the kinase cascade required for signaling [67]. Tyrosine 701 of STAT1

and tyrosine 690 STAT2 are subsequently activated by phosphorylation

[68]. STAT2 must be phosphorylated before docking and

phosphorylation of STAT1 can occur [61, 62]. STAT1 and STAT2 are

then believed to heterodimerize and traffic to the nucleus to initiate

the transcription of type I IFN inducible genes [14, 69]. For the vast

array of cytokines, growth factors, and hormones that utilize this

pathway, only six STATs and four JAKs have been identified [14].

However, despite the overlap in the use of JAKs and STATs, the

cellular response to cytokines differs. This suggests that the specificity









of function elicited by cytokines, growth factors and hormones is only

in part due to the differential roles of JAKs and STATs; differential

receptor binding is probably responsible as well. Homologous type I

IFN subtypes that have slightly different functional properties bind to

the type I IFN receptor differentially [70-72]. These observations may

stimulate more research on the structural differences at the receptor

level that may be responsible for the varying functional abilities of the

type I IFNs.


Cellular Internalization and Processing

The type I IFN receptor not only undergoes lateral movement on

the cell membrane in response to type I IFN stimulation, but it is also

internalized into the cell via receptor-mediated endocytosis (RME) [73,

74]. The effects of RME on the signal transduction of the type I IFNs

are unknown. However, RME may serve as a means of attenuating the

receptor population in response to continual or excessive type I IFN

stimulation [75]. The dynamics of the receptor population expressed

on the cell membranes reflect 1) continual de novo synthesis, 2)

internalization, 3) degradation and/or 4) receptor recycling, especially

after a cell interacts with ligand [77]. After ligand occupancy of a

membrane-bound receptor that undergoes RME, the ligand-receptor

complex is internalized via a clathrin-coated pit [77, 78]. The pits

containing ligand-occupied receptors are engulfed to form clathrin-









coated vesicles [79]. Fusion of a vesicle with an acidic compartment

and loss of the clathrin creates an early endosome (EE) [80-82].

Dissociation between the ligand and the receptor begins in the EE [83,

84]. The pH of the inside of an EE ranges from 6.0 to 6.8 [82].

Therefore, to allow receptor recycling, ligand and receptor sorting can

occur without damaging the proteins [77]. Ligand starts to

accumulate in the central region of the vesicle, while the receptor

tends to gather in peripheral microtubules of the EEs [77, 81, 82, 84,

85]. The secretary pathway can meet the endocytotic pathway at this

point [85, 86]. Receptor recycling has been shown to occur from

microtubular extensions of the EE [84, 86]. The extensions can bud

off into compartments separate from EEs called recycling vesicles

(RVs) to return the dissociated receptors to the plasma membrane

[84, 85]. Within the cytoplasm, microtubular "tracks" guide the EEs

with free ligand into a perinuclear region, where they fuse with

subsequent vesicles to create late endosomes (LEs) [80, 84]. The

interior of LEs has an even lower pH (pH = 5) than that of EEs and

degradation of the ligand often begins here [80]. Ligand degradation

is completed in lysosomes, which have high quantities of degradative

enzymes in addition to the acidic environment, to finish the breakdown

of ligand [80, 85, 87]. If the receptor is not recycled, it may traffic to

the LEs and lysosomes to undergo degradation [77, 80]. Alternatively,









it is believed that receptors may be transferred reversibly to the

perinuclear trans-Golgi network (TGN) from endosomal or lysosomal

compartments [88-90]. The TGN may be another connection to the

secretory/recycling pathway [91-93]. Studies have suggested that the

pH within the TGN is more basic (pH = 6.0 to 6.4) in comparison to

LEs and lysosomal compartments (pH = 5) [82, 94, 95]. The mildly

acidic environment would spare receptors from further degradation for

recycling purposes [77, 88]. However, it is difficult task for

researchers to distinguish between these vesicles morphologically [80,

96]. Many of the vesicles, especially the various types of endosomes,

are microtubular in appearance [88, 96]. In addition, pH is not a good

indicator because the pH of the endosomal compartments may be

equivalent to that of the TGN [82, 94, 95]. Further, proteins that are

found in various endosomal vesicles are also detected in the TGN [88].

Distinguishing lysosomes from the TGN is less difficult because

lysosomes function as terminal degradative compartments for ligand

and sometimes receptor [77, 97]. After degradation of the

macromolecules occurs in the lysosomes, the contents are released

from the cells [77].


Equilibrium Binding of Type I IFNs to the Type IFN Receptor

Biochemical techniques, such as 40C equilibrium-binding studies

of whole cells, give insight into ligand:receptor binding on the cell









surface, allowing the comparison of how various ligands interact with

their membrane-bound receptors [53, 74, 75, 77, 98]. The

internalization of ligand is inhibited at 40C, so all ligand binding occurs

only on the plasma membrane of cells [53, 77]. Cells are treated with

radiolabeled ligand in increasing concentrations and then allowed to

incubate at 40C to achieve equilibrium. Equilibrium is reached when

nearly saturating amounts of added ligand binds to surface-bound

receptor and no further changes in binding are observed. The

unbound ligand is then separated from the bound ligand. A popular

and effective method is using cell suspensions and centrifugation [77].

The cells are pelleted in microfuge tubes within 5 seconds leaving the

unbound ligand in solution. The tip of the microfuge tube is cut off to

count the radioactivity in the cell pellet. The slope of a Scatchard plot,

a plot of bound ligand/free ligand versus bound ligand, reveals the

equilibrium association constant (Ka), the equilibrium dissociation

constant (Kd), and the maximum concentration of surface-bound

receptors (Bmax) [77].

To determine Ka, Kd, and Bmax, the data can be transformed

linearly to use Scatchard analysis; however, a graph of the

concentration of bound ligand versus the concentration of free ligand

can also reveal these values [77, 99]. The Bmax is equivalent to the

concentration on the y-axis when the receptor is saturated [77, 100].









The Kd is the Bmax/2 and is the inverse of Ka. A graph in the latter

form shows a criterion for equilibrium binding; ligand binding to its

receptor must be saturable [75, 77]. Receptor binding is considered

to be saturated when it reaches a plateau on a saturation binding

curve. This plateau is represented by the hyperbolic relationship

between bound and free ligand [77, 100-102]. Affinity is directly

proportional to Ka and inversely proportional to Kd. Therefore, as the

Kd value decreases, the affinity of a ligand for its receptor increases.

The total binding increases hyperbolically as a function of

increasing amounts of free ligand, while nonspecific binding increases

linearly [77, 102]. The linear nature of nonspecific binding is due to

the inability of nonreceptor sites to be saturated in the same form as

the specific receptor sites [77, 103]. Specific binding equals total

binding minus nonspecific binding. Specific binding, therefore, also

assumes the hyperbolic shape in a plot of bound versus unbound

ligand. To determine non-specific binding in an equilibrium-binding

assay, unlabeled ligand is added in excess to the free ligand [53, 54,

74, 75, 77, 104, 105]. Usually, an amount equivalent to 100 times

the Kd is used [77, 104]. The unlabeled and labeled hormones should

have similar biological activity [53, 74, 77]. The previously

determined Kd values of human IFNaA and MDBK cells range from

2.5 x 10-10 M to 6.0 x 1011M [53, 54, 74, 75, 106].









Steady-state Models and Kinetics of the Type I IFNs and the Type I
IFN Receptor in Whole Cells

Although equilibrium-binding studies give insight into surface

binding, physiologically relevant ligand and receptor dynamics on and

within cells must be studied at 370C [107-109]. Kinetic studies in

which ligand-treated cells are placed at 370C for specified time

intervals and then quickly placed at 40C allows the discrimination

between surface-bound ligand, internalized ligand: receptor complexes,

and degraded ligand [74-77, 104, 107-109]. Internalization of ligand-

receptor complexes is prevented by reducing the temperature to 40C

because RME is ATP dependent [77]. A temperature of 40C also slows

further degradation, inhibits changes in surface receptor binding and

prevents changes in receptor population [107,110]. Treating the

supernatant with trichloroacetic acid (TCA) and coprecipitation with

bovine serum albumin (BSA) separates degraded ligand from intact

free ligand that is not associated with the cell [77, 111]. The TCA-

soluble fraction has ligand degraded by the cells exogenously and

endogenously and the TCA-insoluble fraction contains intact ligand.

Surface-bound ligand is retrieved from cells at 40C by treating with an

acidic buffer followed by centrifugation and removal of the supernatant

[110, 112, 113]. Changes in surface binding over time are then

compared to the optimal surface binding that occurs at 40C to reveal









the dissociation rate constant (kd) [104, 110, 113, 114].

Internalization of ligand is inhibited at 40C, so all ligand binding occurs

only on the plasma membrane [77, 99, 106]. After surface-bound

ligand is dissociated from the cells, internalized ligand can be released

by cell solubilization [104, 106]. These kinetic studies have been

performed on human IFNuA treated A549 cells to give insight into the

binding, internalization, and cellular processing of human IFNaA and

the type I IFN receptor [115]. The dissociation rate constant (kd) of

human IFNuA and membrane-bound type I IFN receptor was

determined to be 7.2 x 10-2 min-1 using the kinetic experiments

previously described and the following equation [77]:

kd, (min-1) = Slope of In[LR]s/[LR]o vs. time
[LR]s = Molar concentration of surface ligand: receptor complexes
at 370C
[LR]o = Molar concentration of surface ligand:receptor
complexes at 40C

A straight line indicates that the interaction between the ligand

and its receptor is a first-order, bimolecular reaction that follows the

law of mass action [77, 113]. Only one receptor population is specific

for the ligand and changes in affinities do not occur while the ligand

binds to various regions on the receptor [99, 113, 116].

Changes in affinities as the ligand binds, referred to as positive

and negative cooperativity, result in curvilinear plots [99]. Positive

cooperativity means that as ligand binds to receptor, the affinity for









additional ligand binding increases and does not readily dissociate.

The slope becomes more steep over time. With negative

cooperativity, the slope becomes more shallow because binding of

ligand to receptor decreases subsequent binding. If more than one

receptor is specific for the ligand and the independent forms of

receptors have different binding affinities, a curvilinear plot will result

[77]. The dissociation rate constant has been referred to as k2 in

some reports. The association rate constant (ka), sometimes called kj,

is determined using the results from an equilibrium binding study,

specifically the equilibrium dissociation constant (Kd) and the kd. The

equation is as follows:

ka, (min-M-1) = kd, (min-1)/Kd, (M)

The ka of human IFNuA to type I IFN receptors on MDBK cells was

determined to be 3.3 x 108 min-M-1 based on the Kd of human IFNUA

to MDBK cells and time-concentration points of [LR]s [75].

Further analysis of receptor population turnover at 370C is

accomplished by using steady state models developed by Wiley and

Cunningham [108-110]. Values for rate constants of endocytosis of

ligand-receptor complexes (ke), endocytosis of unoccupied receptors

(kt), degradation of internalized ligand (kh), and the insertion of

receptors into the plasma membrane (Vr) are some of the applications

of steady state models [108-110, 116-118]. Before a cell is









stimulated by a ligand, it is in a steady state in which the rate of

insertion of a receptor population equals the rate of removal of the

unoccupied receptors [77, 110].

Therefore, Vr = kt [R]s
[R]s = the concentration of surface receptors, ([M])
Vr = rate of insertion of receptors into the cell membrane,
([M] min-1)
kt = rate of endocytosis of unoccupied receptors, (min-1)


Ligand stimulation of the cells can then adjust these cellular

dynamics. Ligand occupancy of the cell-surface receptors introduces

another variable into the equation. Often the rate of internalization of

the occupied receptors exceeds that of unoccupied receptors.

Therefore, Vr = kt[R]s + ke[LR]s
[LR]s = the concentration of surface ligand:receptor complexes,
([M])
ke = endocytosis of ligand occupied receptors, (min-1)

If the procedures for a 40C equilibrium binding experiment are

performed at 370C and the results are plotted linearly as in a

Scatchard graph, the slope reveals not -Ka, but the negative steady

state constant (-Kss), and the x-intercept represents Vr/ke instead of

Bmax. The determination of ke and kt is discussed later. The units for

Kss and Vr/ke are the same as Ka and Bmax, respectively. However, Ka

is only the association constant of the ligand and its specific surface-

bound receptor; Kss represents cell dynamics internally and externally

[110]. The Kss of human IFNuA interaction with MDBK cells was









previously determined to be 1.9 x 1010 M-1 [76]. The equation Kss for

is as follows:

Kss = (keka)/kt(kd + ke)
Kss = Steady state constant, (molar concentration-' ([M-1])

The internalization of ligand-receptor complexes can be

expressed using the following equation:

[LR]i = ke[LR]s/kh
[LR]i = the concentration of internalized ligand:receptor
complexes, ([M])
kh = rate of hydrolysis of internalized ligand, (min-1)

When the rates of internalized ligand-receptor complexes and

degradation of ligand internally remain unchanged (i.e. steady state is

achieved), then ke and kh can be accurately determined. The

degraded ligand is monitored by treating with TCA. The TCA soluble

fraction reveals the degraded ligand [119]. If degradation is not

occurring, internal ligand measurements are assumed to be solely

dependent on internalized ligand from the cell surface, isolating a ke

measurement [110]. Therefore, the slope of [LR]/[LR]s versus time

when degradation is not occurring equals ke. A linear plot shows that

degradation is not occurring and that the cell has reached a steady

state. A change in the slope of the line reveals when degradation

occurs. The ke of human IFNacA into MDBK cells was 3.1 x 10-2 min-1

and the kh was 1.4 x 10-2 min-1 [75]. The ratio of ke/kh was therefore









2.2. For every two human IFNu.A molecules endocytosed, only one is

hydrolyzed.

The rate of turnover of unoccupied receptors can be used to

determine the relative change in internalization of receptors into cells

when a ligand is associated with the receptor [110]. Initially, cells are

allowed to reach steady state in the presence of a constant,

subsaturating concentration of ligand, [L]x. The bound and free ligand

quantities are measured and cells with the same concentration of

ligand are placed at 40C. Therefore, the receptor population that was

readjusted by adding subsaturating amounts of ligand is prevented

from changing any further. An equilibrium-binding assay is then

performed on these cells to reveal the new number of cell-surface

receptors. The x-intercept equals [LR]x + [R]x and the value for [LR]x

was previously determined at 370C. So, kt is then determined by the

following equation:

kt = (Vr ke[LR]x)/[R]x

Zoon et al. determined the kt of the type I IFN receptors of MDBK cells

in response to human IFNacA stimulation to be 2.3 x 10-2 min-1 [75].

However, a computer modeling program that calculates steady state

parameters ka, kd, ke, kh, and kt using the time-concentration pairs of

[LR]s and [LR]i in Zoon's studies contended that a kt, of









3.3 x 10-3 min-1 was a better fit [74]. The ratio of ke/kt reported by

Zoon for IFN.A treated MDBK cells was 1.4. When using the best fit

as determined by the computer modeling program, the ratio increased

to 9.7, suggesting that in MDBK cells, ligand-occupied type I IFN

receptors are internalized 10 times faster than unoccupied type I IFN

receptors.

Elucidating the specific structural/functional sites on ovine IFNT

and the type I IFN receptor and determining the cellular binding,

internalization and processing of this ligand and its receptor are very

significant, especially in view of its potent biological activities without

type I IFN associated toxicity. Most importantly, determining the sites

involved in binding and function of ovine IFNT and IFNAR2 may serve

as groundwork for the future design of less toxic and more specific

therapeutics. Further, determination of the specific binding sites and

cellular processing necessary for the ovine IFN-:type I IFN receptor

interaction will also provide important insight into receptor recognition

and signal transduction initialized by type I IFNs.












CHAPTER 2
MATERIALS AND METHODS


Cell Lines

Madin-Darby Bovine Kidney (MDBK) cells, a cell line responsive

to ovine IFNt, were used for all assays involving viable cells. Cells

were grown in a 370C, 5% C02, 100% humidity incubator. Eagle

minimum essential media (EMEM) supplemented with antibiotics, 12

U/mL of penecillin and 125 [tg/mL streptomycin, and 10% fetal bovine

serum (FBS) nourished the cells.


Recombinant Ovine IFNt

Recombinant ovine IFNt was expressed in Pichia pastoris. The

specific antiviral activity of the purified ovine IFNt was determined to

be 1 x 108 antiviral units/mg. The FDA approved IFN, Roferon, was

used as the standard in the antiviral assay. Recombinant ovine IFN-

was received as a gift from two independent sources, Dr. Howard M.

Johnson and Dr. Fuller W. Bazer.


Peptide Synthesis and Purification

Using solid-phase 9-fluorenylmethylcarbonyl (Fmoc) chemistry,

overlapping peptides corresponding to the primary sequence of ovine

IFNt and IFNAR2, the binding subunit of the type I IFN receptor were









synthesized on a PE Biosystems 9050 Peptide Synthesizer [121].

Completed peptides were removed from the resin by adding 2.5:44:1

phenol: trifluoroacetic acid (TFA): triispropylsilane. Resin-conjugated

peptides in this solution were placed on a nutator for two hours to

separate the peptides from the resin. The peptides remain in solution;

the resin is insoluble. The TFA solution containing the soluble peptides

was then treated with ethyl ether at a 10 to 1 ratio to precipitate the

peptide product. The peptides were then resolubilized with deionized

water and lyophilized to remove moisture. Molecular weight and purity

of the synthesized peptides were analyzed using mass spectrometry

(Alfred Chung, Protein Core, University of Florida). Amino acid

analysis was used to determine if the amino acid composition of the

peptide product was as expected (Alfred Chung, Protein Core,

University of Florida).


Biotin Labeling of IFNT Peptides and Ovine IFNT for Synthetic Peptide
Studies

Ovine IFNT and ovine IFNT peptides were biotinylated by using a

biotin-bound, reactive N-hydroxysuccinimide ester [122]. The N-

hydroxysulfosuccinimide (NHS) ester cross-links biotin to lysine

epsilon-amino groups. Ovine IFNT (2 mg) or ovine IFNT peptides (4.5

mg) were dissolved in 500 [IL of pH 7.2 phosphate buffered saline

(PBS). NHS-LC-Biotin (Pierce, Rockford, IL) (2 mg) was dissolved in 1









mL of DMF and then 20 [pL were added to either the ovine IFNT or

ovine IFNT peptide solution. The reaction mixture was then placed on

ice for 2 hours and agitated every 30 minutes to maximize

biotinylation. Free biotin was removed from the biotinylated ovine

IFNT by centrifugation for at least 15 minutes in a 3000 molecular

weight cut-off (MWCO) Amicon microconcentrator. This process was

performed three times. Biotinylated ovine IFNT peptides were

removed from the free biotin by gel filtration using either a pre-packed

1800 MWCO column (Pierce, Rockford, IL) or a column packed with

Sephadex G-10, whichever was more appropriate for the mass of the

peptide. The protein concentration was then determined using a

bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) [123].


Radioiodination of Ovine IFNt for Synthetic Peptide Studies

Ovine IFNt was radiolabeled with 125-Iodine using the Bolton and

Hunter monoiodoester and the Bolton and Hunter protocol [124]. The

solvent on the monoiodoester was removed by a gentle stream of

nitrogen gas transferred by a 22-gauge needle inserted into the cap of

the vial. Ovine IFNT (5 [pg) in 10 pl of 0.1 M borate buffer, pH 8.5, was

added to the 500 pCi of monoiodoester in the solid-phase, agitated,

and placed on ice for 15 minutes. Further reactions between ovine

IFN- and the monoiodoester were stopped by a 5-minute incubation at

0C with 500 pl of 0.2 M glycine in 0.1 M borate buffer. Ovine 125,-








IFNc was separated from unwanted radiolabeled products by a gel

filtration column. A Sephadex G-10 packed column was equilibrated

and eluted using 0.05 M phosphate buffer with 0.25% gelatin w/v.

Specific activity of the ovine 125I-IFN- was determined by placing 5 |l

of the radiolabeled proteins in a 1275 Minigamma gamma counter

(LBK Wallac).


Direct Binding Assays

Synthetic IFNAR2 peptides (1.5 [tg) were bound to 96-microwell

plates using a pH 9.6, 0.1 M bicarbonate binding buffer [125]. Solvent

was removed by evaporation using a gentle stream of warm air.

Nonspecific binding was inhibited by the addition of 1% bovine serum

albumin (BSA) in phosphate buffered saline (PBS), pH 7.5, to the

solid-phase IFNAR2 peptides for 2 hours. The BSA solution was then

removed and the IFNAR2 peptides were treated with biotinylated-

intact ovine IFNT or biotinylated-synthetic ovine IFNT peptides at a

final concentration of 0.5 [!M. The peptides were then incubated with

anti-biotin antibodies conjugated to alkaline phosphatase (1:5000

dilution) for 1 hour. At a volume of 50 [iL/well, p-Nitrophenyl

phosphate (PNP) (1 mg/mL) was added. Receptor peptide binding was

determined colormetrically by measuring the alkaline phosphatase

activity at a wavelength of 405 nM (Bio-Rad, Hercules, CA).









Competitive Binding Assays on Viable Cells

Viable MDBK cells were grown to near confluency on microwell

plates. The cells were treated with a final concentration of 1 nM of

125I-ovine IFN- in the presence of increasing concentrations of

synthetic ovine IFNT or IFNAR2 peptide competitors [125, 126].

Radioiodine counts from bound 125I-ovine IFN- were measured after a

2-hour incubation at room temperature. Wells were removed from the

microwell strip plates and placed in the 1275 Minigamma gamma

counter (LBK Wallac).


Solid-phase Competition Assays

Synthetic IFNAR2 peptides (1.5 [tg) were bound to 96-microwell

plates using a pH 9.6, 0.1 M bicarbonate binding buffer [125]. Solvent

was removed by evaporation using a gentle stream of warm air.

Solid-phase peptides were treated with a final concentration of 5 nM of

125I-ovine IFN- in the presence of increasing concentrations of

synthetic ovine IFNT or IFNAR2 peptide competitors. Radioiodine

counts from bound 125I-ovine IFNT were measured after a two-hour

incubation at room temperature. Wells were removed from the

microwell strip plates and placed in the 1275 Minigamma gamma

counter (LBK Wallac).









Vesicular Stomatis Virus (VSV) Propagation

MDBK cells were grown to confluency in 75-cm2 tissue culture-

treated flasks in EMEM supplemented with antibiotics, 12 U/mL of

penecillin and 125 [tg/mL streptomycin, and 10% FBS, respectively, in

a 370C, 5% carbon dioxide, 100% humidified incubator. To increase

the yield of VSV and increase specificity of the virus for each cell line,

confluent monolayers were infected with approximately 3 x 105 plaque

forming units of VSV in 3 mL of 370C of EMEM. The VSV was supplied

by Dr. Janet K. Yamamoto at the University of Florida. The flasks were

incubated at 370C for a total of 1 hour. Every 15 minutes the flasks

were gently rocked for approximately 30 seconds to maximize viral

absorption to cells. The maintenance medium, EMEM supplemented

with 2% FBS, was warmed to 370C and added to the virally infected

flasks at a volume of 30 mL. The flasks were placed back in the 370C,

5% CO2, humidified incubator until greater than 50% cytopathic effect

(CPE) occurred, requiring at least a 24-hour incubation time. The

supernatants, which contained the virus, were then removed and

centrifuged at approximately 1000 rpm for 5 minutes to remove

residual cellular material. The supernatants of the centrifugation step

were then placed on ice, 10% DMSO was added, and virus was stored

at -700C in 1 mL aliquots.









Antiviral Cytopathic Effect Inhibition Assay

MDBK cells in microwell plates at near confluency were incubated

with 10 U/mL of ovine IFNT in the presence of extracellular competitor

receptor peptides for 18 hours [8, 39]. Ovine IFNT reaches optimum

antiviral activity by 18 hours. The MDBK cells monolayers were then

infected with VSV. After 36 hours the remaining fixed cells were

stained with crystal violet, washed, and air-dried. The dye was

extracted from each well with 2-methoxyethanol [53]. The

absorbance of dye was then measured at 570 nM to reveal the ability

of the receptor peptides to inhibit the antiviral activity of ovine IFNT.


Localization of IFNAR2 in MDBK Cells Using Immunofluorescence

MDBK cells (2.5 x 105/ slide) were grown overnight in

chambered glass slides (Lab-Tek). Cells were stimulated with 5000

U/mL of ovine IFNT and incubated at 40C for 2 hours. Cells were then

transferred to a 370C incubator for the time intervals, 0, 8, 16, 32, 60,

and 120 minutes. Cells were fixed immediately after the completion of

each time interval with 2% paraformaldehyde at 40C for 30 minutes.

Polyclonal rabbit antibody against IFNAR2 (Santa Cruz, Santa Cruz,

CA) in 0.1% saponin (Sigma, St. Louis, MO) was placed on the cells for

1 hour at room temperature. Saponin, derived from quillaja bark,

permeabilizes the cells. After three vigorous washings with the

saponin:1% BSA: PBS solution, cells were treated with goat anti-rabbit









secondary antibodies conjugated to Texas Red (Molecular Probes,

Eugene, OR), for 1 hour at room temperature, also in the presence of

saponin as the permeabilization is temporary. The goat anti-rabbit

secondary immunoglobulin G (IgG) antibodies were highly cross-

absorbed against bovine, goat, human, mouse, and rat IgG serum.

Cells were viewed using a Zeiss fluorescent microscope at a 40X

magnification. Pictures of the cells were taken using a Spot digital

camera (Diagnostic Instrumental, Inc.) and the Spot version 3.0 digital

imaging software.


Localization of Ovine IFNT with respect to the Lysosomes in MDBK Cells
Using Immunofluorescence

MDBK cells (2.5 x 105/slide) were grown overnight in chambered

glass slides (Lab-Tek). Cells were stimulated with 5000 U/mL of ovine

IFNT and incubated at 40C for 2 hours; the units per milliliter were

standardized by the antiviral activity of the FDA approved human

IFNa, Roferon. Cells were then transferred to a 370C incubator for

various time intervals. After fixation with 2% paraformaldehyde at 40C

for 30 minutes, cells were treated with a mouse monoclonal antibody

against ovine IFNT and a highly cross-absorbed rabbit polyclonal

antibody against the lysosomal associated matrix protein 1 (LAMP-1)

(Santa Cruz, Santa Cruz, CA) in 0.1% saponin to permeabilize the

cells. After three vigorous washings with the saponin:BSA:PBS









solution, cells were treated with a highly cross-absorbed polyclonal

goat anti-mouse secondary antibody coupled to Alexa Green

(Molecular Probes, Eugene, OR) and highly cross-absorbed polyclonal

goat anti-rabbit secondary antibody coupled to Texas Red (Molecular

Probes, Eugene, OR), also in the presence of saponin as the

permeabilization is temporary. The goat anti-rabbit secondary

immunoglobulin G (IgG) antibodies were highly cross-absorbed against

bovine, goat, human, mouse, and rat IgG serum; the goat anti-mouse

secondary IgG antibodies were highly cross-absorbed against bovine,

goat, human, rabbit, and rat IgG serum. The anti-mouse antibody:

Alexa Green conjugate allows the detection of ovine IFNT and the anti-

rabbit: Texas Red conjugate allows the detection of LAMP-1. Cells

were viewed using a Zeiss fluorescent microscope at 40X and 63X

magnification. Pictures of the cells were taken using a Spot digital

camera (Diagnostic Instrumental, Inc.) and the Spot version 3.0 digital

imaging software.


Assays to Determine Lysosomal Degradation

MDBK cells grown to near confluency in 150 cm2 flasks were

treated with a final concentration of 100 [M chloroquine in 10 mL of

10% FBS MEM or 10 mL of 10% FBS MEM with no chloroquine and

incubated for 4 hours at 37oC [77, 109, 119]. Cells were scraped from

the flasks and resuspended in 35 mLs of MEM. The cells were then








treated with a final concentration of 0.5 nM of ovine 125I-IFNT. Cells

were incubated on a rocker for 7 hours at 40C to allow ovine 125I-IFNT

to bind. Cells were aliquoted at a quantity of 2.7 x 106 cells/mL to

represent the time intervals of 37oC incubation. The specific time

intervals were 0, 15, 60, and 120 minutes. The sets of cells were then

placed into a 370C water bath for the specified times. Cells were then

removed from the water bath at appropriate times and were pelleted

using centrifugation. Following centrifugation the supernatant fluids

from each 1 mL replicate were aspirated and treated with 200 itg of

BSA followed by a 10% final volume of TCA to precipitate intact ligand,

leaving degraded ligand in solution [109]. The TCA treated

supernatants were incubated overnight at 4C to ensure proper

separation of degraded and intact ligand.


Iodogen Radioiodination of Ovine IFNT and Human IFNaA for
Equilibrium Binding and Kinetic Assays

Iodogen (75 [L_) was placed in a glass vial under a gentle stream

of air to evaporate the chloroform solvent from the solid-phase [127].

Foil was placed over the apparatus to protect iodogen from exposure

to light, as it is light sensitive. Evaporation time was approximately 15

minutes. Ovine IFNt or human IFNuA (10 [IL of 1 mg/mL) in 65 [iL of

0.05 phosphate buffer pH 7.5 was then placed on the solid-phase

iodogen [129, 130]. Free 125Iodine (0.5 [[Ci) was added to the vial and









incubated at room temperature for 5 minutes. Radiolabeling of the

interferon molecules was slowed with the addition of excess 0.25%

gelatin in 0.05 M phosphate buffer, pH 7.5, which dilutes the reaction

mixture. The reaction was stopped when this solution was aspirated

from the solid phase lodogen. The solution was then placed on a

column packed with Sephadex G-25. The Sephadex G-25 was

previously equilibrated and eluted with 0.25% gelatin in 0.05 M

phosphate buffer, pH 7.5. One milliliter fractions were removed and

counted on the gamma counter to determine fractions containing

radiolabeled ligand. Radiolabeled ligand fractions were then tested for

their biological potency using an antiviral cytopathic assay.


Cellular Binding and Internalization Kinetic Assays

MDBK cells grown to near confluency were rinsed 2 times with

40C FBS-free EMEM in the cold room. FBS-free EMEM (5 mL) was then

added to flasks, to allow the manual removal of cells. Cells were spun

down and resuspended in 35 mLs of MEM with 0.1% BSA to reduce

non-specific binding in the experiment. The viable cells population

was determined with a hemocytometer and trypan blue treatment to

detect dead cells. The cells were then split into two sets. Set one, the

experimental set, was treated with a final concentration of 0.5 nM of

either 1251- ovine IFNt or 125I-human IFNuA [105]. In addition to the

final concentration of 0.5 nM of either 1251-ovine IFNt or 125I-human









IFNuA, set two was treated with a 100 nM final concentration of

unlabeled ovine IFNT or human IFNuA, respectively [77, 105]. Still in

the cold room, the tubes were placed on a rocker to increase

homogeneity and cellular contact and binding with ligand. Cells were

incubated on the nutator for 7 hours. Duplicates of the experimental

and control cell sets were then agitated and aliquoted at a quantity of

2.7 x 106 cells/mL into 5 sets of 3 mLs to represent the 5 time

intervals of 370C incubation. Triplicate one mL aliquots of the

radiolabeled IFN treated cells were then placed into microcentrifuge

tubes for each time point. The specific time intervals were 0, 16, 32,

60, and 120 minutes. The sets of cells were then placed into a 370C

water bath for the specified times. The cells were agitated while in the

water bath to maintain homogeneity. Cells were then removed from

the bath at appropriate times and transferred to the cold room where

the surface bound ligand could be separated from the internalized

ligand by centrifugation for 10 seconds [77]. Following centrifugation,

the supernatant was aspirated and 200 itg of BSA was added per mL of

supernatant followed by a 10% final volume of TCA, which precipitated

intact ligand, leaving degraded ligand in solution [77, 111]. The TCA

treated supernatants were incubated overnight at 40C. The cell pellets

were treated with a glycine buffer (100 mM NaCI/ 50 mM glycine) to

remove surface-bound ligand [105]. The cells were solubilized in 0.1









M NaOH at room temperature for 10 minutes to release internalized

ligand [75, 105]. The specific activities of the radiolabeled human

IFNuA and ovine IFNT ranged from 157 to 161 [tCi/[g and 122 to 129

[lCi/[g, respectively. Radioiodinations were performed using solid-

phase iodogen as previously described.


Equilibrium Binding Assay

MDBK cells grown to near confluency were rinsed 2 times with

40C FBS-free MEM in the cold room. Fetal bovine serum-free MEM was

then added to flasks, so flasks could be scraped to collect cells. Cells

were pelleted and resuspended in MEM with 0.1% BSA to reduce non-

specific binding in the experiment. The viable cells population was

determined with a hemocytometer and trypan blue treatment to detect

dead cells. The cells were aliquoted at a quantity of 2.5 x 106 cells/mL

into 5 duplicate sets of 3 mLs, one experimental set and one control

set. At 40C, the duplicate sets of cells were treated with increasing

concentrations of 1251-ovine IFNT or 125I-human IFNuA [75, 77]. The

control sets were treated with the same concentrations of radiolabeled

ligand and 100 nM of unlabeled ovine IFNT or human IFNuA to reveal

non-specific binding. Still in the cold room, the tubes were placed on a

nutator to increase homogeneity and cellular contact and binding with

ligand. Cells were incubated on a rocker for 9 hours to ensure

equilibrium was reached. The cells were then transferred to microfuge









tubes and surface bound ligand was separated from the free ligand by

centrifugation for 10 seconds. The supernatant was removed to

determine [L]f. The tips of the microfuge tubes were cut off at the 0.1

mL mark to measure [LR]s [77]. Non-specific binding was subtracted

from total binding to reveal specific binding. The specific activities of

the radiolabeled human IFNoCA and ovine IFNT ranged from 157 to 161

[lCi/[g and 122 to 129 [tCi/[g, respectively. Radioiodinations were

performed using solid-phase iodogen as previously described [127].

Linear regressions of the Scatchard plots used to determine the

equilibrium constants and total concentrations of surface receptor were

derived using the Sigmaplot version 2000 scientific graphing software.

GraphPad Prism software version 3.0, a program specifically designed

for ligand:receptor binding analysis, calculated the binding constants

and receptor concentrations using nonlinear regression analysis.


Steady State Binding Assays

MDBK cells grown to near confluency were rinsed 2 times with

40C FBS-free MEM. Flasks were scraped to collect cells. Cells were

pelleted and resuspended in MEM with 0.1% BSA to reduce non-

specific binding in the experiment. The viable cell population was

determined with a hemocytometer. Trypan blue was added to the

suspension for the detection of dead cells. The cells were aliquoted at

a quantity of 2.0 x 106 cells/mL into 5 duplicate sets of 3 mL volumes,









one experimental set and one control set. The duplicate sets of cells

were treated with increasing concentrations of 1251-ovine IFN- or 125-

human IFNuA [77, 105, 110]. The control sets were treated with the

same concentrations of radioligand and 100 nM of unlabeled ovine

IFNT or human IFNuA to reveal non-specific binding. The cells were

incubated for 8 hours at 370C to achieve steady state conditions [75,

110]. The cells were then transferred to microfuge tubes in one mL

aliquots and surface bound ligand was separated from the free ligand

by centrifugation. The supernatant was removed to determine [L]f.

Using a hot scalpel, the tips of the microfuge tubes were cut off at the

0.1 mL mark to measure [LR]T [77]. Non-specific binding was

subtracted from total binding to reveal specific binding. The specific

activities of the radiolabeled human IFNuA and ovine IFNT ranged from

157 to 161 [tCi/[tg and 122 to 129 [tCi/[tg, respectively.

Radioiodinations were performed using solid-phase iodogen as

previously described [127]. Sigmaplot version 2000 scientific graphing

software derived the steady state constants and total receptor

concentrations by calculating the slope and x-intercept of the

Scatchard graphs modified for steady state assays. GraphPad Prism

software version 3.0 eliminated the need of the linear transformation

of nonlinear raw data by calculating the binding constants and receptor

concentrations using nonlinear regression analysis.








Unoccupied Receptor Turnover Binding Assays

MDBK cells grown to near confluency were rinsed two times with

40C FBS-free MEM. FBS-free MEM was then added to flasks, so flasks

could be scraped to collect cells. Cells were pelleted and resuspended

in MEM with 0.1% BSA to reduce non-specific binding in the

experiment. The viable cells population was determined with a

hemocytometer and trypan blue treatment to detect dead cells.

Human 125I-IFNaA (6 x 105 CPM) or ovine 125I-IFNt (6 x 105 CPM) was

added to each 3 mL aliquot of 1.0 x 106 cells/mL in one experimental

group and one control group [77, 110]. Concentrations of unlabeled

human IFNaA or ovine IFN- that were equivalent to the amounts of

radiolabeled IFN were added to 3 mL aliquots of 1.0 x 106 cells/mL in 5

experimental group and 5 control sets of cells. Each set of cells

represented a concentration point for the equilibrium-binding assay

that was performed after these cells reached steady state. The cells

were incubated for 8 hours at 370C for steady state to be achieved.

Cells with 125I-human IFNaA of 1251-ovine IFNT were then immediately

pelleted by centrifugation to separate free ligand from receptor bound

ligand. The tips of the microfuge tubes were cut off with a hot scalpel

to reveal [LR]x [77, 110]. The constant subsaturating concentration of

ligand added is represented by the subscript, x. The concentration of

free ligand was determined by counting the supernatant in the gamma









counter. The remaining cells, which were previously treated with

unlabeled ligand, were transferred to the cold room. The duplicate

sets of cells were treated with increasing concentrations of 125_-ovine

IFN- or 125I-human IFNuA. The control sets were treated with the

same concentration of radiolabeled ligand and 100nM of unlabeled

ovine IFNT or human IFNuA to reveal non-specific binding [77]. The

cells were incubated at 40C on a rocker for 9 hours to ensure that

equilibrium was reached. The cells were then transferred to microfuge

tubes in one mL aliquots and surface bound ligand was separated from

free ligand by centrifugation. The supernatant fluid was removed to

determine [L]f. Using a hot scalpel, the tips of the microfuge tubes

were cut off at the 0.1 mL mark to measure [LR]T [77]. Non-specific

binding was subtracted from total binding to reveal specific binding.

The specific activities of the radiolabeled human IFNuA and ovine IFNT

ranged from 157 to 161 [tCi/[g and 122 to 129 [tCi/[g, respectively.

Radioiodinations were performed using solid-phase iodogen as

previously described [127]. The total receptor concentrations were

determined as the x-intercept of the Scatchard plots as revealed by

Sigmaplot version 2000 scientific graphing software. GraphPad Prism

software version 3.0, a program specifically designed for

ligand:receptor binding analysis, calculated the total receptor

concentrations using nonlinear regression analysis.













CHAPTER 3
DETERMINATION OF STRUCTURAL AND FUNCTIONAL REGIONS OF
OVINE IFNt AND THE TYPE I IFN RECEPTOR


Direct Binding of Biotinylated Ovine IFNc to IFNAR2 Extracellular
Domain Peptides

To determine the major ligand binding sites on IFNAR2 for ovine

IFNt, long overlapping peptides of the extracellular domain of IFNAR2

were synthesized [134]. The sequences of the synthesized IFNAR2

peptides are shown in Table 3-1. As an initial approach, direct binding

studies entailed treating immobilized extracellular domain IFNAR2

peptides with biotin-labeled ovine IFNt. The biotinylated, biologically

active ovine IFNc bound directly and most significantly to peptides

IFNAR2(1-38) and IFNAR2(34-67) of the 7 synthetic peptides of the

extracellular domain (Figure 3-1). Direct binding of biotinylated ovine

IFN- to overlapping synthetic peptides of the extracellular domain of

IFNAR2. Biotinylated ovine IFNc was used at a final concentration of

0.5 [M. Receptor peptide binding was determined colormetrically by

alkaline phosphatase activity. Bars represent the standard

significantly to peptides IFNAR2(1-38) and IFNAR2(34-67) of the 7

synthetic peptides of the extracellular domain (Figure 3-1).








Table 3-1. Sequences and secondary structure predictions of the
synthetic extracellular domain peptides of IFNAR2. Secondary
structure predictions were formulated by comparing Chou-Fasman
(CF) secondary prediction results to those obtained using the Garnier-
Osguthorpe-Robson (GOR) secondary structure prediction method.
Chou-Fasman and GOR predictions were calculated using the
PeptideStructure computer program (Genetics Computer Group, Inc.)
[129-131]. Lower case letters denote regions where the structure
prediction models did not coincide or predictions were weak.


Peptide Name


Peptide Sequence


Secondary Structure


IFNAR2(1-38) ISYDSPDYTDES TURN/BETA/
CTFKISLRNFRSI helix/BETA
LSWELKNHSIVP
T
IFNAR2(34-67) SIVPTHYTLLYTI BETA/turn/
MSKPEDLKVVK BETA/turn
NCANTTRSFC
IFNAR2(63-99) TRSFCDLTDEW turn/helix/
RSTHEAYVTVLE turn/BETA/
GFSGNTTLFSCS TURN/BETA
IFNAR2(95-133) LFSCSHNFWLAI BETA/TURN/
DMSFEPPEFEIV HELIX/TURN/
GFTNHINVMVK BETA
FPSI
IFNAR2(129-164) FPSIVEEELQFD BETA/HELIX/
LSLVIEEQSEGI turn
VKKHKPEIKGN
IFNAR2(160-196) EIKGNMSGNFT turn/BETA/
YIIDKLIPNTNYC TURN/BETA/
VSVYLEHSDEQ TURN
A
IFNAR2(186-217) LEHSDEQAVIKS TURN/HELIX/
PLKCTLLPPGQE BETA/TURN/
SESAESAK HELIX














2.0






1.5

0O
0









COCt- O D C> t
O 1.0
0
O,




0.5






0.0
00 1-- CO""--

CY) CD

Extracellular IFNAR2 Peptides


Figure 3-1. Direct binding of biotinylated ovine IFN- to overlapping
synthetic peptides of the extracellular domain of IFNAR2. Biotinylated
ovine IFN- was used at a final concentration of 0.5 [pM. Receptor
peptide binding was determined colormetrically by alkaline
phosphatase activity. Bars represent the standard error. All assays
were performed in triplicate.








The remaining five receptor peptides were not recognized by the

biotinylated ovine IFNT (Figure 3-1). These results suggest that the

first 67 amino acids of the N-terminus of IFNAR2 may play an

important role in the recognition of ovine IFNT.


Extracellular Domain Receptor Peptides Block Ovine 125I-IFNt Binding
to Viable MDBK Cells

Specificity of bindings was established using the IFNAR2 peptides

as competitors against type I IFN receptors on MDBK cells for

biologically active ovine 125I-IFNt. On viable and intact MDBK cells, N-

terminal peptides IFNAR2(1-38) and IFNAR2(34-67) were dose-

dependent inhibitors of type I IFN receptor binding of ovine 125I-IFNt

at a final concentration of 5 nM (Figure 3-2). Peptide IFNAR2(34-67)

consistently appeared to be a more effective inhibitor at higher

concentrations, which suggests that a region within 34-67 may be

more structurally important for ovine IFNt binding (Figure 3-2). The

failure of IFNAR2(186-217) to block binding is representative of those

receptor peptides that did not recognize ovine IFNt (Figure 3-2). In

addition, the combination of IFNAR2(1-38) and IFNAR2(34-67), in

which each peptide made up 50% of the total concentration of each

dose, inhibited ovine 125I-IFNt binding by a factor greater than the

additive effect of the individual peptides (Figure 3-2). Therefore, the

binding of ovine IFNt with the distinct ligand binding regions within














110

100 --- Ovine IFNt
---7- IFNAR2(1-38)
-9 IFNAR2(34-67)
90 IFNAR2(1-38 & 34-67)
I--- IFNAR2(186-217)
80 -
Z
UL
S70

() 60
0
50

C 40 -

"0 30

20

10

0
0.0001 0.001 0.01 0.1 1 10 100
Competitor Concentration [pM]


Figure 3-2. Dose-response of unlabeled ovine IFN- and extracellular
IFNAR2 peptide competitors on ovine IFNT binding to the type I IFN
receptor on viable MDBK cells. Ovine 125I-IFN1 was used at a final
concentration of 5 nM. Radioiodine counts from bound ovine 125I-IFNT
were measured after a 2-hour incubation at room temperature. All
assays were performed in triplicate with coefficients of variation not
greater than 10%. IFNAR2(186-217) represents receptor peptides
that did not bind to ovine IFNT.















100-


90-


S-80-


> 70 -


60 -


50- IFNAR2(34-67)
IFNAR2(34-67) Linear Regression
95% Confidence Intervals
40- 0 IFNAR2(1-38)
IFNAR2(1-38) Linear Regression
-- 95% Confidence Intervals
30 I I I
0.001 0.01 0.1 1 10 100

Competitor Concentration [pM]


Figure 3-3. Dose-response of extracellular IFNAR2 peptide
competitors, IFNAR2(1-38) and IFNAR2(34-67) on ovine 125I-IFNt
binding to the type I IFN receptor on MDBK cells. Ovine 125I-IFNt was
used at a final concentration of 1 nM. Radioiodine counts from bound
ovine 125I-IFNT were measured after a 2-hour incubation at room
temperature. Bars represent the standard error. All assays were
performed using at least four replicates to increase accuracy.








residues 1-67 in combination appears to establish the property of

avidity, thereby strengthening the interaction.


Differential Binding Affinities of IFNAR2(1-38) AND IFNAR2(34-67) for
Ovine 125I-IFNT

The more effective inhibition of ovine 125I-IFNt binding to MDBK

cells by IFNAR2(34-67) than IFNAR2(1-38) at concentrations above 1

[[M indicates that the affinity of ovine IFNT for these independent

binding regions may differ. Therefore, to further elucidate the

differential affinities of ovine 125I-IFNt for IFNAR2(1-38) and

IFNAR2(34-67), IFNAR2(1-38) and IFNAR2(34-67) served as

competitors against receptor on MDBK cells for a reduced,

subsaturating concentration of ovine 125I-IFNt (Figure 3-3). When

concentrations of radioligand approach saturation, low affinity binding

sometimes cannot be distinguished from that of high affinity because

of the shear quantity of radioligand that binds to low affinity sites.

Therefore, the final concentration of ovine 125I-IFNt to 1 nM was based

on previous Kd determinations for ovine IFNT binding to MDBK cells

[53]. The concentration of IFNAR2(1-38) required to block just 25%

of receptor binding was approximately 100-fold more than the

necessary concentration of IFNAR2(34-67) (Figure 3-3). This further

suggests that the sequence of the extracellular domain that

corresponds to peptide IFNAR2(1-38) may contain a low affinity














100



80 -
z
U-

w 60


040




20



0 I I
0.01 0.1 1 10 100
Competitor Concentration [pM]


Figure 3-4. Extracellular IFNAR2 peptide, IFNAR2(186-217), does not
effect ovine 125I-IFNt binding to the type I IFN receptor on MDBK cells.
Ovine 125I-IFN- was used at a final concentration of 1 nM. Radioiodine
counts from bound ovine 125I-IFNt were measured after a 2-hour
incubation at room temperature. Bars represent the standard error.
All assays were performed using at least four replicates to increase
accuracy.








binding site, whereas amino acids 34-67 may contain a site more

significant for ovine IFN- recognition. As observed in previous

experiments using a final concentration of 5 nM ovine 125I-IFNT,

extracellular IFNAR2(186-217) showed no dose-dependent inhibition of

receptor binding, indicating that this region is not crucial for direct

interactions between ovine IFNT and the type I IFN receptor (Figure

3-4).


Structurally Important Extracellular IFNAR2 Peptides Block Antiviral
Activity of Ovine IFNT

The physiological importance of the structurally required

peptides of the extracellular domain of IFNAR2 was examined using an

antiviral cytopathic effect inhibition assay. Consistent with the

competition binding assays, the structurally important IFNAR2

peptides also blocked antiviral activity of ovine IFNt (Figure 3-5).

Again, IFNAR2(34-67) was the more potent inhibitor of antiviral

activity when compared to IFNAR2(1-38) (Figure 3-5). In addition,

IFNAR2(1-38) and IFNAR2(34-67) in combination enhanced the

inhibition of function when compared to the individual performance of

either peptide (Figure 3-5). Peptide IFNAR2(186-217), previously

shown not to block binding of ovine 125I-IFN-, failed to block function

(Figure 3-5). Collectively, the dose-dependent inhibition of function as

well as inhibition of receptor binding by extracellular IFNAR2 peptides









IFNAR2(1-38) and IFNAR2(34-67) indicate that amino acids 1-67 of

the type I IFN receptor P subunit comprise a region or regions that are

structurally and functionally important.


Human IFNuD Antagonizes Biotinylated Ovine IFNT Binding to
IFNAR2(34-67)

Solid phase competition assays revealed that human IFNaD, like

ovine IFNT, specifically recognizes peptide IFNAR2(34-67) (Figure 3-6).

Unlabeled human IFNuD appeared to bind slightly more effectively

than unlabeled ovine IFNT to IFNAR2(34-67). These data further

demonstrate that ovine IFNT specifically binds IFNAR2(34-67) and may

require a region within amino acids 34-67 of IFNAR2 to bind to the

type I IFN receptor. However, it also suggests that the residues

corresponding to IFNAR2(34-67) do not recognize ovine IFNT

exclusively, but may interact with more than one type I IFN.


Direct Binding of Biotinylated Ovine IFNT to IFNAR2 Intracellular
Domain Peptides

To determine if possible ligand binding sites for ovine IFNT on

the intracellular IFNAR2 existed, long overlapping peptides of the

intracellular domain of IFNAR2 were synthesized. The sequences of

the synthesized intracellular domain IFNAR2 peptides are presented in

Table 3-2. A potential intracellular ligand binding site on the receptor

was revealed when biotinylated and biologically active ovine IFNT












110

100

90

80

< 70

*> 60

S50
U-
S40
0, 30

20 IFNAR2(1-38)
--- IFNAR2(34-67)
10 -I- IFNAR2(1-38 & 34-67)
i-I- IFNAR2(186-217)
0
0.01 0.1 1
Competitor Concentration [mM]


Figure 3-5. Extracellular IFNAR2 peptides inhibition of ovine IFN-
antiviral activity. After an 18-hour incubation of 10 U/mL ovine IFNT in
the presence of extracellular competitor receptor peptides, MDBK cell
monolayers were infected with VSV. After 36 hours, cells were stained
with crystal violet, washed, and air-dried. The remaining dye was
extracted with 2-methoxyethanol [53]. The absorbance was measured
at 570 nM. All assays were performed in triplicate.











110

100\

90. v

z 80 .

S70 ".
-o 60
-.
S50 \

M 40 .

30 -

o 20 0 Human IFND .
V Ovine IFNt
10 Linear Regression
- 95% Confidence Lines
0 I 'I
0.01 0.1
Competitor Concentration [pM]


Figure 3-6. Dose-dependent inhibition of biotinylated ovine IFNT
binding to IFNAR2(34-67) by unlabeled ovine IFNt and human IFNaD.
Biotinylated ovine IFNt was used at a final concentration of 0.5 [iM. All
assays were performed in triplicate with coefficients of variation not
greater than 10%.









Table 3-2. Sequences and secondary structure predictions of the
synthetic intracellular domain peptides of IFNAR2. Secondary
structure predictions were formulated by comparing Chou-Fasman
(CF) secondary prediction results to those obtained using the Garnier-
Osguthorpe-Robson (GOR) secondary structure prediction method
[129-131]. Chou-Fasman and GOR predictions were calculated using
the PeptideStructure computer program (Genetics Computer Group,
Inc.). Lower case letters denote regions where the structure
prediction models did not coincide or predictions were weak.


Peptide Name


Peptide Sequence


Secondary Structure


IFNAR2(265-300) KWIGYICLRNSLPKVLNFA BETA/turn/BETA/turn/
WPFPNLPPLEAMDM HELIX
IFNAR2(287-315) AWPFPNLPPLEAMDMVVE turn/HELIX/beta
VIYINRKKKVW D
IFNAR2(313-338) VWDYNYDDESDSDTEAA beta/TURN/BETA
PRTSGGGYT
IFNAR2(335-368) GGYTMHGLTVRPLGQASA BETA/HELIX
TSTESQLIDPESEEEP
IFNAR2(366-395) EEPDLPEVDVELPTMPKDS HELIX/TURN/HELIX/TUR
PQQLELLSGPC N
IFNAR2(393-423) GPCERRKSPLQDPFPEED TURN/BETA
YSSTEGSGGRITF
IFNAR2(421-445) ITFNVDLNSVFLRVLDDED BETA/TURN/HELIX
SDDLEA
IFNAR2(442-470) DLEAPLMLSSHLEEMVDP HELIX/TURN/helix
EDPDNVQSNHL
IFNAR2(467-498) SNHLLASGEGTQPTFPSP helix/beta/TURN/HELIX/
SSEGLWSEDAPSDQ TURN
IFNAR2(495-515) PSDQSDTSESDVDLGDG TURN/helix/TURN/beta
YIMR









bound to the intracellular IFNAR2 peptide, IFNAR2(287-315) (Figure

3-7). IFNAR2(265-300), an overlapping receptor peptide that lacks

the C-terminal 15 amino acids of IFNAR2(287-315), was not

recognized by biotinylated ovine IFNT, suggesting that these 15

residues may be particularly important for ovine IFNT binding (Figure

3-7). Peptides corresponding to residues 313-515 of IFNAR2 were not

significantly recognized by biotinylated ovine IFNT (Figure 3-7).

Therefore, similar to results found for IFNy, ovine IFNT appears to

recognize a region localized on the intracellular domain of the binding

subunit of the receptor [126].


An Intracellular Peptide Inhibits Ovine 125I-IFNt Binding to IFNAR2 on
Viable and Intact MDBK Cells

As the concentration of IFNAR2(287-315) was increased, binding

of ovine 125I-IFNt to MDBK cells decreased, revealing that this

synthetic peptide specifically recognized ovine 125I-IFNT (Figure 3-8).

When the final concentration of ovine 1251 -IFN was reduced to 1 nM in

the competition binding studies, IFNAR2(287-315) still effectively

competed with receptors on MDBK cells for ovine 125I-IFN- in a dose-

dependent manner (Figure 3-9). This further implicates the sequence

of the intracellular domain that corresponds to peptide IFNAR2(287-

315) as a potential binding site for ovine IFNt.













1.5








c 1.0 -
O
0
0
C
0


c 0.5 -
0)








0.0 ---- g-------
0 LO 1- O0 LO CY LO 0 O0 LO
0 V- CY CD 0T. 0 It 1- 0 I -

CD 0 0 V- CO C.0 0') Cq I- C. 0')
I I I I I I I I I I

Intracellular IFNAR2 Peptides



Figure 3-7. Direct binding of biotinylated ovine IFN- to overlapping
synthetic peptides of the intracellular domain of IFNAR2. Biotinylated
ovine IFN- was used at a final concentration of 0.5 [M. Receptor
peptide binding was determined colormetrically by alkaline
phosphatase activity. Bars represent the standard error. All assays
were performed in triplicate.












110
--- Ovine IFNt
100 -v-v- IFNAR2(287-315)
9 -- IFNAR2(186-217)
90

z 80
U-
u- 70

.E 60

50

40

30

20

10


0.0001 0.001 0.01 0.1 1 10 100
Competitor Concentration [pM]


Figure 3-8. Dose-response of unlabeled ovine IFNT and extracellular
IFNAR2 peptide competitors on ovine IFNT binding to the type I IFN
receptor on MDBK cells. Ovine 125I-IFNt was used at a final
concentration of 5 nM. Radioiodine counts from bound ovine 125I-IFNt
were measured after a 2-hour incubation at room temperature. All
assays were performed in triplicate with coefficients of variation not
greater than 10%. IFNAR2(186-217) represents receptor peptides
that did not bind to ovine IFNT.











110


100 -


S90 -
z


^ 80

0
S70 -


60 -


50 0 Ovine IFNc
Linear Regression
-- 95% Confidence Lines

40 r T T T-
0.1 1
Competitor Concentration [pM]


Figure 3-9. Dose-response of intracellular IFNAR2 peptide competitor,
IFNAR2(287-315) on ovine 125I-IFNt binding to the type I IFN receptor
on MDBK cells. Ovine 125I-IFNt was used at a final concentration of
1 nM. Radioiodine counts from bound ovine 125I-IFNt were measured
after a 2-hour incubation at room temperature. All assays were
performed using at least four replicates to increase accuracy.












110

100 v v v

90 -

80
Z
I-
1 70


0
| 50

40

0 -30

20

10 -- IFNAR2(287-315)
-V-- IFNAR2(186-217)

0.01 0.1 1

Competitor Concentration [mM]


Figure 3-10. Intracellular IFNAR2 peptide inhibition of ovine IFNt
antiviral activity. After an 18 hour incubation of 10 U/mL ovine IFNt in
the presence of varying concentrations of IFNAR2(287-315), MDBK cell
monolayers were infected with VSV. After 36 hours, cells were stained
with crystal violet, washed, and air-dried. The remaining dye was
extracted with 2-methoxyethanol [53]. The absorbance was measured
at 570 nM. All assays were performed in triplicate.








An Intracellular Peptide Inhibits Ovine IFNT Antiviral Activity

Like the structurally important extracellular receptor peptides

IFNAR2(34-67) and IFNAR2(1-38), the intracellular receptor peptide

IFNAR2(287-315) significantly inhibited ovine IFNT function in a

cytopathic antiviral assay (Figure 3-10). The potency of IFNAR2(287-

315) was most comparable to that of IFNAR2(34-67) and more

effective as an inhibitor than IFNAR2(1-38). This study suggests that

IFNAR2(287-315) represents a functionally important region on

IFNAR2.


Direct Binding of Biotin-conjugated Ovine IFNT Peptides to Extracellular
IFNAR2 Peptides

Three ovine IFNT peptides, IFNc(1-38), IFNT(118-138), and

IFNT(153-168), were labeled with biotin and used to reveal potential

sites of interaction between regions on ovine IFNT and IFNAR2.

Immobilized extracellular and intracellular domain IFNAR2 peptides

were treated with these biotinylated ovine IFNT peptides. In general,

on the extracellular domain IFNAR2 peptides, the biotin-labeled ovine

IFNT peptides bound to IFNAR2(1-38) and IFNAR2(34-67) (Figure

3-11). However, the recognition of the receptor peptides was not as

strong as that of ovine IFNT. IFNt(1-38) bound approximately as

strongly as ovine IFNT to IFNAR2(1-38) and one third as strongly as

ovine IFNT to IFNAR2(34-67) (Figure 3-11). Ovine IFNT peptide,








IFNT(118-138), bound significantly to IFNAR2(1-38) (Figure 3-11).

IFNAR2(34-67) was highly recognized by IFNT(118-138) (Figure 3-11).

The C-terminal ovine IFNT peptide, IFNT(153-168) did not significantly

bind IFNAR2(1-38); however, it did have some recognition of

IFNAR2(34-67) (Figure 3-11). The lack of recognition of

IFNT(153-168) concurs with the findings that the C-terminus of ovine

IFNT is not largely responsible, if at all, for extracellular receptor

recognition of the ovine IFNT molecule. It appears that the internal

amino acids of the ovine IFNT sequence, and possibly the N-terminus

of ovine IFNT, may play the pivotal role in ovine IFNT receptor

recognition. These results also further suggest that the first 67 amino

acids of the N-terminus of IFNAR2 recognize regions on ovine IFNT.


Direct Binding of Biotin-conjugated Ovine IFNT Peptides to Intracellular
IFNAR2 Peptides

Interestingly, IFNT(153-168) was attracted to the intracellular

receptor peptide IFNAR2(287-315) (Figure 3-12). IFNT(118-138)

significantly recognized IFNAR2(287-315) and IFNc(1-38) also bound

IFNAR2(287-315), although less effectively. These results suggest

that sites within the internal and C-terminal regions of ovine IFNt may

be recognizing an intracellular, transmembrane proximal region of

IFNAR2. These results are similar to those in studies that have






61







1.4 IFNT(1-38)
W IFNT(118-138)
/ IFNT(153-168)
1.2


a 1.0
LO
0

,-o
cc 0.8 -

O
I 0.6
C
(0
0.4-


0.2


0.0 I
O0 ) CT0 CV CO C)
0 CD N
CD CD LO 0) 0 CD
0) N CD 00

Extracellular IFNAR2 Peptides


Figure 3-11. Binding of biotinylated ovine IFN-, and ovine IFN-
peptides, IFN-c(1-38), IFN-c(118-138), and IFN-c(153-168) to
overlapping synthetic peptides corresponding to the extracellular
domain of IFNAR2. Biotinylated ovine IFN- and ovine IFN- peptides
are at a final concentration of 0.5 jpM. Receptor peptide binding was
determined colormetrically by alkaline phosphatase activity. Bars
represent the standard deviations. All assays were performed in
triplicate.












1.2

M IFNtc(1-38)
m IFNT(118-138)
1.0 IFNt(153-168)



S0.8-


C
LO



| 0.6
0
o0


c 0.4
0)



0.2 -



0.0 I I
o LO 1- 00 LO Ce LO 0 00 LO
o M Cle CO O I t I'- O LO
CDO 0O CO C.O O' N 1- C.O O'
0 N0 CO C C I I t It
Intracellular IFNAR2 Peptides


Figure 3-12. Binding of biotinylated ovine IFN-, and ovine IFN-
peptides, IFNt(1-38), IFNT(118-138), and IFNT(153-168) to
overlapping synthetic peptides corresponding to the intracellular
domain of IFNAR2. Biotinylated ovine IFN- and ovine IFN- peptides
are at a final concentration of 0.5 [M. Receptor peptide binding was
determined colormetrically by alkaline phosphatase activity. Bars
represent the standard deviations. All assays were performed in
triplicate.








determined that the C-terminus of IFNy is recognizing an intracellular

receptor site [128].


Ovine IFNT Peptides Compete with Ovine 125I-IFNt for IFNAR2(1-38)

The remaining ovine IFNT peptides to complete the molecule

were synthesized. Each of the five helices inclusive of surrounding

turns and structurally random regions were represented, as recently

determined by x-ray crystallography are shown in Table 3-3. The

ovine IFNT peptides were initially used as competitors against ovine

125I-IFNt for immobilized extracellular receptor peptide IFNAR2(1-38)

in solid-phase competition assays. Five of the six ovine IFNT peptides

blocked ovine 125I-IFNt binding to IFNAR2(1-38) in a concentration-

dependent manner; only IFNT(136-158) did not compete (Figure

3-13A-F). However, although some dose-dependent inhibition of

binding of ovine 125I-IFNt to IFNAR2(1-38) was evident in the

saturation experiments, ovine IFNt did not appear to interact strongly

with this receptor peptide (Figure 3-14). The structural integrity of the

immobilized IFNAR2(1-38) was ensured by the competition between

soluble IFNAR2(1-38) and ovine 125I-IFNt (Figure 3-15). Receptor

peptide IFNAR2(186-217), a receptor peptide that did not bind ovine

IFNt in previous experiments, served as a negative control and did not

compete with ovine 125I-IFNt for immobilized IFNAR2(1-38).













Table 3-3. Sequences and secondary structure of the synthetic ovine
IFN- peptides. Secondary structure of ovine IFN- was determined with
x-ray crystallography [9].


Peptide Name


Peptide Sequence


Secondary Structure


IFNt(1-38) CYLSRKLMLDAREKLLDRM HELIX A, AB LOOP
NRSPHSCLQDRKDFGL (cys 29 bound to cys
139 of helix e), AB
LOOP 2 (note: loops
are turns that
connect the helices.)
IFNt(36-79) KDFGLPQEMVEGDQLQKD AB LOOP 2, AB LOOP
QAFPVLYEMLQQSFNLFYT 3, HELIX B, BC LOOP
EHSSAADT
IFNT(77-119) WDTTLLEQLCTGLQQQLD BC LOOP, HELIX C,
HLDTCRGQGMGEEDSELG CD LOOP
NMDPIVT
IFNT(118-138) LFSCSHNFWLAIDMSFEPP HELIX D, DE LOOP
EFEIVGFTNHINVMVKFPSI
IFNT(136-158) FPSIVEEELQFDLSLVIEEQ HELIX E
SEGIVKKHKPEIKGN
IFNT(153-168) EIKGNMSGNFTYIIDKLIPN HELIX E, RANDOM
TNYCVSVYLEHSDEQA REGION (i.e. little
electron density)






65




110

100

90

S80
LL-
O 70 -

60-

o) 50

._ 40-

30-

20

10 -- IFNt(1-38)

0 Iii I I I I I II I I I I I I I I I I I I I I I I I I I
0.1 1 10 100
Competitor Concentration [pM]


Figure 3-13. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(1-
38) in the presence of unlabeled ovine IFNT peptides, (A) IFN-(1-38),
(B) IFNt(36-79), (C) IFNT(77-119), (D) IFNT(118-138), (E) IFNt(136-
158) and (F) IFNT(153-168). Ovine 125I-IFNT was used at a final
concentration of 5 nM. All assays were performed in triplicate with
coefficients of variation not greater than 10%.











110

100

90

80

70

60

50

40

30

20

10


-0- IFNT(36-79)


100


Competitor Concentration [pM]


Figure 3-13B.













100

90

80

z 70
U-
S60-
0
- 50 -5
0)
0 40

o 30

20

10 -
-0 IFNT(77-119)


0.1 1 10 100
Competitor Concentration [[iM]


Figure 3-13C.













100

90

80

z 70
U-
LI.
c 60
0
u- 50
0)
0 40

o 30

20

10

0


1 10


Competitor Concentration [iM]


Figure 3-13D.


100













110

100-

90 -

S80
z
U-
Il_
- 70

O 60

0 50

Ec 40

30

20

10 - IFN-(136-158)

0*
0.1 1 10 100

Competitor Concentration [pM]


Figure 3-13E.













110

100-

90

80
z
U-
70

o 60

- 50
.C_
Ec 40

30

20

10 -*- IFN-(153-168)

0*
0.1 1 10 100
Competitor Concentration [pM]


Figure 3-13F.











100

90 -

80 1

z 70
U-

60

50

5 40-

S30-

20 -
Ovine IFN-c
10 -- Linear Regression
_- 95% Confidence Lines


0.001 0.01 0.1 1
Competitor Concentration [pM]


Figure 3-14. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(1-
38) in the presence of unlabeled ovine IFNT. Ovine 125I-IFNt was used
at a final concentration of 5 nM. Radioiodine counts from bound ovine
125I-IFN- were measured after a 2-hour incubation at room
temperature. All assays were performed in triplicate with coefficients
of variation not greater than 10%.











110

100 C0 0

90

80 o

70

60 -

50

40

30-

20 0 IFNAR2(1-38)
0 IFNAR2(186-217)
10 Linear Regression
-- 95% Confidence Lines

0.01 0.1 1 10
Competitor Concentration [pM]



Figure 3-15. Dose-response of extracellular IFNAR2 peptide
competitors, IFNAR2(1-38) and IFNAR2(186-217), on ovine 125I-IFNt
binding to the type I IFN receptor on MDBK cells. Ovine 125I-IFNt was
used at a final concentration of 5 nM. Radioiodine counts from bound
ovine 125I-IFNT were measured after a 2-hour incubation at room
temperature. All assays were performed using at least four replicates
to increase accuracy.








Ovine IFNT Peptides Compete with Ovine 125I-IFNt for IFNAR2(34-67)

In solid-phase competition assays, ovine 125I-IFNT binding to

IFNAR2(34-67) was displaced by three of the six unlabeled ovine IFNT

peptides, IFNt(1-38), IFNT(77-119), and IFNT(118-138) (Figure 3-16A,

C, and D). Therefore, both IFNAR2(1-38) and IFNAR2(34-67) were

specifically recognized by ovine IFNT peptides IFNt(1-38), IFNt(77-

119), and IFNT(118-138), suggesting that the N-terminal extracellular

domain of IFNAR2 recognizes multiple sites on ovine IFNT (Figures

3-13A, C, and D and 3-16A, C, and D). Although previously shown to

be functionally important [39], the C-terminus of ovine IFNt, does not

appear to be important for ovine IFN- interactions with the structurally

and functionally important extracellular regions of IFNAR2 (Figure

3-16F). It appears that the N-terminus of IFNAR2 specifically

recognizes sites within amino acids 1-38 of the N-terminus of ovine

IFNt and regions corresponding to residues 77-138. Unlabeled ovine

IFNt strongly inhibited ovine 125I-IFN- interaction with IFNAR2(34-67)

(Figure 3-17). These results provide further evidence for the

interaction of ovine IFNt and a region within residues 34-67 on

IFNAR2. Soluble IFNAR2(34-67) competitor inhibited ovine 125I-IFNt

binding to immobilized IFNAR2(34-67), suggesting that immobilization











110

100

90

H 80
z
LL
-- 70-

.c 60 -
0
50

40-

30

20

10 -*- IFN-(1-38)


0.1 1 10
Competitor Concentration [pM]


Figure 3-16. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(34-
67) in the presence of unlabeled ovine IFN- peptides, (A) IFNt(1-38),
(B) IFNt(36-79), (C) IFNT(77-119), (D) IFNT(118-138), (E) IFNt(136-
158) and (F) IFNT(153-168). Ovine 1251-IFN- was used at a final
concentration of 5 nM. Radioiodine counts from bound ovine 125I-IFNT
were measured after a 2-hour incubation at room temperature. All
assays were performed in triplicate with coefficients of variation not
greater than 10%.












110

100

90 -

H 80
z
U-
-- 70
0)
. 60-

50 -

40-

30

20

10 -A- IFNt(36-79)


0.1 1 10
Competitor Concentration [pM]


Figure 3-16B.













100

90 -

80

z 70
U-

S 60-






30

20

10
- IFNT(77-119)

0.1 1 10
Competitor Concentration [gM]


Figure 3-16C.











100

90

80

70

60

50

40

30

20

10

0


0.1 1 10
Competitor Concentration [pM]


Figure 3-16D.












110

100

90-

80 -

70 -

60-

50-

40-

30-

20 -

10-

0 -


-II


Competitor Concentration [pM]


Figure 3-16E.


-- IFNt(136-158)


I I I I I "


I I I I I I I















100

90

80

70

60

50

40

30

20

10

0 -


III


Competitor Concentration [pM]


Figure 3-16F.


-0- IFNt(153-168)


I ~ ~ I I I I I


S . . I I








of the receptor peptide did not significantly alter the structure of

IFNAR2(34-67) (Figure 3-18). In addition, the competition between

ovine 125I-IFNt and IFNAR2(34-67) complements other results showing

that ovine IFNt specifically interacts with sites within extracellular

IFNAR2 residues 34-67 (Figure 3-18). IFNAR2(186-217), an

extracellular IFNAR2 peptide that does not interact with ovine IFNt, did

not compete effectively for IFNAR2(34-67) (Figure 3-18).


Ovine IFNt and Ovine IFNt Peptides Specifically Recognize
IFNAR2(287-315)

Biotinylated ovine IFNt binding to immobilized intracellular

receptor peptide IFNAR2(287-315) was inhibited dose-dependently by

ovine IFNt peptide IFNT(118-138) (Figure 3-19B). Although IFNt(153-

168) recognized IFNAR2(287-315) in direct binding assays, IFNC(153-

168) did not effectively inhibit biotin-labeled ovine IFNt binding in the

competition assays, suggesting that the C-terminus of ovine IFN- does

not bind to an intracellular region of IFNAR2 (Figure 3-19C).

Therefore, it appears that IFNAR2(287-315) may interact with an

internal region of ovine IFNt, but not regions on the N- or C-terminus.

The binding of intact, biotinylated ovine IFNt to IFNAR2(287-315) was

effectively inhibited by unlabeled ovine IFNt (Figure 3-20). This

suggests that ovine IFNt specifically recognizes IFNAR2(287-315).

Competition binding assays in which soluble IFNAR2(287-315)













100

90 -

80 -


70 -

60 -

50 -

40 -

30 -

20 -

10 -


0 -
0.001


0.01


Competitor Concentration [pM]


Figure 3-17. Dose-dependent binding of ovine 125I-IFNt to IFNAR2(34-
67) in the presence of unlabeled ovine IFNT. Ovine 125I-IFNt was used
at a final concentration of 5 nM. Radioiodine counts from bound ovine
125I-IFNt were measured after a 2-hour incubation at room
temperature. All assays were performed in triplicate with coefficients
of variation not greater than 10%.


* Ovine IFN-c
-- Linear Regression
- 95% Confidence Lines


* ".






82



110

100 - -- --

90

z 80
U-
S70 \

60

O 50

40

o 30
IFNAR2(34-67) \
20 0 IFNAR2(186-217) *
10 Linear Regression
- 95% Confidence Lines

0.01 0.1 1 10
Competitor Concentration [pM]


Figure 3-18. Dose-response of extracellular IFNAR2 peptide
competitors, IFNAR2(34-67) and IFNAR2(186-217), on ovine 125I-IFNt
binding to IFNAR2(34-67). Ovine 125I-IFNt was used at a final
concentration of 5 nM. Radioiodine counts from bound ovine 125I-IFNT
were measured after a 2-hour incubation at room temperature. All
assays were performed using at least four replicates to increase
accuracy.













100

90

80


70

60

50

40

30

20


100
Competitor Concentration [pM]


1000


Figure 3-19. Dose-dependent binding of biotinylated ovine IFNT
peptides, IFNt(1-38), IFNT(118-138), and IFNT(153-168) to
IFNAR2(287-315) in the presence of unlabeled ovine IFNT peptides.
Biotinylated ovine IFNT was used at a final concentration of 0.5 |pM.
Receptor peptide binding was determined colormetrically by alkaline
phosphatase activity. Bars represent the standard deviations. All
assays were performed in triplicate with coefficients of variation less
than 10%.


S -~---.-






0


* IFNt(1-38)
Linear Regression
- 95% Confidence Lines


' ' ' ' '"'






84




100

90 -

80-
z
U-
"70-

O 60

_50

o 40

330
20 -

IFNt(118-138)
10 -- Linear Regression
-- 95% Confidence Lines

0-
10 100 1000
Competitor Concentration [pM]


Figure 3-19B.






85




100

90 -

80

- 70 -

O 60

- 50

0
E 40

- 30
c
20 _
IFNt(153-168)
10 -- Linear Regression
-- 95% Confidence Lines


10 100 1000
Competitor Concentration [pM]


Figure 3-19C.




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