JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 16:813-817 (1996)Mary Ann Liebert, Inc.

Structural Requirements for Agonist Activity of a MurineInterferon- Peptide

B.E. SZENTE,1·* I.J. WEINER,1 M.J. JABLONSKY,2 N.R. KRISHNA,2 B.A. TORRES,1 and H.M. JOHNSON1

ABSTRACT

We have demonstrated previously that murine interferon- (MuIFN- ) binds to the extracellular domain ofthe receptor a chain through its N-terminus and subsequently to the cytoplasmic domain of the receptor viaits C-terminus. Binding of the C-terminus to the cytoplasmic domain of the receptor is thought to occur fol¬lowing endocytosis of the IFN-y-receptor complex. In fact, the MuIFN- C-terminus peptide, MuIFN-y(95-133), has full agonist activity on macrophages where it is internalized through pinocytosis. Here we examinethe structural elements required for the agonist activity of MuIFN-y(95-133). Disruption of the a helical struc¬ture of the peptide by proline substitutions or truncation of the helix resulted in significant loss of binding or

loss of antiviral activity or both and induction of MHC class II molecules. Further, removal of the polycationicsequence RKRKR in the tail beyond the helical structure also resulted in loss of agonist activity. Thus, we

have isolated the functional site on MuIFN-y to the C-terminus and have shown that its helical structure andpolycationic tail are required for binding to the cytoplasmic domain of the receptor and induction of biologicactivity.

INTRODUCTION

Interferon- (IFN- ) is a pleiotropic cytokine product of ac¬tivated cells and natural killer (NK) cells. Its many bio¬

logic effects include, but are not limited to, the induction of a

number of antiviral proteins, upregulation of major histocom-patibility complex (MHC) antigen expression, and roles in cell maturation and immunoglobulin isotype switching/" Theseactivities are induced as the IFN- molecule interacts with a

single class of cell surface receptor consisting of a ligand-bind-ing subunit a chain and an associated cofactor protein neces¬

sary for efficient signal transduction/2;3'The N-terminal and C-terminal domains of - have been

identified as being important for receptor binding and biologicactivity of the molecule, using synthetic peptide competition andspecific antibody neutralization of - activity/4^6' We havedemonstrated previously that murine - (MufFN- ) binds toa recombinant soluble form of its receptor's ligand binding sub-unit via both the N-terminal and C-terminal IFN- domains/7'Whereas the N-terminus has been shown to bind to the extra¬cellular domain of the receptor (amino acids 95-120)/8' the C-terminus binds to the membrane proximal region of the cyto¬

plasmic domain/9' Further, a C-terminal peptide of MufFN- , - (95-133), possessed - agonist activity on

macrophages, where it is internalized/10' These findings have al¬lowed us to focus on the C-terminus of - in order to deter¬mine the structural basis for its biologic activity. We show herethat the a helical structure of - (95-133) is necessary forreceptor binding and biologic- activity. The polycationic tailRKRKR beyond the a helix is also necessary for function/10'Thus, we have identified critical structures in - that are re¬

quired for its antiviral and immunomodulatory activity.

MATERIALS AND METHODS

Synthetic peptidesMufFN-7 peptides were synthesized on a PerSeptive Biosystems9050 automated peptide synthesizer using fluorenylmethyloxy-carbonyl (Fmoc) chemistry/11' Peptides were cleaved from theresin using trifluoroacetic acid/phènol/water/triisopropylsilane ata ratio of 88:5:5:2. The cleaved peptides were then extracted inether and ethyl acetate and subsequently dissolved in water and

'Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611.Comprehensive Cancer Center and Department of Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294.*Present address: Vascular Research Division, Brigham and Women's Hospital, Boston, MA 02115.

813

814 SZENTE ET AL.

lyophilized. Reverse-phase HPLC analysis of crude peptides in¬dicated one major peak in each profile. Amino acid analysis ofthese peptides showed that the amino acid composition corre¬

sponded closely to theoretical values. The sequences of the pep¬tides synthesized and their relationship to the C-terminal peptideMurFN--y(95-133) and -· are shown in Table 1.

Circular dichroismCircular dichroism measurements of peptides were deter¬

mined at 25°C using a temperature-controlled AVIV 62 DSspectropolarimeter. Scans were conducted with a 0.01 cm path-length cell with a 1 sec averaging time. The wavelength rangemeasured was from 260 to 180 nm at a scan step rate of 0.5nm/step. Scans were conducted on peptides at neutral pH in wa¬

ter with or without the helix-stabilizing agent trifluoroethanol(TFE) (final TFE concentration 30%). The mean values of ninerepetitive scans were expressed in terms of related to themean residue ellipticity at a given wavelength ([ ]\) for eachpeptide. The following formula was used to generate /12'

Mean residue ellipticity ([<?]) 1OO[0] observed

cl

where [0]observed is expressed in degrees, c is equal to the mean

residue concentration in moles/liter, and / is the path length ofthe cell in centimeters.

Biotinylation of MuIFN-y(95-133)MuIFN^95-133) was labeled with an excess of NHS-LC-

biotin (Pierce) in 50 mM bicarbonate buffer (pH 9.6) for 2 h at4°C. Unreacted biotin was removed by dialysis.

Solid-phase binding assaysThe receptor peptide MrR(253-287) was plated in carbonate

buffer at a final concentration of 1.5 ¿ag/well in a 96-well mi¬crotiter plate. The peptide was allowed to incubate overnightand was subsequently dried to the wells of the plate. Plates were

incubated for 2 h at room temperature with phosphate-bufferedsaline (PBS) containing 1% BSA in order to block nonspecificbinding. Competitor peptides were added to the wells for a 2-h incubation, after which biotinylated - (95-133) was

added to a final concentration of 10 mM for a further 2-h in¬cubation. Plates were then rinsed three times with PBS/1%BSA. Next, a 1:1000 dilution of streptavidin-alkaline phos-

phatase conjugate (Sigma) was added for a 90-min incubation.Plates were rinsed and blotted to dryness. Detection of boundligand was accomplished by adding 50 µ /well of a 1 mg/mlsolution ofp-nitrophenyl phosphate for 1 h. Color developmentwas halted by the addition of 50 µ, /well of 3 M NaOH.Absorbance was measured at 405 nm in a model 450 MicroplateReader (Bio-Rad).

Virus yield reduction assayWEHI-3 cells (2 X 106) were preincubated with either pep¬

tide at a concentration of 100 µ or medium alone for a pe¬riod of 18-24 h. Medium was removed from the cells, and theywere subsequently washed three times before infection withvesicular stomatitis virus (VSV) on an moi of 0.05. The cellswere incubated with the virus for an initial period of 45 min,after which the virus was removed, and the cells were rinsedthree times with medium. An additional 2 ml of medium was

added to the cells, and they were incubated for 24 h (37°C, 5%CO2). Supernatants were harvested, and the VSV produced was

titered in a standard viral plaque assay on murine L929 cells/13'

Induction of la expressionCells (1 X 105) were incubated for 24 h with MuIFN- pep¬

tides at a final concentration of 100 µ , after which the cellswere physically dislodged from culture plates, washed withFACS buffer (PBS containing 0.5% BSA and 10 mM NaN3),and incubated with a biotinylated anti-la monoclonal antibody,MKD6, for 1 h at 37°C/10' Cells were then washed and incu¬bated with streptavidin-phycoerythrin (TAGO Inc., Burlingame,CA) for 15 min at room temperature. Analysis was performedon a FACScan (Becton-Dickinson, Mountain View, CA) as

10,000 events per sample.

RESULTS AND DISCUSSION

To determine the role of a helical structure in - (95-133) agonist activity, we synthesized the - (95-133) with praline substituted at residues 108 and 113 (Table 1) anddetermined the effect of the proline substitution on helical struc¬ture. CD analysis in both water and 30% TFE in water showedrelatively reduced helical structure in the praline-substituted ana¬

log (Fig. 1). As expected, greater helical structure was observedin TFE, as it is a helix stabilizer/14' However, the reduced heli¬cal structure of the proline analog was clearly evident.

Table 1. Sequences of Murine - and - Receptor Peptides

Peptide3 Sequence Comment

MuIFN^95-133)MuIFN-r(95-133)pj» - (108-133)MuIFN^l-39)MfR(253-287)

AKraVNNPQVQROAFNELIRVVHQLLPESSLRKRKRSRCAKFEVNNPQVQRQPFNELPRVVHQLLPESSLRKRKRSRC

AFNELIRVVHQLLPESSLRKRKRSRCHGTVffiSLESLNNYFNSSGrDVEEKSLFLDrWRNWQKDGTKKNSFKRKSrMLPKSLLSVVKSATLETKPESKYS

C-terminal peptideC-terminal peptide with proline

substitution at residues 108 and 113Truncated C-terminal peptideN-terminal peptideRegion on the cytoplasmic domain

of receptor that binds IFN-

aPeptides were synthesized as described in Materials and Methods.

AGONIST ACTIVITY OF A MuIFN-y PEPTIDE 815

0) m.

O)

> m.

-10

-20

MuIFNy (95-133)-·— MuIFNy (95-1 33)

200 220 240

Competitor concentration (µ )260

-O— MuIFNy (95-133)-·— MuIFNy (95-133)

180 200 220 240

Competitor concentration (µ )260

FIG. 1. Circular dichroism spectra of MuIFN-t(95-133) andMuIFN-7(95-133)p?p peptides. Peptides were analyzed in (A)water or (B) a 30% aqueous solution of TFE.

The two peptides, MurFN-7(95-133) and MurFN-7(95-133)p,p, were then compared for their ability to bind to the re¬

ceptor peptide MfR(253-287) in a competition assay againstbiotinylated - (95-133) (Fig. 2). MfR(253-287) corre¬

sponds to the membrane proximal segment of the cytoplasmicdomain of the MufFN- receptor's ligand binding subunit andhas been shown to contain the binding site for the C-terminal re¬

gion of - /9·10' The praline-substituted analog demonstrateda decrease in its ability to bind to receptor peptide MfR(253-287)

0.3

0.25

cCD0)S

0.15

0.05

MuIFNy peptides:-O— (95-133)-O— (95-133)piP-·— (1-39)-m— (108-133)

10 15 20 25 30

Competitor concentration (µ )

FIG. 2. Dose-response competition of biotinylated MuIFN-7(95-133) binding to MIR(253-287) vs. MurFN- peptides.Biotinylated MuIFN-t(95-133) was used at a final concentra¬tion of 10 nM.

relative to peptide MuIFN-7(95-133). Peptide MuIFN-7(108-133), which is truncated at the N-terminus where the a helix be¬gins, had slightly decreased helical structure by CD (data not

shown) but competed as well as intact MuIFN-7(95-133) for re¬

ceptor. The N-terminal MufFN-7 peptide, MurFN-7(l-39),which binds to the extracellular domain of the receptor, did not

compete for binding to peptide MIR(253-287). The data pre¬sented here show that the a helix of MurEN-7(95-133) is im¬portant for binding to the cytoplasmic domain of the receptor.However, the N-terminal end of the helix as per known struc¬tural data(15·16' does not play as important a role as other partsof the helix when one observes the relatively poor competitionof the MurFN-7(95-133)p?p analog with helix-breaking pralinesat positions 108 and 113. ^

To assess the effect of altered secondary structure of MuIFN- (95-133) on the biologic activity of the MuIFN- peptides, we

performed a standard virus yield reduction assay using VSV/13'MuIFN-7<95-133) at a concentration of 100 µ reduced virusyield by a factor of greater than 6.5 X 10' ' (Table 2). The dou-

Table 2. IFN- Peptide Reduction of VSV Yield

Peptide and concentration Virus yield (PFUImlf Fold reduction

MuIFN-7(95-133) 100 µ MuIFN- (95-133) , 100 µ MuIFN-7(108-133)' 100 µ MuIFN-7(1-39) 100 µ MuIFN-7 200 U/ml (1.3 nM)Virus controlCell control

<102.1 X2.0 X6.5 X5.0 X6.5 X0.0

101010710121051012

6.5 X 10"3.0 X 1023.3 X 1050.01.3 X 107

aWEHI-3 cells were infected with VSV (moi = 0.5), and the virus produced was

harvested and assayed on L929 cells in a standard viral assay.

816 SZENTE ET AL.

ble-proline-substituted analog, MuIFN^95-133)pp, reducedvirus yield by 3.0 X 102-fold, which was greater than 109-foldless than MuIFN- (95-133). MuIFN- (108-133) reduced virusyield by a factor of approximately 3.3 105. The N-terminalMuIFN-7 peptide, MuIFN-t(1-39), had no effect on virus yield.Therefore, truncations or substitutions in the a helical regionof MurFN-7(95-133) have a profoundly negative effect on itsagonist properties. the case of MufFN-7(95-133)^, this isreflected by significantly reduced competition for receptor,whereas for MuIFN-t(108-133), competition for receptor was

not reduced, but this peptide was greater than 106-fold less ef¬fective than MuIFN- (95-133) in inhibiting virus replication.

As a second measure of the function of the MuIFN- C-ter¬minal peptide and its analogs, we determined their ability to in¬crease the level of surface expression of MHC class II (la) mol¬ecules on the murine macrophage cell line WEHI-3. Treatmentof WEHI-3 cells with MuIFN-7(95-133) resulted in an increasein Ta expression of greater than 5-fold above that of untreatedcells (Fig. 3). MurFN-7(95-133)p>p exhibited a small increasein fa expression, less than 25%. The truncated peptide, MufFN-7(108-133), was somewhat more effective and caused roughlya 2.5-fold increase in WEHI-3 surface la expression. The neg¬ative control, N-terminal rFN-7 peptide MurFN-7(l-39),showed no capacity to upregulate la expression. These data con¬

firm the observations made using virus yield reduction assay.Thus, gross or subtle alterations of the a helical secondary struc¬ture of the MuIFN-7 agonist peptide, MufFN-7(95-133), haveprofound effects on its biologic activity.

We think that the praline substitutions at positions 108 and113, replacing alanine and isoleucine, altered the peptide func¬tion primarily by disrupting the helical structure. The truncatedanalog, MurFN-7(108-133), had reduced function similar to

MurFN-7(95-133)p>p, but the alanine and isoleucine residueswere not altered. Thus, helical disruption by two approaches re¬

sulted in significant reduction in MuIFN-t(95-133) function.We previously showed that truncation of MufFN-7(95-133)

at the C-terminus with removal of the polycationic residuesRKRKR outside of the helix also had profound inhibitory ef¬fects on the C-terminal peptide function/10' We have confirmed

Untreated mu(95-133) mu(1-39) mu(l08-133)MuIFNy peptides

mu(95-133)p

FIG. 3. la induction of WEHI-3 by MuTFN-7 peptides. Allpeptides were used at a final concentration of 100 µ . Resultswere assayed by flow cytometry and are expressed as mean flu¬orescence values.

this observation (data not shown). Thus, an intact C-terminalhelical structure as well as the polycationic sequence RKRKRbeyond the helix are required for binding of rFN-7 t0 me m_

tracellular domain of the a chain of its receptor with inductionof IFN-7 activity. These findings are important for structurallybased approaches to the development of small molecule non-

protein agonists and antagonists of rFN-7 activity.

ACKNOWLEDGMENTS

This research was supported by grant CA 38779 (to HMJ)and grants CA 13148 and MCB 9118503 (to NRK) from theNational Tnstitutes of Health. The manuscript is FloridaAgriculture Experiment Station Tournai Series No. R-05343.

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AGONIST ACTIVITY OF A MuIFN-y PEPTIDE 817

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Address reprint requests:Dr. Howard M. Johnson

Department of Microbiology and Cell ScienceBox 110700

University of FloridaGainesville, FL 32611

Received 3 April 1996/Accepted 30 May 1996