JOURNAL OF INTERFERON RESEARCH 10:621-626 (1990)Mary Ann Liebert, Inc., Publishers

Identification of a Decapeptide Region of HumanInterferon-a with Antiproliferative Activity and

Homology to an Irnmunosuppressive Sequence of theRetroviral Transmembrane Protein P15E

CURTIS L. RUEGG and METTE STRAND

ABSTRACTWe have identified a 10-amino acid sequence (amino acid, 9-18) from interferon-a (IFN-a)that when presented as a synthetic peptide inhibits both the proliferation of the Daudilymphoblastoid cell line and the antigen receptor-stimulated proliferation of fresh human Tlymphocytes. This sequence, which was identified by virtue of its sequence similarity (70%identity) to the irnmunosuppressive sequence of the retroviral transmembrane proteinpl5E, represents the smallest fragment of IFN-a that has been shown to date to bebiologically active.

Efforts from several laboratories have been directed toward the derivation of structure-activityrelationships for interferon-a (IFN-a), a cytokine with antiviral, antiproliferative, and immunoregula-

tory activities/l) Several bioactive regions of the protein have been identified by use of the technique ofantibody blocking'2,3' and by the construction of chimeric IFNs, in which selected amino acids aresubstituted for their respective counterparts from a non-cross-reactive IFN species.(4,5) The complexstructure-activity relationship that has emerged from these studies has made it extremely difficult to identifysubfragments of IFN-a with biologic activity; the smallest fragment of IFN-a that has been shown to bebiologically active (i.e., to mediate antiviral and antiproliferative activity) is one containing the amino-terminal 110 amino acids of the ~ 166-amino acid protein.(6) Although shorter fragments containing residues1-60 and 113-149 have been shown to antagonize the activity of intact IFN-a, these peptides are devoid ofbiologic activity/7'

Cianciolo and his colleagues'8' previously have shown that a highly conserved sequence within theretroviral transmembrane protein pl5E mediates antiproliferative effects on lymphocytes, and we haveextended these studies by demonstrating that the minimum amino acid sequence required for inhibitoryactivity consists of 10 residues within this conserved region.'9' To identify proteins with similarantiproliferative activity, we used the 10-amino acid sequence as a probe in a computer-assisted sequencesimilarity analysis.'10' One of the highest scoring entries (70% identity) was a short and highly conservedregion of IFN-a, amino acids 9-18. Alignment of the inhibitory pl5E sequence (designated MOLV. 23) with

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore,MD 21205.

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RUEGG AND STRAND

the homologous sequence (designated IFNA.l) of the IFN-a consensus sequence'11' showed sequenceidentity in 7 of the 10 residues (Table 1).

We synthesized the IFNA.l peptide containing this conserved IFN-a sequence and tested it forantiproliferative activity. As had previously been observed for the synthetic peptides derived from retroviraltransmembrane protein sequences,'8'9,12' the IFNA. 1 peptide required conjugation to a carrier protein forbiologic activity. Thus, all of the peptides used in the present study were coupled to bovine serum albumin(BSA) using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-hydrochloride (EDC).'9' The antiprolifera-tive activity of the IFNA. 1 peptide was determined by a [3H]thymidine uptake assay'9' using the Daudi cellline.

The IFNA. 1 peptide specifically inhibited proliferation of Daudi cells with a half-maximal inhibitoryconcentration (IC50) of —10 pM (Fig. 1 A); the potency of this peptide was slightly greater than that of thehomologous pl5E peptide MOLV.23 (IC50 ~20 pM). The inhibition produced by IFNA.l was sequence-specific, since neither an irrelevant control peptide (amino acids 655-671)'12' nor BSA treated with EDC inthe absence of peptide (BSA[edc]) showed any inhibitory activity. Recombinant human IFN-a2 (kindlyprovided by Dr. Peter F. Sorter, Hoffmann-LaRoche Inc., Nutley, NJ) inhibited Daudi cell proliferationwith substantially greater potency (IC50 ~10 pM) than did the synthetic IFNA.l peptide (Fig. IB). Thisdifference is consistent with our previous results, which showed that the native retroviral transmembraneprotein pl5E was ~ 1,000-fold more potent than the pl5E synthetic peptide MOLV.23.'9' A possibleexplanation for the decreased potency of the synthetic peptide is that conjugation of the peptide to carrierprotein results in only a limited fraction of the peptide molecules adopting an active conformation.

We further characterized the biologic activity of the IFNA.l peptide by assessing its effect on theproliferation of human T lymphocytes stimulated via the CD3/T-cell antigen receptor complex."2' TheIFNA. 1 peptide inhibited T-cell proliferation (Fig. 2A) in a sequence-specific and dose-dependent mannersimilar to that seen for intact IFN-a (Fig. 2B). As was observed in the case of Daudi cells, the syntheticpeptide was less potent than intact IFN-a.

Our demonstration that a peptide containing amino acids 9-18 of IFN-a is capable of inhibitinglymphocyte proliferation is consistent with previous experiments in which other investigators used indirectmethods to map a critical determinant in the activity of IFN-a to the amino-terminal region of the protein(amino acids 5-15,'7' to 10-44,'4' to 10-35,'2' and to 1-22'5'). The results of the present study suggest thatthe amino acid 9-18 region of IFN-a is sufficient for antiproliferative activity; however, additional residuesappear to contribute to the adoption of the active conformation and thus the much higher potency of intactIFN-a.

Pitha and co-workers'13' have used a series of computer algorithms to model the three-dimensionalstructure of IFN-a and predicted that amino acids 1-13 adopt a random coil type structure followed by a

ß-turn and that amino acids 13-24 exist in the form of an a-helix. Furthermore, amino acids 9-18 representa peak of hydrophilic character. The high degree of hydrophilicity and the presence of a ß-turn are consistentwith the prediction that the amino acids 9-18 region is accessible on the surface of the IFN-a molecule.

It is interesting to note that the IFNA. 1 peptide inhibited both Daudi and T-cell proliferation with equalpotency (IC50 ~10u.M), whereas intact IFN-a was ~20-fold more potent in its inhibition of Daudi cellproliferation than of T-cell proliferation. This difference suggests that the IFNA.l peptide, in contrast tointact IFN-a, may not depend on the relative expression of high-affinity IFN-a receptors (Daudi cells have~ 10,000 high-affinity receptors per cell, whereas T lymphocytes express only —200 receptors per cell.'14'

Table 1. Sequence Similarity Between Immunosuppressive Retroviral andIFN-a Amino Acid Sequences

MOLV.23 (pl5E) LQNRRGLDLL

IFNA.l (IFN-aC011) 9LGNRRALILL18

The sequence shown for IFN-a represents the consensus sequence of 10 distinct IFN-a genes.' ' ' Sequence identityis designated by (:) and conservative substitutions are designated by (.).

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DECAPEPTIDE WITH IFN-a ACTIVITY

120

100

80

60.

40

20

0

.2 "201

- — IFNA.1- — MOLV.23-*— aa655-671-O— BSA(edc)

P^¥10 100

Peptide Cone. (p.M)1000

.0001 .001 100 1000

IFN Cone. (nM)FIG. 1. Inhibition of Daudi cell proliferation by an IFN-a-derived synthetic peptide (A) and recombinanthuman IFN-a2 (B). Data represent the mean ± SEM of quadruplicate samples. Percent inhibition wascalculated using the following formula: % Inhibition = [1 -(exp cpm)/(total cpm)] x 100, where total cpm(336,958 ± 21,814) is uptake in the absence of peptide and exp cpm is uptake in the presence of peptide inthe concentration specified.

In agreement with this hypothesis are the results we have obtained from competitive inhibition assays, inwhich the IFNA.l peptide did not antagonize the binding of 125I-IFN-a to Daudi cells (data not shown).Taken together, these findings suggest that the peptide does not include the regions of IFN-a that are requiredfor receptor binding. We have demonstrated previously'15' that the inhibitory retroviral peptides requireendocytosis for activity; it is possible that the IFNA.l peptide may enter the cell by a similar route, thusbypassing the IFN-a high-affinity receptor.

Pfeffer and colleagues have reported that the antiproliferative activity of intact IFN-a is not blocked byincubation with cells at 15°C, suggesting that endocytosis of IFN-a is not required for activity.'16' However,they noted that maximum antiproliferative activity is observed at low receptor occupancy, thus allowing forthe possibility that either (i) endocytosis is not completely blocked at 15°C and some IFN-a enters the cell toelicit its biologic effect or (ii) that our competitive inhibition assays were not sufficiently sensitive to detecta low degree of competitive binding of the amino acids 9-18 peptide to the IFN-a receptor that may be

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RUEGG AND STRAND

sufficient to elicit the antiproliferative signal. Alternatively, it the amino acids 9-18 peptide requiresintemalization similar to the retroviral peptides then the antiproliferative activity observed for amino acids9-18 may involve a mechanism that is distinct from that utilized by intact IFN-a.

Several groups have reported that the biologic activities of IFN-a can be assigned to distinct domains ofthe protein,'17-19' thus it would not be surprising if the amino acids 9-18 sequence is subsequently found notto mediate all of the biologic activities of intact IFN-a. Ortaldo et a/.'17' have found that IFN-a does notboost natural killer (NK) cell activity but does mediate antiviral and antiproliferative activities. Greiner andcolleagues have described differential effects of some IFN-a species on induction ofHLA Class I expressionand antiproliferative activity.'18' Cebrian and co-workers have defined distinct epitopes that contribute toantiviral and NK cell boosting activities.'19' Pestka has reviewed'" the differential activity of various IFN-asubspecies with respect to their antiviral and antiproliferative activities as well as their ability to boostcytotoxic T lymphocyte and NK cell activities. These differences in the biologic activities of IFN-a subtypes

co

100

1 10 100

Peptide Cone. (|j.M)1000

1 10

IFN Cone. (nM)100

FIG. 2. Inhibition of proliferation of OKT3-stimulated human PBMC by an IFN-a-derived syntheticpeptide (A) and recombinant human IFN-a2 (B). Data represent the mean ± SEM of quadruplicate samples.Percent inhibition was calculated using the following formula: % Inhibition = [l-(exp cpm - bkgdcpm)/(total cpm

-

bkgd cpm)] x 100, where bkgd cpm (2,536 ± 2,009) is [3H]TdR uptake in the absenceof OKT3 and synthetic peptide, total cpm (98,399 ± 15,850) is uptake in the presence of OKT3 withoutpeptide, and exp cpm is the uptake in the presence of OKT3 and peptide in the concentration specified.

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DECAPEPTIDE WITH IFN-a ACTIVITY

are almost certainly represented by correlative differences in the primary structure of these proteins. We havenot determined whether the amino acids 9-18 peptide mediates any of these additional activities ascribed toIFN-a, however, during the course of this investigation, it was reported that a synthetic peptide containingthe highly conserved irnmunosuppressive sequence from the retroviral transmembrane protein pl5E(analogous to the peptide MOLV. 1 of this study) inhibited the release ofmurine leukemia virus from infectedcells similar to that observed for IFN-a.'20' This finding suggests that the amino acids 9-18 sequence fromIFN-a which is homologous to pl5E may also mediate antiviral activity, but confirmation of this hypothesiswill require further study.

The fact that the antiproliferative pl5E sequence exhibited a high degree of similarity to a conservedregion present in virtually all (> 15) IFN-a sequences of both human and animal origin' ' ' ' suggests that theactivity observed in the present study is functionally relevant. Although the origin of this sequence similarityis unknown, it is possible that the IFN-a sequence was incorporated into the retroviral genome via geneticrecombination. Further investigation should help to clarify the relationship between the homologousretroviral and IFN-a sequences identified in this study and to elucidate the role of the amino acids 9-18region in other biologic functions of IFN-a, including its antiviral activity.

ACKNOWLEDGMENTS

We wish to thank Dr. Peter F. Sorter of Hoffmann-LaRoche Inc. (Nutley, NJ) for kindly providingrecombinant human IFN-a and Dr. Deborah McClellan for editorial assistance. This work was supported byPublic Health Service grant AI-28206 from the National Institutes of Health (M.S.).

REFERENCES

1. PESTKA, S., LANGER, J.A., ZOON, K.C., and SAMUEL, CE. (1987). Interferonsand their actions. Annu. Rev.Biochem. 56, 727-777.

2. LYDON, N.B., FAVRE, C, BOVE, S., NEYRET, O., BENUREAU, S., LEVINE, A.M., SEELIG, CF.,NAGABHUSHAN, T.L., and TROTTA, P.P. (1985). Immunochemical mapping of alpha-2 interferon. Biochem-istry 24, 4131-4141.

3. TYMMS, M.J., BEILHARZ, M.W., NISBET, LT., CHAMBERS, P.J., MCINNES, B., TURTON, J.C.,HERTZOG, P.J., and LINNANE, A.W. (1987). Amino acid residues affecting antiviral and antiproliferativeactivity in interferon-alpha, in: Handbook ofExperimental Pharmacology, Vol. 71: The Biology of the InterferonSystem. K. Cantell and H. Schellekens (eds.). Dordrecht: Martinus Nijhoff. pp. 3-9.

4. FISH, E.N., BANERJEE, K., and STEBBING, N. (1989). The role of three domains in the biological activity ofhuman interferon-alpha. J. Interferon Res. 9, 97-114.

5. SHAFFERMAN, A., VELAN, B., COHEN, S., LEITNER, M., and GROSFELD, H. (1987). Specific residueswithin an amino-terminal domain of 35 residues of interferon-a are responsible for recognition of the humaninterferon-a cell receptor and for triggering biological effects. J. Biol. Chem. 262, 6227-6237.

6. ACKERMAN, S.K., ZUR NEDDEN, D., HEINTZELMAN, M., HUNKAPILLER, M., and ZOON, K. (1984).Biologic activity in a fragment of recombinant human interferon alpha. Proc. Nati. Acad. Sei. USA 81, 1045-1047.

7. HOROVITZ, 0., RUBINSTEIN, M., and REVEL, M. (1985). Two regions of the human IFN-alphaC moleculeinvolved in binding to human cell receptor, in: The Biology of the Interferon System, W.E. Stewart, II and H.Schellekens (eds.). Netherlands: Elsevier. pp. 157-162.

8. CIANCIOLO, G.J., COPELAND, T.D., OROSZLAN, S., and SNYDERMAN, R. (1985). Inhibition oflymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins. Science 230, 453-455.

9. RUEGG, C.L., MONELL, C.R., and STRAND, M. (1989). Identification, using synthetic peptides, of theminimum amino acid sequence from the retroviral transmembrane protein pl5E required for inhibition oflymphoproliferation and its similarity togp21 of human T-lymphotropic virus types I and II. J. Virol. 63,3250-3256.

10. DEVEREUX, J., HAEBERLI, P., and SMITHIES, O. (1984). A comprehensive set of sequence analysis programsfor the VAX. Nucleic Acids Res. 12, 387-395.

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11. HENCO, K., BROSIUS, J, FUJISAWA, A., FUJISAWA, J.-L, HAYNES, JR., HOCHSTADT, J., KOVACIC,T., PASEK, M., SCHAMBOCK, A., SCHMID, J., TODOKORO, K., WALCHLI, M., NAGATA, S., andWEISSMANN, C. (1985). Structural relationship of human interferon alpha genes and pseudogenes. J. Mol. Biol.185, 227-260.

12. RUEGG, C.L.,MONELL,C.R., and STRAND, M. (1989). Inhibition of lymphoproliferation by a synthetic peptidewith sequence identity to gp41 of human immunodeficiency virus type 1. J. Virol. 63, 3257-3260.

13. RAJ, N.B.K., ISRAELI, R., KELLEY, K.A., LEACH, S.J., MINASIAN, E., SIKARIS, K., PARRY, D.A.D.,and PITHA, P.M. (1988). Synthesis, antiviral activity, and conformational characterization of mouse-humana-interferon hybrids. J. Biol. Chem. 263, 8943-8952.

14. LANGER, J.A., and PESTKA, S. (1988). Interferon receptors. Immunol. Today 9, 393-400.15. RUEGG, C. L., and STRAND, M. ( 1990). Inhibition of protein kinase C and anti-CD3-induced Ca2+ influx in Jurkat

T cells by a synthetic peptide with sequence identity to HIV-1 gp41. J. Immunol .14, 3928-3935.16. PFEFFER, L.M., STEBBING, N., and DONNER, D.B. (1987). Cytoskeletal association of human a-interferon-

receptor complexes in interferon-sensitive and -resistant lymphoblastoid cells. Proc. Nati. Acad. Sei., USA 84,3249-3253.

17. ORTALDO, JR., HERBERMAN, R.B., HARVEY, C, OSHEROFF, P., PAN, Y-C.E., KELDER, B., andPESTKA, S. (1984). A species of human a interferon that lacks the ability to boost human natural killer activity.Proc. Nati. Acad. Sei. USA 81, 4926-4929.

18. GREINER, J.W., FISHER, P.B., PESTKA, S., and SCHLOM, J. (1986). Differential effects of recombinanthuman leukocyte interferons on cell surface antigen expression. Cancer Res. 46, 4984-4990.

19. CEBRIAN, M., YAGUE, E., DE LANDAZURI, M.O., RODIGUEZ-MOYA, M., FRESNO, M., PEZZI, N.,LLAMAZARES, S., and SANCHEZ-MADRID, F. (1987). Different functional sites on rIFN-a2 and their relationto the cellular receptor binding site. J. Immunol. 138, 484-490.

20. WEGEMER, D.E., KABAT, K.G., and KLOETZER, W.S. (1990). Biological activities of a synthetic peptidecomposed of two unlinked domains from a retroviral transmembrane protein sequence. J. Virol. 64, 1429-1436.

Address reprint requests to:Dr. Mette Strand

Department of Pharmacology and Molecular SciencesThe Johns Hopkins University School of Medicine

725 North Wolfe StreetBaltimore, MD 21205

Received 19 March 1990/Accepted 13 July 1990

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