Abstract
To develop a small peptide antagonist for a cytokine, an oligopeptide ligand interacting with interleukin-6 (IL-6) has been screened by affinity panning from a random heptapeptide displaying phage library. After biopanning five times against IL-6, ten phages displaying heptapeptides with consensus sequence, K(L/V)WXIPQ, were selected out of nucleotide sequencing of 21 phages. A synthetic oligopeptide, KLWTIPQ (P-1), was prepared but it did not inhibit growth of MH60, an IL-6 dependent cell line. To stabilize the structure of oligopeptide and bring stronger molecular hindrance In interaction between IL-6 and its two receptors (IL-6R and gp130), a peptide with two cysteine residues at each end of consensus sequence, GGCKLWTIPQCGG (PC), was also synthesized, which significantly inhibited cell growth of MH60 at over 100 μM and phosphorylation of Stat3, which is primary signal transducer and activator of transcription, phosphorylated by IL-6 signal. These results strongly suggest that PC is specifically bound to IL-6 and hinders formation of IL-6/IL-6R/gp130 complex.
Similar content being viewed by others
References
Akeson AL, Woods CW, Hsieh LC, Bohnke RA, Ackerman BL, Chan KY, Robinson JL, Yanofsky SD, Jacobs JW, Barrett RW, Bowlin TL (1996) AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin-1 (IL)-1 receptor and blocks in vitro response to IL-1. J. Biol. Chem. 271: 30517–30523.
Cwirla SE, Balasubramanian P, Duffin DJ, Wagstrom CR, Gates CM, Singer SC, Davis AM, Tansik RL, Mattheakis LC, Boytos CM, Schatz PJ, Baccanari DP, Wrighton NC, Barrett RW, Dower WJ (1996) Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine. Science 276: 1696–1699.
Darnell JE Jr, Kerr IM, Stark GR (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415–1421.
David M, Petricoin E 3rd, Benjamin C, Pine R, Weber MJ, Larner AC (1995) Requirement for MAP kinase (ERK2) activity in interferon alpha-and interferon beta-stimulated gene expression though STAT proteins. Science 269: 1721–1723.
Devlin JJ, Panganiban LC, Devlin PE (1990) Random peptide libraries: A source of specific protein binding molecules. Science 249: 404–406.
Hirano T, Matsuda T, Turner M, Miyasaka N, Buchan G, Tang B, Sato K, Shierfelder WE, Kreider B, Sivennoinen O (1994) Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem. Sci. 19: 222–227.
Kishimoto T, Akira S, Narazaki M, Taga T (1995) Interleukin-6 family of cytokines and gp130. Blood 86: 1243–1254.
Krebs B, Griffin H, Winter G, Rose-John S (1998) Recombinant human single chain Fv antibodies recognizing human interleukin-6. J. Biol. Chem. 273: 2858–2865.
Livnah O, Stura EA, Johnson DL, Middleton SA, Mulcahy LS, Wrighton NC, Dower WJ, Jolliffe LK, Wilson IA (1996) Functional mimicry of a protein hormone by a peptide agonist: The EPO receptor complex at 2.8 A. Science 273: 464–471.
Matsuda T, Hirano T, Kishimoto T (1988) Establishment of an interleukin-6 (IL-6)/B cell stimulation factor 2–dependent cell line and preparation of anti-IL-6 monoclonal antibodies. Eur. J. Immunol. 18: 951–956.
Murakami M, Hibi M, Nakagawa N, Nakagawa T, Yasukawa K, Yamanishi K, Taga T, Kishimoto T (1993) IL-6–induced homodimerization of gp130 and associated activation of tyrosine kinase. Science 260: 1808–1810.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Satofuka H, Amano S, Atomi H, Takagi M, Hirata K, Miyamoto K, Imanaka T (1999) Rapid method for detection and detoxification of heavy metal ions in water environments using phytochelatin. J. Biosci. Bioeng. 88: 287–292.
Wen Z, Zhong Z, Darnell JE Jr (1995) Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82: 241–250.
Yasukawa K, Saito T (1990) A new system for expression of human interleukin-6 in Escherichia coli. Biotechnol. Lett. 12: 419–424.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mizuguchi, H., Kubomi, T., Nomura, R. et al. Screening of an oligopeptide antagonist for interleukin-6 from a random phage library. Biotechnology Letters 22, 1015–1020 (2000). https://doi.org/10.1023/A:1005605808186
Issue Date:
DOI: https://doi.org/10.1023/A:1005605808186