STRUCTURE-FUNCTION ANALYSIS OF HUMAN IL-6 - IDENTIFICATION OF 2 DISTINCT REGIONS THAT ARE IMPORTANT FOR RECEPTOR-BINDING

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an impor tant role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despi te a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although h uman IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrate d that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472- 1481). To further probe the structure-function relationship of this cy tokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys- 65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructe d, expressed, and purified to homogeneity. The conformational integrit y of the IL-6 hybrids was assessed by far-UV CD. Analysis of their bio logical activity in a human bioassay (using the HepG2 cell line), a mo use bioassay (using the 7TD1 cell line), and receptor binding properti es indicates that at least 2 regions of hIL-6, residues 178-184 in hel ix D and residues 63-113 in the region incorporating part of the putat ive connecting loop AB through to the beginning of helix C, are critic al for efficient binding to the human IL-6 receptor. For human IL-6, i t would appear that interactions between residues Ala-180, Leu-181, an d Met-184 and residues in the N-terminal region may be critical for ma intaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a sig nificant loss of alpha-helical content and a 200-fold reduction in act ivity in the mouse bioassay. A homology model of mIL-6 based on the X- ray structure of human granulocyte colony-stimulating factor is presen ted.