|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Site-directed mutagenesis of three leucine residues in the C-terminal region of human interleukin-6(IL-6) was performed. Both receptor-binding and immunoglobulin-induction activities of a triple mutant Leu168, 175, 182->Val were only 1 % compared with those of wild-type IL-6. We also prepared the peptide fragments. A significant binding activity was observed for the peptides Leu168-Met185 and Ile88-Lys121, althouth the activity was estimated at 10^4-fold less than that of intact IL-6. These results suggest that these peptides compose a part of the receptor-binding region and that Leu168, Leu175, and Leu182 existing in the region of Leu168-Met185 play an important role in the receptor-binding. Solution structure of the peptide Ile88-Lys121 was analyzed by using two-dimensional nuclear magnetic resonance spectroscopy. The results indicate that the peptide Ile88-Lys121 forms two alpha-helical rods (Leu93-Phe106 and Glu110-Ser119) with a kink in the region of Glu107-Ser109. Proton nuclear magnetic resonance studies of wild-type and mutant IL-6 were also performed. Using a deuterium-labeling method in combination with the site-directed mutagenesis, the methyl proton resonances of Leu152, 168, and 175 in wild-type IL-6 and Val152, 159, 166, 168, and 175 in mutated IL-6 were assigned. On the basis of NOE data and predicted secondary structure of IL-6, the proton resonances of Phe95, Val97, Ile167, Phe171, and Phe174 were assigned. On the basis of these signal assignments, two long-range NOE cross-peaks were observed between Phe95 in the helix B and Phe171 in the helix D, and between Tyr98 and 101 in the helix B and Leu152 in the helix C-D loop.