ABSTRACT
In order to locate the receptor-binding region of human interleukin-6 (IL-6), twelve peptide fragments were prepared by digestion of IL-6 with lysylendopeptidase. A significant activity of the receptor-binding was observed only for a peptide Ile88-Lys121, although the activity was estimated at 10(4)-fold less than that of intact IL-6. Solution structure of the peptide Ile88-Lys121 was analyzed by using two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The results indicate the presence of alpha-helices in the regions Leu93-Phe106 and Glu110-Ser119. On the basis of the NMR data, we also prepared two peptides. Four-fold less binding activity than that of the peptide Ile88-Lys121 was observed for the peptide Ile88-Arg105, but no activity for the peptide Glu110-Lys121. These results suggest that the helical peptide Ile88-Arg105 composes a part of the receptor-binding region.
Subject(s)
Interleukin-6/chemistry , Interleukin-6/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Receptors, Immunologic/metabolism , Amino Acid Sequence , Binding Sites , Chromatography, High Pressure Liquid , Circular Dichroism , Humans , Kinetics , Magnetic Resonance Spectroscopy , Models, Structural , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Protein Conformation , Protein Folding , Receptors, Interleukin-6 , Serine EndopeptidasesABSTRACT
Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.
Subject(s)
Interleukin-6/genetics , Interleukin-6/pharmacology , Leucine , Mutagenesis, Site-Directed , Amino Acid Sequence , Antibody Formation/drug effects , B-Lymphocytes/drug effects , Base Sequence , Binding, Competitive , Cell Line, Transformed , Cloning, Molecular , Escherichia coli/genetics , Herpesvirus 4, Human , Humans , Interleukin-6/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides , Peptide Fragments/chemical synthesis , Receptors, Immunologic/metabolism , Receptors, Interleukin-6 , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Structure-Activity RelationshipABSTRACT
Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.
