Disulfide bond assignment and identification of regions required for functional activity of oncostatin M.
Oncostatin M is a polypeptide cytokine having unique structure and diverse biological activities, including the ability to inhibit growth of certain cultured tumor cells. Here we have determined the disulfide bonding pattern of recombinant oncostatin M and have used site-directed mutagenesis to identify regions of this molecule necessary for receptor binding and growth inhibitory activities. Two intramolecular disulfide bonds, C6-C127 and C49-C167, were identified in recombinant oncostatin M. Analysis of mutations at each of the five cysteines in oncostatin M indicated that mutants C49S and C167S were inactive (less than 1/10 wild type activity) in growth inhibitory assays and radioreceptor assays. Carboxyl-terminal deletion mutations terminating at S185 and beyond were active, but further shortening abolished activity in both assays. Two deletion mutants proximal to C49 (delta 22-36 and delta 44-47) and insertion mutant GAG77 also were inactive. One deletion mutant, delta 87-90, had significantly (approximately 3-fold) increased activities in both growth inhibitory assays and radioreceptor assays. A potential amphiphilic domain was identified beginning at C167 and extending toward the carboxyl terminus. Two mutants having altered hydrophobic residues within this domain (F176G and F184G) were inactive, suggesting that these residues are required for proper conformation of the receptor binding site. Taken together, these results indicate that biological activity of oncostatin M requires discontinuous regions of the molecule, including residues near the essential disulfide bond, C49-C167, and within a putative amphiphilic helix at the carboxyl terminus. Oncostatin M thus belongs to a growing family of cytokines whose interactions with their respective receptors are mediated in part by known or predicted carboxyl-terminal amphiphilic helices.
Kallestad
J C
JC
Oncogen Division, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Shoyab
M
M
Linsley
P S
PS
D000818:Animals; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004220:Disulfides; D066298:In Vitro Techniques; D053683:Oncostatin M; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; D053683:Oncostatin M