title: Effects of site-specific mutations on biologic activities of recombinant human IL-6.
abstract: To examine structure-activity relationships of human IL-6, we have determined the effects of specific mutations on the biologic activity of a human rIL-6 expressed in bacteria. Three types of mutants were examined: 1) a variant that contains serines in place of the four naturally occurring cysteines; 2) a series of cysteine-containing deletion mutants, each having a single internal 20 amino acid deletion; and 3) a cysteine-free variant containing a single 20 amino acid deletion. The mutants of the second type constitute a set of nonoverlapping, adjacent deletions spanning amino acids 4 through 183 of the 184 amino acids in natural human IL-6. All of the mutants were expressed, along with the full length, cysteine-containing analogue, in Escherichia coli as fusion proteins, joined to beta-galactosidase through a collagen linker. This system allows microgram quantities of the rIL-6 variants to be partially purified from small bacterial cultures without chromatographic or refolding steps. Each of the rIL-6 variants was released from the beta-galactosidase fusion protein with collagenase, and the recovered rIL-6 was quantitated by laser densitometry of Coomassie-stained, SDS polyacrylamide gels. The sp. ac. of each of the rIL-6 variants was determined using four assays: induction of IgM secretion from an EBV transformed human B cell line, induction of fibrinogen secretion from a human hepatoma cell line, induction of fibrinogen secretion from a rat hepatoma cell line, and induction of proliferation of a murine hybridoma cell line. Replacement of cysteines with serines reduced activity relative to cysteine-containing rIL-6 to about 20% in the rat hepatoma assay and about 3% in the mouse hybridoma assay, whereas activity in both of the human cell lines was reduced to less than 0.1%. These data suggest that the murine and rat cell lines are less selective than the human cell lines in their requirements for recognition of biologically active IL-6. Each of the deletions, except that of amino acids 4 through 23, resulted in loss of activity in all four assays. These results suggest that the information necessary for activity is not contained within any one portion of the IL-6 molecule, but rather that multiple segments of the protein are required for each of the biologic activities that we tested.
authors.affiliation: Curriculum in Neurobiology, University of North Carolina, Chapel Hill 27599.
authors.forename: J N
authors.initials: JN
authors.lastname: Snouwaert
authors.forename: K
authors.initials: K
authors.lastname: Kariya
authors.forename: D M
authors.initials: DM
authors.lastname: Fowlkes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology; D001483:Base Sequence; D001681:Biological Assay; D002454:Cell Differentiation; D002455:Cell Division; D002872:Chromosome Deletion; D003545:Cysteine / Q000502:physiology; D006801:Humans; D006825:Hybridomas / Q000166:cytology; D015850:Interleukin-6 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008099:Liver / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D015850:Interleukin-6; D011994:Recombinant Proteins; D003545:Cysteine