Characterization of high molecular weight glycosylated forms of murine tumor necrosis factor.
Tumor necrosis factor (TNF) is synthesized as a prohormone with an unusually long and atypical signal sequence which is absent from the mature secreted cytokine. In addition to mature 17 kDa TNF, LPS-stimulated murine macrophages secrete at least seven TNF-like proteins (isoforms) of differing electrophoretic mobility which appear as a "ladder" on SDS-PAGE. We here present data indicating that these isoforms derive not from sequential clipping of propiece fragments, but rather from differential glycosylation at sites on the mature hormone. Selected isoforms have been isolated and purified by sequential chromatographic and electrophoretic steps. NH2-terminal sequence analysis of two of these isoforms reveal sequences identical to that of mature 17 kDa murine TNF. Characterization of the secretory products of tunicamycin-treated. LPS-stimulated murine macrophages indicate that the "ladder" complex reflects differential glycosylation of mature 17 kDa TNF. Digestion of purified isoforms with a battery of glycosidic enzymes indicate that secreted forms of murine TNF contain both sialic acid and asparagine(N)-linked chains. The biological significance of this heterogeneity is not known.
Sherry
B
B
Laboratory of Medical Biochemistry, Rockefeller University, New York, NY 10021.
Jue
D M
DM
Zentella
A
A
Cerami
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001216:Asparagine / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007536:Isomerism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D008070:Lipopolysaccharides; D012794:Sialic Acids; D014409:Tumor Necrosis Factor-alpha; D001216:Asparagine; D019158:N-Acetylneuraminic Acid