Structural properties of a soluble bioactive precursor for transforming growth factor-alpha.
The precursor for transforming growth factor-alpha, proTGF-alpha, is synthesized as an integral membrane glycoprotein with the mature TGF-alpha sequence located in the extracellular domain. Retrovirally transformed rat embryo fibroblasts (FeSV-Fre cells) expressing the endogenous proTGF-alpha gene release and accumulate in the medium mature TGF-alpha as well as a heterogeneous (17-19 kDa) group of soluble, bioactive TGF-alpha precursor forms. These precursors correspond to the heterogeneously glycosylated extracellular domain of proTGF-alpha which is released from the membrane by proteolytic cleavage. They are designated mesoTGF-alpha to denote their intermediate position in the proTGF-alpha processing pathway. The nature of the carbohydrate linked to mesoTGF-alpha has been examined by treatment with glycosidases and the use of metabolic inhibitors of glycosylation. The results indicate that the TGF-alpha precursors from FeSV-Fre cells contain O-linked carbohydrate as well as sialylated N-linked carbohydrate. Heterogeneous N-linked glycosylation of an 11-kDa core polypeptide accounts for the heterogeneous nature of mesoTGF-alpha. MesoTGF-alpha released by cells treated with inhibitors of N-linked carbohydrate processing appears as a 17-kDa species. Treatment with these inhibitors does not alter significantly the production of mesoTGF-alpha or mature TGF-alpha by the cells. However, treatment of cells with an inhibitor of co-translational N-linked glycosylation, tunicamycin, reduces the accumulation of mesoTGF-alpha in the medium and blocks the production of mature TGF-alpha under conditions in which overall protein synthesis is only minimally affected. These findings suggest that the proTGF-alpha processing activity is limiting in FeSV-Fre cells and other transformed cells that accumulate mesoTGF-alpha in the medium and that proTGF-alpha processing depends on a component whose function may require N-linked glycosylation.
Teixidó
J
J
Department of Biochemistry, University of Massachusetts Medical School, Worcester 01605.
Massagué
J
J
D017485:1-Deoxynojirimycin; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006133:Growth Substances / Q000235:genetics*; D007212:Indolizines; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D017026:Swainsonine; D016211:Transforming Growth Factor alpha; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D000470:Alkaloids; D000900:Anti-Bacterial Agents; D002241:Carbohydrates; D005944:Glucosamine; D006133:Growth Substances; D007212:Indolizines; D008562:Membrane Glycoproteins; D011498:Protein Precursors; D017026:Swainsonine; D016211:Transforming Growth Factor alpha; D014415:Tunicamycin; C052268:protransforming growth factor alpha; C037806:castanospermine; D054836:Indolizidines