Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein.
In order to study the temporal and topological events involved in the processing and assembly of chondroitin sulfate proteoglycan, a fractionation scheme involving differential centrifugation and discontinuous sucrose density gradient centrifugation was developed for homogenates of chick embryo sternal chondrocytes. The precursors in these subcellular fractions were examined by a series of pulse, pulse-chase, and continuous labeling experiments. When chondrocytes were pulsed for 20 min with [35S]methionine, an immunoprecipitable core protein precursor with an approximate molecular size of 376,000 Da was localized to the rough endoplasmic reticulum fractions. Further incubation under chase conditions showed the presence of the 376,000-Da species as well as two additional polypeptides of higher molecular masses in the smooth membrane-enriched fractions within the next 2 h. This translocation did not occur in the presence of the energy transfer inhibitor carbonyl cyanide-m-chlorophenylhydrazone. The labeling pattern of the newly synthesized core protein precursor with either [3H] mannose or [3H]glucosamine showed that N-linked oligosaccharide addition was found on the earliest synthesized product in the rough endoplasmic reticulum, and the addition of this oligosaccharide was inhibited by co-incubation with tunicamycin. Furthermore, the high mannose oligosaccharide was susceptible to cleavage by endo-beta-N-acetylglucosaminidase H, while upon chase approximately 56 and 31% of the glucosamine- and mannose-labeled oligosaccharides, respectively, were processed to resistant forms, presumably in the Golgi complex. Both direct assay of glycosyl- and sulfotransferases requisite for addition of chondroitin sulfate chains and sensitivity of intracellular precursors to chondroitinase, keratanase , and endoglycosidase H suggest that only the N-linked oligosaccharides are added in the rough endoplasmic reticulum and glycosaminoglycan chain addition occurs predominantly in smooth membranes.
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002356:Cartilage / Q000648:ultrastructure*; D002458:Cell Fractionation; D002642:Chick Embryo; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008970:Molecular Weight; D011509:Proteoglycans / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D002241:Carbohydrates; D011508:Chondroitin Sulfate Proteoglycans; D011509:Proteoglycans; D014415:Tunicamycin