title: Biosynthesis of proteodermatan sulfate in cultured human fibroblasts.
abstract: Biosynthesis and secretion of proteodermatan sulfate produced by cultured human skin fibroblasts were investigated employing immunological procedures. During an incubation period of 10 min in the presence of [3H]leucine, two core protein forms of Mr = 46,000 and 44,000, respectively, were synthesized. They were converted to mature proteodermatan sulfate with a half-time of approximately 12 min. Fifty per cent of total mature proteodermatan sulfate were found in the culture medium after a 35-min chase. Six to eight per cent remained associated with the cell layer after a chase of 6 h. In the presence of tunicamycin, fibroblasts synthesized a single core protein of Mr = 38,000 that was converted to mature proteodermatan sulfate and secreted with similar kinetics as the N-glycosylated species. Subtle differences in the molecular size of core proteins were noted when cell-associated and secreted proteodermatan sulfate were degraded with chondroitin ABC lyase, but core proteins free of N-linked oligosaccharides were identical. Labeling with [3H]mannose revealed that secreted proteodermatan sulfate contains two or three complex-type or two complex-type and one high-mannose-type N-linked oligosaccharide chains. The N-glycans are bound to a 21-kDa fragment of the core protein. After incubation in the presence of [3H]glucosamine, the [3H]galactosamine/[3H]glucosamine ratio was 3.76 and 3.30 for secreted and cell-associated proteodermatan sulfate, respectively. Evidence for the presence of O-linked oligosaccharides could not be obtained. Small amounts of core protein free of dermatan sulfate chains were secreted when the cultures were treated with p-nitrophenyl-beta-D-xyloside.
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D002478:Cells, Cultured; D002807:Chondroitin / Q000031:analogs & derivatives*; D003871:Dermatan Sulfate / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011509:Proteoglycans / Q000096:biosynthesis* / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D011509:Proteoglycans; D013431:Sulfates; C028774:proteodermatan sulfate; D014415:Tunicamycin; D003871:Dermatan Sulfate; D002807:Chondroitin; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D007930:Leucine; D005944:Glucosamine