Biosynthesis of proteodermatan sulfate in cultured human fibroblasts.
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.
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
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