Biosynthetic mechanisms for the addition of polylactosamine to chondrocyte fibromodulin.
The cartilage matrix glycoprotein fibromodulin contains four N-linked glycosylation sites which act as acceptors for the addition of sulfated polylactosamine (keratan sulfate). In the present study we examined the biosynthetic processing of these N-linked oligosaccharides for subsequent addition of polylactosamine. Chondrocytes were treated with castanospermine, 1-(+)deoxymannojirimycin, and swainsonine, radiolabeled with [3,4,5-3H]leucine, [2-3H]mannose, or [6-3H]glucosamine, and newly synthesized fibromodulin was immunoprecipitated for analysis. Castanospermine and 1-(+)deoxymannojirimycin inhibited polylactosamine addition, whereas swainsonine was not effective. This indicated that the linkage regions must be processed to GlcNAc(Man)5(GlcNAc)2Asn but do not require further modification to GlcNAc(Man)3(GlcNAc)2Asn. In both control and swainsonine-treated cells one or two N-linked oligosaccharides per molecule were modified with polylactosamine containing 4-6 repeating disaccharide units. Moreover, a single short chain was added either to the C-3 or the C-6 branch in control cultures, whereas only the C-3 branch was substituted in the presence of swainsonine. Analysis of endo-beta-galactosidase and keratanase II digestion products of the polylactosamine chains synthesized in both culture conditions showed that only about 25% of the hexosamine residues and less than 5% of the adjacent galactose residues were substituted with sulfate. These findings are discussed in relation to the regulation of fibromodulin glycosylation and the likely influence of polylactosamine structure on the extracellular interactions and turnover of fibromodulin.
Plaas
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AH
Shriners Hospital for Crippled Children, Tampa, Florida 33612.
Wong-Palms
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S
D000606:Amino Sugars / Q000378:metabolism*; D000818:Animals; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D016326:Extracellular Matrix Proteins; D000071235:Fibromodulin; D005643:Fucose / Q000378:metabolism; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D008361:Mannosidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011509:Proteoglycans; D043323:alpha-Mannosidase; D001616:beta-Galactosidase
D000606:Amino Sugars; D002352:Carrier Proteins; D016326:Extracellular Matrix Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011509:Proteoglycans; C066929:polylactosamine; D000071235:Fibromodulin; D005643:Fucose; D006026:Glycoside Hydrolases; D008361:Mannosidases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D043323:alpha-Mannosidase