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 sulfate d polylactosamine (keratan sulfate). In the present study we examined the biosynthetic processing of these N-linked oligosaccharides for sub sequent addition of polylactosamine. Chondrocytes were treated with ca stanospermine, 1-(+)deoxymannojirimycin, and swainsonine, radiolabeled with [3,4,5-H-2]leucine, [2-H-1]mannose, or [6-H-3]glucosamine, and n ewly synthesized fibromodulin was immunoprecipitated for analysis. Cas tanospermine and 1-(+)deoxymannojirimycin inhibited polylactosamine ad dition, 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 oligosacchar ides per molecule were modified with polylactosamine containing 4-6 re peating disaccharide units. Moreover, a single short chain was added e ither to the C-3 or the C-6 branch in control cultures, whereas only t he C-3 branch was substituted in the presence of swainsonine. Analysis of endo-beta-galactosidase and keratanase II digestion products of th e polylactosamine chains synthesized in both culture conditions showed that only about 25% of the hexosamine residues and less than 5% of th e adjacent galactose residues were substituted with sulfate. These fin dings are discussed in relation to the regulation of fibromodulin glyc osylation and the likely influence of polylactosamine structure on the extracellular interactions and turnover of fibromodulin.