Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin.
Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.
D000818:Animals; D002813:Chondrosarcoma / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000096:biosynthesis; D007700:Kinetics; D046911:Macromolecular Substances; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology*
D046911:Macromolecular Substances; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; D014415:Tunicamycin; D006820:Hyaluronic Acid; D005944:Glucosamine