Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis.
We examined the ability of Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan synthesis to metabolize 125I-labeled thrombospondin (TSP). Wild type CHO cells bound and degraded 125I-TSP with kinetics similar to those reported for endothelial cells. Both binding and degradation were saturable (half-saturation at 20 micrograms/ml). When the concentration of labeled TSP was 1-5 micrograms/ml, mutant 745, defective in xylosyltransferase, and mutant 761, defective in galactosyltransferase I, bound and degraded 6- to 16-fold less TSP than wild type; mutant 803, which specifically lacks heparan sulfate chains, bound and degraded 5-fold less TSP than wild type; and mutant 677, which lacks heparan sulfate and has increased levels of chondroitin sulfate, bound and degraded 2-fold less TSP than wild type. Binding and degradation of TSP by the mutants were not saturable at TSP concentrations up to 100 micrograms/ml. Bound TSP was localized by immunofluorescence to punctate structures on wild type and, to a lesser extent, 677 cells. Heparitinase pretreatment of wild type cells caused a 2- to 3-fold decrease in binding and degradation, whereas chondroitinase pretreatment had no effect. Chondroitinase pretreatment of the 677 mutant (deficient heparan sulfate and excess chondroitin sulfate) caused a 2-fold decrease in binding and an 8-fold decrease in turnover, whereas heparitinase pretreatment had no effect. Treatment of wild type cells with both heparitinase and chondroitinase resulted in a 6- to 8-fold decrease in binding and turnover. These results indicate that cell surface proteoglycans mediate metabolism of TSP by CHO cells and that the primary effectors of TSP metabolism are heparan sulfate proteoglycans.
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D005260:Female; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006497:Heparan Sulfate / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010053:Ovary / Q000378:metabolism*; D013499:Surface Properties; D019699:Thrombospondins
D002809:Chondroitin Sulfates; D006023:Glycoproteins; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D019699:Thrombospondins