Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells.
The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse-chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from 'high-mannose' to 'complex' structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N-glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa. Pulse-chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio-Gel P-4 and concanavalin-A-Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.
D000118:Acetylglucosaminidase; D001792:Blood Platelets / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006026:Glycoside Hydrolases; D006801:Humans; D006868:Hydrolysis; D007120:Immunochemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D011402:Pronase; D011499:Protein Processing, Post-Translational; D019699:Thrombospondins; D014415:Tunicamycin / Q000494:pharmacology
D006023:Glycoproteins; D019699:Thrombospondins; D014415:Tunicamycin; D006026:Glycoside Hydrolases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D011402:Pronase