Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface.
After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.
D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D006025:Glycosaminoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D014357:Trypsin
D000596:Amino Acids; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D011509:Proteoglycans; D014357:Trypsin