Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis.
We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.
Iozzo
R V
RV
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003110:Colonic Neoplasms / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000096:biosynthesis* / Q000302:isolation & purification*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000302:isolation & purification*; D006801:Humans; D011509:Proteoglycans / Q000096:biosynthesis*; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D011508:Chondroitin Sulfate Proteoglycans; D005944:Glucosamine; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; C047142:N,O-desulfated heparan sulfate