Modulation of cell surface heparan sulfate structure by growth of cells in the presence of chlorate

Abstract

Swiss mouse 3T3 cells, when grown in the presence of 5 mM chlorate, an inhibitor of PAPS synthesis, produce heparan sulfate glycosaminoglycan chains containing only about 8% of the sulfate normally present and which have lost the ability to bind to fibronectin. These undersulfated chains are sensitive to nitrous acid at pH 4.5, indicating that many glucosaminyl residues have unsubstituted amino groups. The iduronic acid content of the heparan sulfate produced in the presence of chlorate is reduced to less than 7% as compared to the 36% in that from untreated cells. The chlorate-treated cells do not demonstrate any alterations in their growth control. However, the spreading behavior of these cells is altered to a flat rounded morphology compared to the more typical fibroblastic appearance of the untreated cell. The sulfation of chondroitin chains is also inhibited, but at a lower chlorate concentration which does not alter growth control or the spreading ability of the cells. These data indicate that (a) 3T3 cell surface heparan sulfate proteoglycan is not involved in growth control but may be involved in cell spreading, (b) the use of chlorate should be a valuable method for the study of the biosynthesis and structure/function relationships of sulfated glycosaminoglycans, and (c) the temporal sequence of the heparan sulfate chain modification reactions predicted from results of studies with cell-free extracts also operates in the cell.

Stimulation of DNA Synthesis and Cell Proliferation of Human Mammary Myoepithelial-like Cells by Hepatocyte Growth Factor/Scatter Factor Depends on Heparan Sulfate Proteoglycans and Sustained Phosphorylation of Mitogen-activated Protein Kinases p42/44

“…5A). As reported in other systems (25,31,32), no change in cell morphology or cell death (assessed by exclusion of Trypan Blue and cell numbers using a hemocytometer) was detected (data not shown). In controls, HGF/SF still failed to stimulate DNA synthesis in chlorate-treated Huma 109 cells plated on plastic (Fig.…”

“…Following trypsinization, the cells were seeded in 24-well plates or 10-cm dishes as described for DNA synthesis or Western blot analysis, respectively, except that sulfate-free Dulbecco's modified Eagle's medium supplemented with 15 mM NaClO 3 was used throughout. As others have described, cells can be kept viable in sulfate-free medium containing chlorate for up to 1 month (25,31,32). Checks were always made on cell death and protein synthesis.…”

Location of N-Unsubstituted Glucosamine Residues in Heparan Sulfate

“…It may further be noted that no significant increase in GlcNH 2 residues was found neither in a Chinese hamster ovary cell mutant deficient in N-sulfation (41), nor upon overexpression of an N-sulfotransferase-deficient NDST1 mutant in 293 cells. 5 Depletion of PAPS, on the other hand, induced by treatment of cells with chlorate led to formation of HS with accumulated GlcNH 2 residues (41,42).…”

Cellular Heparan Sulfate Participates in the Metabolism of Prions

“…Sc -Sodium chlorate, an inhibitor of ATP-sulfurylase and hence of the production of phosphoadenosine phosphosulfate (the active sulfate donor for sulfotransferases) (54), reduces the sulfation of proteins and carbohydrates in intact cells without inhibiting cell growth or protein synthesis (54,55). Treatment with chlorate decreases the amount of protease-resistant PrP Sc in ScN2a cells (22).…”