N-Unsubstituted Glucosamine in Heparan Sulfate of Recycling Glypican-1 from Suramin-treated and Nitrite-deprived Endothelial Cells

Abstract

We have analyzed the content of N-unsubstituted glucosamine in heparan sulfate from glypican-1 synthesized by endothelial cells during inhibition of (a) intracellular progression by brefeldin A, (b) heparan sulfate degradation by suramin, and/or (c) endogenous nitrite formation. Glypican-1 from brefeldin A-treated cells carried heparan sulfate chains that were extensively degraded by nitrous acid at pH 3.9, indicating the presence of glucosamines with free amino groups. Chains with such residues were rare in glypican-1 isolated from unperturbed cells and from cells treated with suramin and, surprisingly, when nitrite-deprived. However, when nitrite-deprived cells were simultaneously treated with suramin, such glucosamine residues were more prevalent. To locate these residues, chains were first cleaved at linkages to sulfated L-iduronic acid by heparin lyase and released fragments were separated from core protein carrying heparan sulfate stubs. These stubs were then cleaved off at sites linking N-substituted glucosamines to D-glucuronic acid. These fragments were extensively degraded by nitrous acid at pH 3.9. When purified proteoglycan isolated from brefeldin A-treated cells was incubated with intact cells, endoheparanasecatalyzed degradation generated a core protein with heparan sulfate stubs that were similarly sensitive to nitrous acid. We conclude that there is a concentration of N-unsubstituted glucosamines to the reducing side of the endoheparanase cleavage site in the transition region between unmodified and modified chain segments near the linkage region to the protein. Both sites as well as the heparin lyase-sensitive sites seem to be in close proximity to one another.

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

“…The polyzwitterionic character of HS is enhanced, as there is an increased number of both the NO-sensitive GlcNH 3 ϩ residues and the highly sulfated high affinity sites for polyamines. Normally, GlcNH 3 ϩ residues are scarce in HS (35) and are predominantly located close to the linkage region to protein (27,35). The GlcNH 3 ϩ residues formed upon polyamine depletion appear to be located inside or near the polyamine-binding sites (15).…”

“…The GlcNH 3 ϩ residues formed upon polyamine depletion appear to be located inside or near the polyamine-binding sites (15). N-Desubstitution could be catalyzed by endo-N-deacetylase or endo-N-sulfamidase or both (15,17,27,35). How polyamine depletion is sensed and conveyed to the HS-modulating enzymes remains unknown.…”

“…We have previously shown that the HS chains in recycling Gpc-1 are subject to degradation and resynthesis (15,17,23,27). Degradation is both enzymatic (heparanase) and non-enzymatic (NO-dependent deaminative cleavage).…”

“…The large BFA-arrested glycoform with long HS chains contains GlcNH 3 ϩ units (27). When the proportion of such units is sufficiently high, the diminished net negative charge of the HSPG will affect its elution position upon ion exchange chromatography (17).…”

“…Heparanase Activity in Normal and Polyamine Uptake-deficient CHO Cells-A Gpc-1 glycoform with long HS chains, suitable as a substrate for heparanase, can be obtained from BFA-treated cells (15,27). The capacity for enzymatic degradation of HS by wild-type and polyamine uptake-deficient CHO cells was compared by incubating radiolabeled large-size HSPG obtained from BFA-treated CHO-K1 or CHO-MGBG cells with the corresponding cells in culture.…”