Selective Effects of Sodium Chlorate Treatment on the Sulfation of Heparan Sulfate

Abstract

We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3-phosphoadenosine 5-phosphosulfate. Metabolically [ 3 H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mM chlorate, overall O-sulfation of HS was inhibited by ϳ70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mM) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlorate reduced both 2-O-and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-Osulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3-phosphoadenosine 5-phosphosulfate availability has distinct effects on the N-and O-sulfation of HS and that O-sulfation is affected in a domain-specific fashion.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

“…The effect of chlorate treatment on reducing NRG1 activity in muscle cells was not as pronounced as that seen previously after removing all of the HSPG GAGs with heparitinase (13) or by ␤-Dxyloside treatment that removes and releases GAGs into the culture media (42). This is perhaps not too surprising, because the desulfation produced by chlorate is often not complete and can be selective for specific sulfate groups (33).…”

“…Therefore, we examined the role of endogenous sulfation by treating L6 muscle cells for 48 h with chlorate, which competitively inhibits the formation of 3Ј-phosphoadenosine 5Ј-phosphosulfate, the sulfate donor for sulfotransferase reactions (33)(34)(35). Using the full-length heparin-binding form of NRG1 (IgEGF), chlorate treatment reduced receptor phosphorylation by 53%, whereas no reduction was seen with FIG.…”

Heparan sulfate Ndst1 gene function variably regulates multiple signaling pathways during mouse development

“…Lastly, Ndst1 is the predominant Ndst expressed in multipotent C3H10T1/2 mouse mesenchymal precursor cells, which, under the induction of Hhs, differentiate into AP-producing osteoblasts (Kinto et al, 1997). The importance of Ndst1 in Hh-induced C3H10T1/2 differentiation was shown by siRNAi, and the general importance of HS in that system was confirmed by chlorate treatment or addition of heparin, which resulted in undersulfated cell surface HS (Safaiyan et al, 1999) or competing Hh binding sites in the medium, respectively, resulting in reduced availability of soluble Hh to cell surface HS. Because Ndst1 mutant embryos display some but not all defects seen in Shh mutant embryos (Grobe et al, 2005), Ndst1 does not seem to be always necessary for Hh signaling, but becomes crucial in the absence of other, possibly compensating Ndsts.…”

Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2

“…2). In the first set of experiments, cells were treated with chlorate, a compound that blocks heparan sulfate biosynthesis (49)(50)(51). A second approach made use of a CHO mutant cell line (CHO-745) that lacks the enzyme xylosyl transferase and, therefore, is incapable of synthesizing heparan sulfate chains on the core protein of proteoglycans (52,53).…”