Selective effects of sodium chlorate treatment on the sulfation of heparansulfate

We have analyzed the effect of sodium chlorate treatment of Madin-Darby can ine kidney cells on the structure of heparan sulfate (HS), to assess how th e various sulfation reactions during HS biosynthesis are affected by decrea sed availability of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate . Metabolically [H-3]glucosamine-labeled HS was isolated from chlorate-trea ted and untreated Madin-Darby canine kidney cells and subjected to low pH n itrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide un its, and (iii) the N-acetylated domains were recovered and subjected to com positional disaccharide analysis. Upon treatment with 50 mM chlorate, overa ll O-sulfation of HS was inhibited by similar to 70%, whereas N-sulfation r emained essentially unchanged. Low chlorate concentrations (5 or 2 mM) sele ctively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlor ate reduced both 2-O and 6-O-sulfation. Analysis of saccharides representin g the different domain types indicated that 6-O-sulfation was preferentiall y 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-spec ific fashion.