PRESENCE OF N-UNSUBSTITUTED GLUCOSAMINE UNITS IN NATIVE HEPARAN-SULFATE REVEALED BY A MONOCLONAL-ANTIBODY

Immunohistochemical application of antibodies against heparan sulfate proteoglycan core protein and heparitinase-digested heparan sulfate st ubs showed the presence of heparan sulfate proteoglycan in all basemen t membranes of the rat kidney. However, a monoclonal antibody (JM-403) against native heparan sulfate (van den Born, J., van den Heuvel, L. P. W. J., Bakker, M. A. H., Veerkamp, J. H., Assmann, K. J. M., and Be rden, J. H. M. (1992) Kidney Int. 41, 115-123) largely failed to stain tubular basement membranes, suggesting the presence of heparan sulfat e chains lacking the specific JM-403 epitope, Heparan sulfate preparat ions from various sources differed markedly with regard to JM-403 bind ing, as demonstrated by liquid phase inhibition in enzyme-linked immun osorbent assay, the interaction decreasing with increasing sulfate con tents of the polysaccharide. Mapping of the JM-403 epitope indicated t hat it was dominated by one or more N-unsubstituted glucosamine unit(s ), since treatments that destroyed or altered the structure of such un its in heparan sulfate preparations (cleavage at N-unsubstituted gluco samine units with HNO2 at pH 3.9 and N-acetylation with acetic anhydri de, respectively), abolished antibody binding, Conversely, immunoreact ivity could be induced in a (D-glucuronyl-1,4-N-acetyl-D-glucosaminyl- 1,4) polysaccharide by the generation of N-unsubstituted glucosamine u nits by chemical N-deacetylation, The presence of N-unsubstituted gluc osamine in a JM-403-binding heparan sulfate (preparation HS-II from hu man aorta) was demonstrated by an similar to 3 fold reduction in molec ular size following HNO2 (pH 3.9) treatment. Further characterization of the epitope recognized by JM-403, based on enzyme-linked immunosorb ent assay inhibition tests with chemically/enzymatically modified poly saccharides, indicated that one or more N-sulfated glucosamine units a re invariable present, whereas L-iduronic acid and O-sulfate residues appear to inhibit JM-403 reactivity. It is concluded that the epitope contains one or more N-unsubstituted glucosamine and D-glucuronic acid units and is located in a region of the heparan sulfate chain compose d of mixed N-sulfated and N-acetylated disaccharide units.