Distinct Substrate Specificities of Bacterial Heparinases against N-Unsubstituted Glucosamine Residues in Heparan Sulfate

Abstract

The rare N-unsubstituted glucosamine (GlcNH 3 ؉ ) residues in heparan sulfate have important biological and pathophysiological roles. In this study, four GlcNH 3 ؉ -containing disaccharides were obtained from partially de-N-sulfated forms of heparin and the N-sulfated K5 polysaccharide by digestion with combined heparinases I, II, and III. These were identified as ⌬HexA-GlcNH 3 and ⌬HexA-(2S)-GlcNH 3؉ (6S). Digestions with individual enzymes revealed that heparinase I did not cleave at GlcNH 3 ؉ residues; however, heparinases II and III showed selective and distinct activities. Heparinase II generated ⌬HexA-GlcNH 3 ؉ (6S), ⌬HexA(2S)-GlcNH 3 ؉ , and ⌬HexA-(2S)-GlcNH 3 ؉ (6S) disaccharides, whereas heparinase III yielded only the ⌬HexA-GlcNH 3 ؉ unit. Thus, the action of heparinase II requires O-sulfation, whereas heparinase III acts only on the corresponding non-sulfated unit. These striking distinctions in substrate specificities of heparinases could be used to isolate oligosaccharides with novel sequences of GlcNH 3 ؉ residues. Finally, heparinases were used to identify and quantify GlcNH 3 ؉ -containing disaccharides in native bovine kidney and porcine intestinal mucosal heparan sulfates. The relatively high content of O-sulfated GlcNH 3 ؉ -disaccharides in kidney HS raises questions about how these sequences are generated.

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

“…Here, we demonstrate an increased 10E4 antibody binding to the IPF HLF when compared with donor HLF, an effect that was reduced following treatment with heparinase I and III. The specificity of heparinase I directs its activity toward a linkage between N-and 6-Osulfated glucosamine and 2-O-sulfated iduronic acid, whereas heparinase III cleaves between non-sulfated or N-sulfated glucosamine and iduronic acid or glucoronic acid (18). Because immunoreactivity of IPF, but not donor, HLF was reduced after combined treatment with heparinase I and III, we concluded that IPF HLF contain more N-sulfated and 6-O-and 2-O-sulfated HS chains at the cell surface.…”

“…Next, we investigated the role of differently sulfated HS structures in FXIIa binding by using heparinase I and heparinase III. N-and 6-O-sulfated glucosamine linked to 2-O-sulfated iduronic acid is a preferred dissacharide recognized by heparinase I, whereas heparinase III cleaves glycosidic linkage between N-acetylated or N-sulfated glucosamine and glucu- ronic acid (18). This distinct substrate specificity dictates degradation of high-or low-sulfated HS regions by heparinase I or heparinase III, respectively.…”

Comparison of the effects of differentially sulphated bovine kidney- and porcine intestine-derived heparan sulphate on breast carcinoma cellular behaviour

“…To investigate this further, we repeated the above experiment but added porcine intestinederived heparan sulphate to the chlorate-containing culture medium instead of the bovine kidney-derived species. Heparan sulphate from porcine intestines possesses a lower degree of sulphation compared to that obtained from bovine kidney (33,34). We hypothesised that it may thus be less effective in blocking the effect of chlorate on cell adhesion.…”

Coagulation Factor IX Mediates Serotype-Specific Binding of Species A Adenoviruses to Host Cells

“…Also, Hep I does not cleave the domains of heparan sulfate that are in proximity to the protein backbone (the N-acetylated domains and transition zones), which are less sulfated. These regions, however, are cleaved by heparinase III (Hep III), which cleaves specifically between uronic acids (iduronic acid [IdoA] or glucuronic acid [GlcA]) and N-sulfated or N-acetylated glucosamine (GlcNSO 4 and GlcNAc, respectively) (17,70). These motifs are relatively common in heparan sulfate, and therefore, Hep III cleaves heparan sulfate more efficiently than Hep I (70).…”

“…These regions, however, are cleaved by heparinase III (Hep III), which cleaves specifically between uronic acids (iduronic acid [IdoA] or glucuronic acid [GlcA]) and N-sulfated or N-acetylated glucosamine (GlcNSO 4 and GlcNAc, respectively) (17,70). These motifs are relatively common in heparan sulfate, and therefore, Hep III cleaves heparan sulfate more efficiently than Hep I (70). As expected, we found that Hep III treatment of FHs74Int cells efficiently prevented FX-mediated HAdV-5 binding, but FIX-mediated HAdV-31 binding was also efficiently reduced.…”