POST-BIOSYNTHETIC MODIFICATION OF HEPARAN SULFATE BY REACTIVE NITROGEN SPECIES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparan sulfate (HS) chains on cell surfaces and in extracellular matrices are acted upon by enzymes and chemical species to alter their biological activities. Reactive nitrogen species (RNS) give rise to conditions for deaminative cleavage of HS chains in vascular tissue. It is likely that aldehyde-containing HS chains so liberated become reactive toward cellular proteins. Structural investigations to elucidate these mechanisms are the subject of this project. Our results using a mass spectrometry glycomics platform show the presence of a disaccharide of structure (¿HexA-GlcNH26S). These results are consistent with the presence of HS repeats corresponding to HexA-GlcNH26S that are not detectable using other methods. It is likely that disaccharides with free amino groups are difficult to detect using standard disaccharide analysis methods because its zwitterionic nature causes poor retention on ion exchange resins or weak pairing with ion-pairing agents. The presence of disaccharide units containing free glucosamine amino groups in mature HS chains seems to be related to the mechanisms of action of N-deacetylase/N-sulfotransferase enzymes and the availability of 3'-phosphoadenosine-5'-phosphosulfate. Our work shows that disaccharide units containing HexA-GlcNH26S are widely expressed in organ tissue. HS chains containing such disaccharide units would be susceptible to NO-mediated cleavage as has been described for glypican-1 cycling. Conditions for NO-mediated cleavage may exist in other biological processes. Glucosamine amino groups of HS are also known to react rapidly with oxidizing agents such as HOCl under conditions that are likely to be present at inflammation. It is clear that the HexA-GlcNH26S repeat is a widely expressed and functionally relevant HS sub-structure.