title: Hydrogen/deuterium exchange-LC-MS approach to characterize the action of heparan sulfate C5-epimerase.
abstract: Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on HS precursors, converts glucuronyl residues into iduronyl residues, and modulates subsequent biosynthetic steps in vivo. Previously, the ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA-(A)Man(R) and IdoA-(A)Man(R) disaccharides using a tedious paper chromatography technique. This radioactive assay, based on measuring either the release or incorporation of (3)H at C5 carbon of uronyl residues of (3)H-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA-(A)Man(R) and IdoA-(A)Man(R) disaccharides on high-performance liquid chromatography in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive-based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reactions on the same substrate catalyzed by C5-epimerase. The results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi.
authors.affiliation: Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Ponnusamy
authors.initials: P
authors.lastname: Babu
authors.forename: Xylophone V
authors.initials: XV
authors.lastname: Victor
authors.forename: Emily
authors.initials: E
authors.lastname: Nelsen
authors.forename: Thao Kim Nu
authors.initials: TK
authors.lastname: Nguyen
authors.forename: Karthik
authors.initials: K
authors.lastname: Raman
authors.forename: Balagurunathan
authors.initials: B
authors.lastname: Kuberan
mesh_terms: D000818:Animals; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D003903:Deuterium; D041961:Deuterium Exchange Measurement / Q000379:methods; D004187:Disaccharides / Q000302:isolation & purification; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006859:Hydrogen; D007313:Insecta / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods*; D011509:Proteoglycans / Q000378:metabolism
chemical_list: D002238:Carbohydrate Epimerases; D003903:Deuterium; D004187:Disaccharides; D006493:Heparin; D006497:Heparan Sulfate; D006859:Hydrogen; D011509:Proteoglycans