title: Hyaluronan synthase assembles chitin oligomers with -GlcNAc(α1→)UDP at the reducing end.
abstract: Class I hyaluronan synthases (HASs) assemble a polysaccharide containing the repeating disaccharide [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP and vertebrate HASs also assemble (GlcNAc-β1,4)n homo-oligomers (chitin) in the absence of GlcUA-UDP. This multi-membrane domain CAZy GT2 family glycosyltransferase, which couples HA synthesis and translocation across the cell membrane, is atypical in that monosaccharides are incrementally assembled at the reducing, rather than the non-reducing, end of the growing polymer. Using Escherichia coli membranes containing recombinant Streptococcus equisimilis HAS, we demonstrate that a prokaryotic Class I HAS also synthesizes chitin oligomers (up to 15-mers, based on MS and MS/MS analyses of permethylated products). Furthermore, chitin oligomers were found attached at their reducing end to -4GlcNAc(α1→)UDP [i.e. (GlcNAcβ1,4)nGlcNAc(α1→)UDP]. These oligomers, which contained up to at least seven HexNAc residues, consisted of β4-linked GlcNAc residues, based on the sensitivity of the native products to jack bean β-N-acetylhexosaminidase. Interestingly, these oligomers exhibited mass defects of -2, or -4 for longer oligomers, that strictly depended on conjugation to UDP, but MS/MS analyses indicate that these species result from chemical dehydrogenations occurring in the gas phase. Identification of (GlcNAc-β1,4)n-GlcNAc(α1→)UDP as HAS reaction products, made in the presence of GlcNAc(α1→)UDP only, provides strong independent confirmation for the reducing terminal addition mechanism. We conclude that chitin oligomer products made by HAS are derived from the cleavage of these novel activated oligo-chitosyl-UDP oligomers. Furthermore, it is possible that these UDP-activated chitin oligomers could serve as self-assembled primers for initiating HA synthesis and ultimately modify the non-reducing terminus of HA with a chitin cap.
authors.affiliation: Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA paul-weigel@ouhsc.edu.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
authors.affiliation: Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602-4712, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602-4712, USA.
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.affiliation: Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Baggenstoss
authors.affiliation: Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Washburn
mesh_terms: D000117:Acetylglucosamine / Q000032:analysis / Q000378:metabolism*; D002686:Chitin / Q000096:biosynthesis* / Q000737:chemistry*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D000076002:Hyaluronan Synthases; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018502:Streptococcus equi / Q000201:enzymology; D014530:Uridine Diphosphate / Q000032:analysis / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D002686:Chitin; D014530:Uridine Diphosphate; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases; D000117:Acetylglucosamine
keywords: Class I hyaluronan synthase; activated oligosaccharide; chitobiosyl; polymer synthesis; reducing end elongation