title: Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.
abstract: Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2-5[GlcUA(β1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3-4].
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
authors.affiliation: Department of Biochemistry & Molecular Biology, 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 & Molecular Biology, 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 / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002686:Chitin / Q000737:chemistry*; D006596:Hexosaminidases / Q000737:chemistry / Q000378:metabolism; D000076002:Hyaluronan Synthases / Q000737:chemistry / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology; D014530:Uridine Diphosphate / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D010940:Plant Proteins; D002686:Chitin; D014530:Uridine Diphosphate; D006820:Hyaluronic Acid; D000076002:Hyaluronan Synthases; D006596:Hexosaminidases; D000117:Acetylglucosamine
keywords: Class I hyaluronan synthase; UDP-activated; chitin oligosaccharide; polymer synthesis; reducing end elongation