title: Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.
abstract: The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.
authors.affiliation: The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA, USA.
authors.forename: James
authors.initials: J
authors.lastname: Kang
authors.forename: William
authors.initials: W
authors.lastname: Low
authors.forename: Thomas
authors.initials: T
authors.lastname: Norberg
authors.forename: Jill
authors.initials: J
authors.lastname: Meisenhelder
authors.forename: Karin
authors.initials: K
authors.lastname: Hansson
authors.forename: Johan
authors.initials: J
authors.lastname: Stenflo
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhou
authors.forename: Julita
authors.initials: J
authors.lastname: Imperial
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Alan C
authors.initials: AC
authors.lastname: Rigby
authors.forename: A Grey
authors.initials: AG
authors.lastname: Craig
mesh_terms: D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins / Q000138:chemical synthesis* / Q000737:chemistry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D020916:Conotoxins