title: Mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of Vibrio cholerae, serotype Ogawa and the recombinant tetanus toxin C-fragment carrier.
abstract: We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT-Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate-protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT-Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein.
authors.affiliation: Memorial University, Biochemistry Department, St John's, NL, Canada, A1B 3X7.
authors.forename: Farid
authors.initials: F
authors.lastname: Jahouh
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
authors.forename: Willie F
authors.initials: WF
authors.lastname: Vann
authors.forename: Pavol
authors.initials: P
authors.lastname: Kováč
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Banoub
mesh_terms: D000595:Amino Acid Sequence; D002771:Cholera / Q000382:microbiology; D022121:Cholera Vaccines / Q000737:chemistry*; D006001:Glycoconjugates / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013744:Tetanus Toxin / Q000737:chemistry*; D041241:Vibrio cholerae O1 / Q000737:chemistry*
chemical_list: D022121:Cholera Vaccines; D006001:Glycoconjugates; D010446:Peptide Fragments; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C
keywords: GluC V8 digestion; MALDI-TOF-MS; Trypsin digestion; nano-LC-ESI-QqTOF-MS/MS; neoglycoconjugate vaccine; recombinant tetanus toxin C-fragment (rTT-Hc)