Characterization of glycosyltransferases and glycosidases using electrospray mass spectrometry

  • Author / Creator
    Soya, Naoto
  • Carbohydrates are one of the major classes of organic compounds found in nature and living organisms, and the diverse roles of carbohydrates are crucial for most life forms. The transformation and degradation of glycans are effectively regulated by carbohydrate-modifying enzymes. This thesis describes the characterization of carbohydrate-modifying enzymes, glycosyltransferases and glycosidases, using electrospray mass spectrometry (ES-MS) to gain a better understanding of these enzymes. The ES-MS binding assay was applied to quantify the affinities of the human blood group synthesizing glycosyltransferases (GTA and GTB) for their H-antigen substrate in the absence and presence of bound uridine 5'-diphosphate (UDP) and divalent metal cofactor Mn2+. The presence of UDP and Mn2+ in the binding site had a marked influence on the association constant (Ka), enthalpy (ΔHa) and entropy (ΔSa) for the association of H-antigen to GTs. Moreover, the interactions between GT and nucleotide-sugar donor were investigated. Our results revealed that Mn2+ enhances the affinities of donors by 20 ~ 100 times. However, donors undergo enzyme-catalyzed hydrolysis in the presence of Mn2+ resulting in monosaccharide and UDP. The catalytic mechanism of GTA and GTB was also investigated using ES-MS. To trap the glycosyl-enzyme intermediates in their enzymatic reaction, GT mutants, in which the putative catalytic nucleophile Glu303 was replaced with Cys, were utilized. The formation of intermediates was observed by incubation of GT mutants with donor substrates. Tandem MS analysis confirmed Cys303 as the site of glycosylation. Incubation of the purified intermediates with H-antigen resulted in the decrease of intermediates and the formation of the trisaccharide products. Our results suggest that the GT mutants could operate by a double displacement mechanism. The rate of substrate cleavage by the human neuraminidase 3 (NEU3) was measured using ES-MS. The kinetic analysis using synthetic substrates revealed that NEU3 activity depended upon the hydrophobicity of the aglycone. In addition, the substrates with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were cleaved by NEU3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Klassen, John (Chemistry)
  • Examining committee members and their departments
    • Le, Chris (Chemistry)
    • Szymanski, Christine (Biological Sciences)
    • Peters, Thomas (University of Luebeck)
    • Cairo, Christopher (Chemistry)
    • Serpe, Michael (Chemistry)