title: Acceptor specificities and selective inhibition of recombinant human Gal- and GlcNAc-transferases that synthesize core structures 1, 2, 3 and 4 of O-glycans.
abstract: BACKGROUND
  Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior.
  
  
  METHODS
  We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors.
  
  
  RESULTS
  Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme-substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-d-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs.
  
  
  CONCLUSIONS
  This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4.
  
  
  CONCLUSIONS
  These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors.forename: Yin
authors.initials: Y
authors.lastname: Gao
authors.forename: Rajindra P
authors.initials: RP
authors.lastname: Aryal
authors.forename: Tongzhong
authors.initials: T
authors.lastname: Ju
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.forename: Gagandeep
authors.initials: G
authors.lastname: Gahlay
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
authors.forename: Khushi L
authors.initials: KL
authors.lastname: Matta
authors.forename: Jason Z
authors.initials: JZ
authors.lastname: Vlahakis
authors.forename: Walter A
authors.initials: WA
authors.lastname: Szarek
authors.forename: Inka
authors.initials: I
authors.lastname: Brockhausen
mesh_terms: D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D005700:Galactosyltransferases; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase