title: A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research.
abstract: Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37 degrees C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well beta-counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 beta1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis(x) in O-glycans. A glycopolymer acceptor for beta1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5-6 fold increase in throughput compared to the corresponding solution-phase assay.
authors.affiliation: GlycoDesign Inc., Toronto, ON, Canada.
authors.forename: R S
authors.initials: RS
authors.lastname: Donovan
authors.forename: A
authors.initials: A
authors.lastname: Datti
authors.forename: M G
authors.initials: MG
authors.lastname: Baek
authors.forename: Q
authors.initials: Q
authors.lastname: Wu
authors.forename: I J
authors.initials: IJ
authors.lastname: Sas
authors.forename: B
authors.initials: B
authors.lastname: Korczak
authors.forename: E G
authors.initials: EG
authors.lastname: Berger
authors.forename: R
authors.initials: R
authors.lastname: Roy
authors.forename: J W
authors.initials: JW
authors.lastname: Dennis
mesh_terms: D000180:Acrylic Resins / Q000378:metabolism; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010969:Plastics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D012371:Robotics; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D004187:Disaccharides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D017351:N-Acetylglucosaminyltransferases; D010969:Plastics; D011994:Recombinant Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D000099166:Polyacrylamides