title: Glycan microarray screening assay for glycosyltransferase specificities.
abstract: Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.
authors.affiliation: Glycan Microarray Synthesis Core, Consortium for Functional Glycomics, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Peng
authors.forename: Corwin M
authors.initials: CM
authors.lastname: Nycholat
authors.forename: Nahid
authors.initials: N
authors.lastname: Razi
mesh_terms: D019921:Biotinylation; D057075:Enzyme Assays / Q000379:methods*; D016695:Glycosyltransferases / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism; D013379:Substrate Specificity; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase