Characterization of sialyltransferase mutants using surface plasmon resonance.
Sialyltransferases are enzymes responsible for the important sialylation of glycoconjugates. Since crystal structures are not available, other tools are needed to study enzymatic mechanisms. As a model, we used human alpha2,6-sialyltransferase. A putative acceptor-binding domain containing the small and the very small sialyl motifs was randomly mutated. This resulted in enzymes with altered enzymatic activity. Affinity chromatography demonstrated that their binding to donor substrate was maintained. To illustrate the role of the mutated domain in acceptor binding, a method based on surface plasmon resonance was set up. Only at low salt and high acceptor concentration was association of wild-type ST6GalI with asialofetuin demonstrated. As expected, this interaction was affected by cytidine 5'-monophospho-N-acetylneuraminic acid, the donor substrate, which proves the specificity of the interaction. Different types of mutants were found. For some, the drop in activity could be explained by loss in affinity for the acceptor. For others, the catalytic center, but not the acceptor-binding site, was affected. Neither acceptor binding nor catalytic activity were limited to the sialyl motifs. To our knowledge, this is the first example in which surface plasmon resonance is successfully used to demonstrate the binding of a glycosyltransferase to its natural acceptor.
Laroy
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W
Department of Molecular Biology, Unit of Fundamental and Applied Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, B-9000 Ghent, Belgium.
Ameloot
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P
Contreras
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R
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D015342:DNA Probes; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D015342:DNA Probes; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase