Endoglycosidase assay using enzymatically synthesized fluorophore-labeled glycans as substrates to uncover enzyme substrate specificities.

ABSTRACT: Glycan synthesis and degradation are not template but enzyme only driven processes. Substrate specificities of glyco-enzymes determine the structures of specific natural glycans. Using endoglycosidases as examples, we describe methods to study these enzymes. Endoglycosidase S/S2 specifically deglycosylates the conserved N-glycans of human immunoglobulin G. Endo-β-Galactosidase hydrolyzes internal β-galactosyl linkage in polylactosaminoglycan structures. To assay these enzymes, eleven fluorophore-labeled N-glycans and one polylactosamine ladder are synthesized. Digestion of these glycans result in mobility shift in gel electrophoresis. Results on Endo S/S2 assays reveal that they are most active on the agalactosylated biantennary N-glycans with decreased activity on galactosylated and sialy

SUBMITTER: Wu ZL 

PROVIDER: S-EPMC9132957 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature