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

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 sialylated glycans and little or no activity on branched and bisected glycans. Assays on Endo-β-Gal reveal that the enzyme is active from pH 3.5 to 9.0 and the β3-linked GlcNAc adjacent to the cleavage site is minimal for the enzyme recognition with the optimal recognition motif spanning at least four lactosamine repeats. Our methods will provide an opportunity to understand how specific glycans are synthesized and degraded.