Specificity of enzymatic in vitro glycosylation by PNGase F:a comparison of enzymatic and non-enzymatic glycosylation

摘要

Enzymatic in vitro glycosylation is possible using a reverse reaction of peptide-N-glycosidase F(PNGase F),and non-enzymatic in vitro glycosylation occurs when the sugar residue is one or two units 10ng.To identify the differences between enzymatic and non-enzymatic glycosylation,glycosylation sites were analyzed by the acid hydrolysis of glycopeptides fol10wed by MALDI-TOF mass spectrometric analysis.Pentapeptide(Arg-Lys-Asp-Val-Tyr)and octapeptide(Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr)were used in this study,and the sequence of the octapeptide was appropriately chosen to investigate the specificity of enzymatic glycosylation by considering the characteristics of PNGase F and non-enzymatic glycosylation.N,N'-Diacetylchitobiose was aminated prior to the glycosylation reaction at an amination extent of 60%.The glycosylation site was very specific to the aspartate residue in the enzymatic reaction,while non-enzymatic glycosylation occurred at arginine or lysine residues.PNGases F can be effectively used for the glycosylation of the non-glycosylated recombinant proteins produced in prokaryotic cells.