Abstract
Glycosylation is one of the common modifications in eukaryotic cell and is important for protein structure and function. Therefore, scientists have been tring to figure out the connection between protein glycosylation and human diseases. However, for protein crystallography, the glycans on the protein make it more difficult to get glycoprotein crystals. Our goal is to utilize endoglycosidases to quickly and massively produce deglycosylated glycoprotein under native condition. Furthermore, a suitable method was applied to immobilize these endoglycosidases for rapid isolation of the deglycosylated glycoproteins. In our experiments, we cloned the endoglycosidase F1, F2, F3 and peptide-N-glycosidase F from Elizabethkingia meningosepticum and endoglycosidase H from Streptomyces plicatus. All the endoglycosidase were expressed in E. coli expression system. We adopted two strains of Influenza A hemagglutinin (HA) as the glycoprotein substrates. Each of HA proteins was expressed by two different cell type individually in order to generate identical protein with different glycan form. Three methods were used to exam the deglycosylation reaction level. First, the deglycosylation of glycoprotein makes protein band down shifted in SDS-PAGE analysis. Second, the exactly molecular weight decreasing due to deglycosylation is measured by MALDI-TOF Mass analysis. Finally, the deglycosylation efficiency on each glycosylation site is detected by glycopeptide analysis method with LC MS/MS. The deglycosylation result of high mannose type glycan HA is satisfactory, even when the reaction is performed with immobilized endoglycosidase H. However, the deglycosylation level of the complex type glycan HA is not enough by using endoglycosidases individually or several endoglycosidases at the same time. The results of two different type glycan substrates digested by peptide-N-glycosidase F were similar. We suggest that the uncompleted deglycosylation of different substrate of HA might be result from the steric barrier caused by bulky complex glycan or the conformation of substrate. Our goal is to find a general procedure to generate deglycosylated glycoproteins in a native condition that is suitable for protein crystallography and other biological research.