Identification and characterization of a novel exo-glycosidase activity of NGLY1 on ENGase-digested N-GlcNAc proteins in vitro

N-Glycanase 1 (NGLY1) is known for its activity to completely remove conjugated N-glycans from glycoproteins. Early studies illustrated that NGLY1 defects were associated with a rare disease named NGLY1-related congenital disorder of glycosylation (NGLY1-CDDG). Despite extensive research has been conducted over the past decade on the biological impact of NGLY1’s endo-glycosidase activity in cells and on disease, whether NGLY1 also exhibits an exo-glycosidase activity remains open. In this study, an exo-glycosidase activity of NGLY1 on the N-GlcNAc proteins was firstly reported and characterized in vitro . To distinguish for the conventional endo-glycosidase activity and the newly reported NGLY1’s exo-glycosidase activity, the active sites of exo-NGLY1 were predicted and compared with those of endo-NGLY1. In addition, the correlation between NGLY1’s exo-glycosidase activity in vitro and NGLY1-CDDG’s clinical characterization was investigated and discussed.