Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A

Abstract

Peptide-N4-( N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H ⁺. The gene of PNGase H ⁺ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H ⁺ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H ⁺ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H ⁺ a versatile tool in N-glycan analysis.

Abstract

PNGase H ⁺, from T. roseus was successfully cloned and expressed in E. coli. It shows activity in extremely acidic conditions and can release all types of N-glycans including core α1,3-fucosylated N-glycans from glycoproteins and glycopeptides in high efficiency.