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.

Mass spectrometry for protein sialoglycosylation

“…Due to its excellent ability to releasing N ‐glycans, the enzyme has contributed greatly to the study of glycomics. So far, the activities and specificities of several PNGases have been demonstrated, of which PNGase F and PNGase A are most widely used for glycomics profiling. Using PNGases, carbohydrates are released as glycosylamines with amino groups at the N ‐terminus.…”

“…Moreover, this enzyme is a glycoprotein and can be slightly self‐deglycosylated, resulting in detectable contamination . PNGase H + , a new glycanase for glycomics field, is a non‐glycosylated protein and exhibits higher efficiency over PNGase A . However, because of an extremely low pH (2.6), the reaction may result in dissociation of sialic acids and removal of residue modifications.…”

“…182 PNGase H + , a new glycanase for glycomics field, is a non-glycosylated protein and exhibits higher efficiency over PNGase A. 166 For large-scale glycan analysis, the release procedure requires long times and high enzyme expense, thus an improved method is needed to reduce process costs and complete the release of glycans from a variety of glycoproteins. 183 Several approaches, such as microwave irradiation, pressure-cycling technology, and enzyme immobilization, have been developed to rapidly cleave oligosaccharides from glycoproteins.…”

Glycosylation of viral surface proteins probed by mass spectrometry

“…When the O-glycans from five pathogenic EBOV GP 1,2 s were compared, differences in O-glycan patterns were observed [23 ]. utilized; however, it is less efficient [55,56]. Machupo GP 1 [57], HIV-1 gp120 [45,58], Dengue virus GP E [59], chikungunya virus E1 and E2 proteins [60], and the LASV GP complex (GPC) [6 ] are a few examples of Examples of site-specific glycan profile outputs.…”

“…Glycan site occupancy N-Glycan site occupancy is an additional MS assessment that can be utilized in the VSP glycoprofile (Figure 3b). In order to obtain site occupancy, N-glycans are released using PNGase F (as mentioned above converting N to D) [56]. The resultant peptides containing aspartic acid are used to determine the extent of glycan occupancy at a given NGS.…”