Molecular characterization of acidic peptide:N-glycanase from the dimorphic yeast Yarrowia lipolytica

Abstract

Peptide:N-glycanase (PNGase) A is used preferentially to cleave the glycans from plant and insect glycopeptides. Although many putative PNGase A homologous genes have been found in the plant and fungus kingdoms through sequence similarity analyses, only several PNGases from plants and one from a filamentous fungus have been characterized. In this study, we identified and characterized a PNGase A-like enzyme, PNGase Yl, in the dimorphic yeast Yarrowia lipolytica. The corresponding gene was cloned and recombinantly expressed in Pichia pastoris. The purified enzyme cleaved glycans from glycopeptides with the maximum activity at pH 5. No metal ions were required for full activity, and rather it was repressed by three metal ions (Fe(3+), Cu(2+) and Zn(2+)). Using glycopeptide substrates, PNGase Yl was shown to release various types of N-glycans including high-mannose and complex-type glycans as well as glycans containing core-linked α(1,3)-fucose that are frequently found in plants and insects. Moreover, in comparison with PNGase A, PNGase Yl was able to cleave with higher efficiency the glycans from some denatured glycoproteins. Taken together, our results suggest that PNGase Yl, the first biochemically characterized yeast PNGase A homologue, can be developed through protein engineering as a useful deglycosylation tool for N-glycosylation study.

Mass spectrometry for protein sialoglycosylation

“…N ‐glycans are usually released by the peptide N ‐glycosidase (PNGase), also known as peptide‐ N 4‐( N ‐acetyl‐β‐glucosaminyl) asparagine amidase. PNGase cleaves N ‐linked oligosaccharides between the innermost GlcNAc and Asn from glycoproteins/glycopeptides to yield free N ‐glycan chains . Due to its excellent ability to releasing N ‐glycans, the enzyme has contributed greatly to the study of glycomics.…”

“…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.…”

“…It is worth mentioning that the optimal condition for the enzymatic activity of PNGase F is weakly alkaline which may effectively inhibit the dissociation of sialic acid residues during the digestion process. Compared to PNGase A, PNGase F has higher deglycosylation activity for sialoglycans . Furthermore, PNGase F can be used for on‐tissue N ‐glycan profiling to assess the spatial distribution of oligosaccharides associated with tissue sections …”

“…Compared to PNGase A, PNGase F has higher deglycosylation activity for sialoglycans. 161 Furthermore, PNGase F can be used for on-tissue N-glycan profiling to assess the spatial distribution of oligosaccharides associated with tissue sections. 177 The activity of PNGase F varies depending on the nature of samples.…”

“…PNGase A shows relatively low digestion efficiency in releasing sialylated oligosaccharides. 161 Moreover, this enzyme is a glycoprotein and can be slightly self-deglycosylated, resulting in detectable contamination. 182 PNGase H + , a new glycanase for glycomics field, is a non-glycosylated protein and exhibits higher efficiency over PNGase A.…”