Peptide Specificity Analysis of Peptide: N-glycanases Using Synthetic Chitobiose-pentapeptides

Abstract

Background: Peptide: N-glycanase is a deglycosylation enzyme releasing N-glycan from glycoproteins. Although glycan specificity analysis of this enzyme has been reported, recognition requirements for the peptide sequence have not been precisely elucidated.

Objective: In this study, we carried out peptide specificity analysis of several peptide:N-glycanases.

Methods: Using synthetic chitobiose-pentapeptide substrates having a systematic series of amino acid sequences composed of hydrophobic leucine and hydrophilic serine, we examined the peptide specificities of peptide: N-glycanases comprising yeast cytoplasmic PNGase, bacterial PNGase F, and plant PNGase A by ultra-performance liquid chromatography combined with electrospray ionization mass spectrometry.

Results: We found that each of the PNGases had higher activity for the more hydrophobic (leucinerich) chitobiose-pentapeptides, although the sensitivities of the PNGases for hydrophobicity varied. Cytoplasmic PNGase showed broad specificity. In contrast, PNGase A showed moderate specificity. PNGase F showed the highest specificity.

Conclusion: PNGases from different origins had similar but significantly independent peptide specificities.

Keywords: Peptide: N-glycanase; chitobiose-pentapeptide; peptide sequence; substrate specificity; synthetic substrate; ultraperformance liquid chromatography mass spectrometry.