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

[ Vol. 24 , Issue. 8 ]

Author(s):

Taiki Kuribara , Toshihiro Ishihara , Takaya Kudo , Makoto Hirano and Kiichiro Totani*   Pages 723 - 728 ( 6 )

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, synthetic substrate, chitobiose-pentapeptide, peptide sequence, substrate specificity, ultraperformance liquid chromatography mass spectrometry.

Affiliation:

Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633, Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633, Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633, Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633, Department of Materials and Life Science, Seikei University, 3-3-1 Kichijoji-kita, Musashino, Tokyo 180-8633

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