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Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

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Abstract

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Manα1 → 3(Manα1 → 6)Man, but not by mannose and α-methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such ‘dual’ properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.

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Abbreviations

GLC:

gas liquid chromatography

GlcNAc-Asn:

2-acetamido-1-β-(l-aspartamido)-1,2-dideoxy-d-glucose

BSA:

bovine serum albumin

DTT:

dithiothreitol

EDTA:

ethylenediaminetetraacetic acid

Gal:

d-galactose

GlcNAc:

N-acetyl-d-glucosamine

Man:

d-mannose; triomannose, Manα1 → 3(Manα1 → 6)Man;

MES:

2-(N-morphorino)ethanesulfonic acid

NeuAc:

N-acetyl-neuraminic acid

PNGase:

peptide:N 4-(N-accetylβ-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52)

PNP:

p-nitrophenyl

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Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo-7, 113, Tokyo, Japan

    Tadashi Suzuki, Ken Kitajima & Yasuo Inoue

  2. School of Pharmaceutical Sciences, Showa University, Hatanodai-1, 142, Tokyo, Japan

    Sadako Inoue

Authors
  1. Tadashi Suzuki
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  2. Ken Kitajima
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  3. Sadako Inoue
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  4. Yasuo Inoue
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Suzuki, T., Kitajima, K., Inoue, S. et al. Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?. Glycoconjugate J 11, 469–476 (1994). https://doi.org/10.1007/BF00731283

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  • DOI: https://doi.org/10.1007/BF00731283

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