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Journal of Biochemistry Advance Access published online on October 9, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp157
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Molecular identification and characterization of an acidic peptide:N-glycanase from tomato (Lycopersicum esculentum) fruits *

Md. Anowar Hossain1,2, Ryohei Nakano3, Kosuke Nakamura1,4 and Yoshinobu Kimura1,{dagger}

1 Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
2 Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh
3 Department of Plant Functions and Regulation, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
4 Kagome Research Institute, Kagome Co., Ltd., Nasu-gun, Tochigi 329-27, Japan

Correspondence Professor Yoshinobu KIMURA Ph.D Department of Biofunctional Chemistry Graduate School of Natural Science and Technology Okayama University, Tsushima-Naka 1-1-1 Okayama 700-8530, Japan Tel:+81-86-251-8296 Fax:+81-86-251-8388 E-mail: yosh8mar{at}cc.okayama-u.ac.jp

Received July 16, 2009; Accepted September 9, 2009


  Abstract

Plant acidic peptide:N-glycanase (PNGase) is one of the deglycosylation enzymes and has been considered to be involved in the catabolism of glycoproteins in plant cells. However, the tangible physiological significance involved in plant differentiation or growth is yet unclear. In this study, as a first step to elucidate the physiological role of free N-glycans and the de-N-glycosylation machinery working in developing plant cells, we have succeeded in expressing a cDNA from tomato fruits in Pichia pastoris and identified an acidic peptide:N-glycanase in the culture supernatant. The PNGase-gene-encoded protein is a single polypeptide chain of 588 amino acids with a predicted molecular mass of 65.8 kDa. The deduced amino acid sequence showed 57.9% similarity with almond PNGase A. The recombinant tomato PNGase showed optimum activity at pH 4.5 and 40°C. It did not require any metal ions for full enzymatic activity and could release the complex-type N-glycan from glycopeptides. Our phylogenetic analysis reveals that the plant acidic PNGase is completely different from the ubiquitous cytosolic PNGase and is involved in a different de-N-glycosylation mechanism associated with plant growth and development.

Key Words: Acidic peptide:N-glycanase, Lycopersicum esculentum, plant N-glycan, tomato fruit

* The nucleotide sequence data has been submitted to the DDBJ/EMBL/GenBank databases under accession number FJ804752


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