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Glycobiology Advance Access originally published online on August 20, 2009
Glycobiology 2009 19(12):1452-1461; doi:10.1093/glycob/cwp119
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

A plant class V chitinase from a cycad (Cycas revoluta): Biochemical characterization, cDNA isolation, and posttranslational modification

Toki Taira1,2, Hiroko Hayashi2, Yoshiko Tajiri2, Shoko Onaga3, Gen-ichiro Uechi4, Hironori Iwasaki5, Takayuki Ohnuma6 and Tamo Fukamizo6

2 Department of Bioscience and Biotechnology, Ryukyu University, Okinawa 903-0213
3 The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima 890-0065
4 Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523
5 Center of Molecular Bioscience, Ryukyu University, Okinawa 903-0213
6 Department of Advanced Bioscience, Kinki University, Nara 631-8505, Japan

1 To whom correspondence should be addressed: Tel: +81-98-895-8802; Fax: +81-98-895-8802; e-mail: tokey{at}agr.u-ryukyu.ac.jp

Received on March 26, 2009; revised on August 10, 2009; accepted on August 12, 2009

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)3 from the substrate (GlcNAc)6 through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

Key words: class V chitinase / cycad / family 18 chitinase / posttranslational modification / transglycosylation


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