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

• Published in: Glycobiology (Oxford) 2009-12, Vol.19 (12), p.1452-1461
• Main Authors: Taira, Toki, Hayashi, Hiroko, Tajiri, Yoshiko, Onaga, Shoko, Uechi, Gen-ichiro, Iwasaki, Hironori, Ohnuma, Takayuki, Fukamizo, Tamo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chitinases - chemistry; Chitinases - classification; Chitinases - genetics; Chitinases - isolation & purification; Chitinases - metabolism; Chromatography, High Pressure Liquid; class V chitinase; Cloning, Molecular; cycad; Cycadophyta; Cycas - chemistry; Cycas - genetics; Cycas - metabolism; Cycas revoluta; DNA, Complementary - isolation & purification; family 18 chitinase; Genes, Plant; Molecular Sequence Data; Peptide Mapping; posttranslational modification; Protein Processing, Post-Translational - physiology; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; transglycosylation
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwp119

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.