A plant class V chitinase from a cycad (Cycas revoluta): biochemical characterization, cDNA isolation, and posttranslational modification.
A plant class V chitinase from a cycad (Cycas revoluta): biochemical characterization, cDNA isolation, and posttranslational modification.
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DOI：
10.1093/glycob/cwp119
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发表时间：
2009-12
期刊：
影响因子：
4.3
通讯作者：
T. Taira;H. Hayashi;Yoshiko Tajiri;Shoko Onaga;Gen‐ichiro Uechi;H. Iwasaki;T. Ohnuma;T. Fukamizo
中科院分区：
文献类型：
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作者：
T. Taira;H. Hayashi;Yoshiko Tajiri;Shoko Onaga;Gen‐ichiro Uechi;H. Iwasaki;T. Ohnuma;T. Fukamizo
几丁质酶-A (CrChi-A) 通过柱层析的几个步骤从 Cycas revoluta 的叶轴中纯化。发现它是一种糖蛋白，分子量为40 kDa，等电点为5.6。 CrChi-A 主要通过保留机制从底物 (GlcNAc)(6) 产生 (GlcNAc)(3)。更有趣的是，CrChi-A 表现出转糖基活性，迄今为止在植物几丁质酶中尚未观察到这种活性。通过快速扩增 cDNA 末端和聚合酶链式反应程序，克隆了编码 CrChi-A 的 cDNA。它由 1399 个核苷酸组成，编码一个由 387 个氨基酸残基组成的开放阅读框。序列分析表明CrChi-A属于植物V类几丁质酶组。通过对天然酶和重组酶的肽图谱和质谱分析，我们发现前体 (M1-A387) 中的 N 端信号肽和 C 端延伸被去除，产生成熟的 N-糖基化蛋白 (Q24-G370)。这是关于具有转糖基活性和植物 V 类几丁质酶翻译后修饰的植物几丁质酶的第一份报告。
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.