Characterization of chitin synthases from Entamoeba
Characterization of chitin synthases from Entamoeba
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DOI：
10.1078/1434461041844204
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发表时间：
2004-09-01
期刊：
影响因子：
2.5
通讯作者：
Tannich, E
中科院分区：
文献类型：
--
作者：
Campos-Góngora, E;Ebert, F;Tannich, E
内阿米巴囊壁的主要成分是几丁质，一种β -(1,4)-连接的n -乙酰- d -氨基葡萄糖的均聚物。几丁质聚合需要活性几丁质合成酶（CHS）的存在，这是一组属于β -糖基转移酶家族的酶。已对真菌、昆虫和线虫进行了CHS描述；然而，关于这类酶在原虫（如内阿米巴原虫）中的结构和表达的信息缺乏。本研究通过基因克隆的方法确定了两种推测为溶组织绦虫CHS （EhCHS-1和EhCHS-2）的初级结构，并在dispar和E. invadens爬虫类寄生虫的数据库中鉴定了同源蛋白。后者是研究内阿米巴囊肿发育的广泛使用的模式生物。这两种阿米巴酶与其他生物的CHS序列相似度在23%至33%之间，并且对CHS活性至关重要的所有残基都完全保存。有趣的是，EhCHS-1和EhCHS-2在预测分子量（73 kD vs. 114 kD）和等电点（5.04 vs. 8.05）上存在很大差异，同源性仅限于含有催化结构域的约400个氨基酸残基的中心延伸。在催化结构域外，EhCHS-1预计具有7个跨膜螺旋（TMH），其中大多数位于c端部分，类似于酵母中的情况；而EhCHS-2在结构上与线虫或昆虫几丁质合成酶相关，因为它含有17个预测TMHs，其中大多数位于分子的n端部分。Northern blot分析显示，在滋养体内阿米巴原虫中不表达与CHS-1和CHS-2相对应的基因，但在葡萄糖剥夺诱导侵入棘球蚴形成囊后4至8小时存在大量的CHS-1和CHS-2 RNA。两种变形虫CHS基因的表达过程略有不同，与CHS-1 RNA相比，CHS-2 RNA的表达更为短暂，在胞制8至16小时之间没有观察到平台期。然而，48小时后，当大多数细胞转化为成熟囊肿时，这两种CHS rna不再被检测到。
A major component of the Entamoeba cyst wall is chitin, a homopolymer of beta-(1,4)-linked N-acetyl-D-glucosamine. Polymerization of chitin requires the presence of active chitin synthases (CHS), a group of enzymes belonging to the family of beta-glycosyl transferases. CHS have been described for fungi, insects, and nematodes; however, information is lacking about the structure and expression of this class of enzymes in protozoons such as Entamoeba. In this study, the primary structures of two putative E. histolytica CHS (EhCHS-1 and EhCHS-2) were determined by gene cloning and homologous proteins were identified in databases from E. dispar and the reptilian parasite E. invadens. The latter constitutes the widely used model organism for the study of Entamoeba cyst development. The two ameba enzymes revealed between 23% and 33% sequence similarity to CHS from other organisms with full conservation of all residues critically important for CHS activity. Interestingly, EhCHS-1 and EhCHS-2 differed substantially in their predicted molecular weights (73 kD vs. 114 kD) as well as in their isoelectric points (5.04 vs. 8.05), and homology was restricted to a central stretch of about 400 amino acid residues containing the catalytic domain. Outside the catalytic domain, EhCHS-1 was predicted to have seven transmembrane helices (TMH) of which the majority is located within the C-terminal part, resembling the situation found in yeast; whereas, EhCHS-2 is structurally related to nematode or insect chitin synthases, as it contained 17 predicted TMHs of which the majority is located within the N-terminal part of the molecule. Northern blot analysis revealed that genes corresponding to CHS-1 and CHS-2 are not expressed in Entamoeba, trophozoites, but substantial amounts of CHS-1 and CHS-2 RNA were present 4 to 8 hours after induction of cyst formation by glucose deprivation of E. invadens. The time-courses of expression differed slightly between the two ameba CHS genes, as in contrast to CHS-1 RNA, expression of CHS-2 RNA was more transient and no plateau was observed between 8 and 16 hours of encystation. However, both CHS RNAs were no longer detectable after 48 hours when most of the cells had been transformed into mature cysts.