Cloning and expression of chitinases of Entamoebae
Cloning and expression of chitinases of Entamoebae
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DOI：
10.1016/s0166-6851(96)02817-4
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发表时间：
1997-04-01
影响因子：
1.5
通讯作者：
Samuelson, J
中科院分区：
文献类型：
--
作者：
delaVega, H;Specht, CA;Samuelson, J
溶组织内阿米巴（Eh）和舞帕内阿米巴（艾德）是感染数亿人的原生动物寄生虫。在结肠腔中，阿米巴形成几丁质壁的包囊，这是寄生虫的感染阶段。侵袭内阿米巴（Entamoeba invadens，Ei）是一种寄生于爬行动物的阿米巴包囊模型，在包囊过程中产生几丁质合成酶和几丁质酶。Ei囊肿的形成被几丁质酶抑制剂allosamidin阻断。在此，使用分子克隆技术鉴定编码几丁质酶的Eh、艾德和Ei的同源基因（EC 3.2.1.14）。Eh基因（Eh cht 1）预测了507个氨基酸（aa）的酶，其分别与艾德和Ei几丁质酶具有93%和74%的位置同一性。内阿米巴几丁质酶具有信号序列，随后是由多个串联排列的7-aa重复序列（Eh和艾德）或长度变化的重复序列（Ei）组成的酸性和亲水性序列。几丁质酶重复序列的氨基酸组成与Eh和艾德富丝氨酸蛋白重复序列的氨基酸组成相似。每个几丁质酶的COOH-末端具有催化结构域，其类似于马来丝虫（33%位置同一性）和烟草天蛾（29%）的催化结构域。重组内阿米巴几丁质酶被几丁质沉淀，并显示与壳寡糖底物的几丁质酶活性。与先前的生化数据一致，几丁质酶mRNA在Ei滋养体中不存在，并且在Ei成囊48 h时积累到最大水平。(C)1997年Elsevier Science B.V.
Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cyst formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant Entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h. (C) 1997 Elsevier Science B.V.