Sarić M, Vahrmann A, Bruchhaus I, Bakker-Grunwald T, Scholze H
Department of Biology/Chemistry, University of Osnabrueck, 49069, Osnabrueck, Germany,
Parasitol Res. 2006 Dec;100(1):171-4. doi: 10.1007/s00436-006-0206-z. Epub 2006 Jun 27.
The genome of Entamoeba histolytica contains two genes encoding inhibitors of cysteine proteases of the chagasin family. In contrast to that of EhICP1, the derived primary structure of the second inhibitor, EhICP2, possesses a typical N-terminal signal sequence. Processed EhICP2 is as weakly related to amoebiasin-1 (27% identity) as to chagasin (identity 30%), indicating a different evolutionary origin of both amebic genes. By Northern blots, we confirmed the expression of the ehicp2 gene, and in Western blots, the presence of the 11.5-kDa protein in trophozoite extracts was demonstrated. The inhibitor localized to large intracellular structures clearly differs from those containing EhICP1 as shown by indirect immunofluorescence. Recombinant EhICP2 significantly inhibited the cysteine protease activity of the amebic cell extract but with a lower extent than EhICP1. An overlay assay using a crude trophozoite extract demonstrated binding affinity of the amebic cysteine protease EhCP1 to EhICP2.
溶组织内阿米巴的基因组包含两个编码查加辛家族半胱氨酸蛋白酶抑制剂的基因。与EhICP1不同，第二个抑制剂EhICP2的推导一级结构具有典型的N端信号序列。加工后的EhICP2与阿米巴辛-1的相关性较弱（同一性为27%），与查加辛的相关性也较弱（同一性为30%），这表明这两个阿米巴基因的进化起源不同。通过Northern印迹法，我们证实了ehicp2基因的表达，通过Western印迹法，证明了滋养体提取物中存在11.5 kDa的蛋白质。间接免疫荧光显示，定位于大型细胞内结构的抑制剂与含有EhICP1的结构明显不同。重组EhICP2显著抑制了阿米巴细胞提取物的半胱氨酸蛋白酶活性，但抑制程度低于EhICP1。使用粗制滋养体提取物的覆盖分析表明，阿米巴半胱氨酸蛋白酶EhCP1与EhICP2具有结合亲和力。
