Transcriptional gene silencing reveals two distinct groups of Entamoeba histolytica Gal/GalNAc-lectin light subunits
Transcriptional gene silencing reveals two distinct groups of Entamoeba histolytica Gal/GalNAc-lectin light subunits
复制标题
DOI：
10.1128/ec.00241-07
复制
发表时间：
2007-10-01
期刊：
影响因子：
--
通讯作者：
Mirelman, David
中科院分区：
文献类型：
--
作者：
Bracha, Rivka;Nuchamowitz, Yael;Mirelman, David
溶组织内阿米巴细胞表面Gal/GalNAc-亲和凝集素是重链（170 kDa）亚基与轻链（31至35 kDa）亚基之间通过二硫键连接的异源二聚体。已经鉴定了5个具有高度同源性的轻亚基基因（Ehlgl 1至-5）。我们以前已经表明，在G3滋养体中已经在阿米巴基因（Ehap-a）中沉默的Ehlgl 1表达的沉默也抑制了Ehlgl 2和-3（RBV株）的转录。RBV滋养体中lgl 1至lgl-3亚基的完全缺失影响了它们将表面半乳糖凝集素分子帽合到尿状体区域的能力。我们现在已经发现，在RBV滋养体中，lgl 4和-5亚基（31 kDa）过表达，并且似乎补偿了异二聚体复合物中缺失的lgl 1至-3。通过用含有连接到Ehap-a基因的5'启动子区的Ehlgl 5开放阅读框的质粒转染G3滋养体来实现Ehlgl 5的转录沉默。转染的滋养体（菌株L5）在Ehlgl 5和密切相关的Ehlgl 4中沉默，而较大的lgl 1至-3亚基的表达上调。L5滋养体保留了它们对半乳糖凝集素分子加帽的能力。迄今为止，同时沉默所有Ehlgl基因的尝试失败了，可能是由于它们对Gal-凝集素功能至关重要。我们沉默属于同一家族的部分基因的能力可以作为研究其他基因家族成员的关系和功能的工具。
The Entamoeba histolytica cell surface Gal/GalNAc-inhibitable lectin is a heterodimer between a heavy (170 kDa) subunit linked via a disulfide bond to a light (31 to 35 kDa) subunit. Five light subunit genes with high homology have been identified (Ehlgl1 to -5). We have previously shown that silencing of the expression of Ehlgl1, in the G3 trophozoites which had already been silenced in the amoebapore gene (Ehap-a), also suppressed the transcription of Ehlgl2 and -3 (strain RBV). The total absence of the lgl1 to -3 subunits in the RBV trophozoites affected their ability to cap the surface Gal-lectin molecules to the uroid region. We have now found that in the RBV trophozoites, the lgl4 and -5 subunits (31 kDa) are overexpressed and appear to compensate for the missing lgl1 to -3 in the heterodimer complex. Transcriptional silencing of Ehlgl5 was achieved by transfection of G3 trophozoites with a plasmid containing the open reading frame of Ehlgl5 ligated to the 5' promoter region of the Ehap-a gene. The transfected trophozoites (strain L5) were silenced in Ehlgl5 and the closely related Ehlgl4, while the expression of the larger lgl1 to -3 subunits was upregulated. L5 trophozoites retained their ability to cap the Gal-lectin molecules. Attempts to simultaneously silence all of the Ehlgl genes have failed so far, possibly due to their crucial importance to the Gal-lectin functions. Our ability to silence part of the genes belonging to the same family can serve as a tool to study the relationships and functions of the members of other gene families.