Katz Uriel, Ankri Serge, Stolarsky Tamara, Nuchamowitz Yael, Mirelman David
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Mol Biol Cell. 2002 Dec;13(12):4256-65. doi: 10.1091/mbc.e02-06-0344.
The 260-kDa heterodimeric Gal/GalNAc-specific Lectin (Gal-lectin) of Entamoeba histolytica dissociates under reducing conditions into a heavy (hgl, 170 kDa) and a light subunit (lgl, 35 kDa). We have previously shown that inhibition of expression of the 35-kDa subunit by antisense RNA causes a decrease in virulence. To further understand the role of the light subunit of the Gal-lectin in pathogenesis, amoebae were transfected with plasmids encoding intact, mutated, and truncated forms of the light subunit lgl1 gene. A transfectant in which the 55 N-terminal amino acids of the lgl were removed, overproduced an N-truncated lgl protein (32 kDa), which replaced most of the native 35-kDa lgl in the formation of the Gal-lectin heterodimeric complex and exerted a dominant negative effect. Amoebae transfected with this construct showed a significant decrease in their ability to adhere to and kill mammalian cells as well as in their capacity to form rosettes with and to phagocytose erythrocytes. In addition, immunofluorescence confocal microscopy of this transfectant with anti-Gal-lectin antibodies showed an impaired ability to cap. These results indicate that the light subunit has a role in enabling the clustering of Gal-lectin complexes and that its N-truncation affects this function, which is required for virulence.
溶组织内阿米巴的260 kDa异二聚体Gal/GalNAc特异性凝集素（Gal凝集素）在还原条件下解离为一个重亚基（hgl，170 kDa）和一个轻亚基（lgl，35 kDa）。我们之前已经表明，反义RNA抑制35 kDa亚基的表达会导致毒力下降。为了进一步了解Gal凝集素轻亚基在发病机制中的作用，用编码轻亚基lgl1基因的完整、突变和截短形式的质粒转染阿米巴。一种去除了lgl 55个N端氨基酸的转染体过量产生了一种N端截短的lgl蛋白（32 kDa），该蛋白在Gal凝集素异二聚体复合物的形成中取代了大部分天然的35 kDa lgl，并发挥了显性负效应。用该构建体转染的阿米巴在黏附、杀死哺乳动物细胞的能力以及与红细胞形成玫瑰花结和吞噬红细胞的能力方面均显著下降。此外，用抗Gal凝集素抗体对该转染体进行免疫荧光共聚焦显微镜观察显示帽化能力受损。这些结果表明轻亚基在使Gal凝集素复合物聚集方面发挥作用，其N端截短会影响这种功能，而这种功能是毒力所必需的。
