Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii
Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii
复制标题
DOI：
10.3347/kjp.2008.46.4.209
复制
发表时间：
2008-12-01
影响因子：
--
通讯作者：
Nam, Ho-Woo
中科院分区：
文献类型：
--
作者：
Kim, Ji Yeon;Ahn, Hye-Jin;Nam, Ho-Woo
Tg556克隆（Tg556）的抗弓形虫单克隆抗体印迹了一个29 kDa的蛋白质，该蛋白质位于速殖子的致密颗粒中，感染宿主细胞后分泌到寄生液泡膜（PVM）中。用Tg556筛选刚地弓形虫cDNA表达文库，得到编码该蛋白的cDNA片段，全长由2086bp的5'-RACE完成，其中包含669bp的开放阅读框(ORF)。 ORF编码222个氨基酸的多肽，与修改后的GRA3同源，但与第一个报道的不同。该多肽具有 N 端终止转移序列的 3 个疏水部分和序列后半部分的 2 个跨膜结构域 (TMD)、第二个 TMD 后的细胞质定位基序和 C 端末端的内质网 (ER) 恢复基序，这表明 GRA3 是 III 型跨膜蛋白。利用GRA3的ORF，在HeLa cDNA表达文库中进行酵母双杂交分析，结果发现GRA3与ER的II型跨膜蛋白钙调节配体（CAMLG）相互作用。 GRA3 和 CAMLG 的特异性结合通过谷胱甘肽 S-转移酶 (GST) 下拉和免疫沉淀测定得到证实。 GRA3和CAMLG质粒转染表达的荧光位置在HeLa细胞胞质中完全重叠。免疫荧光检测显示，GRA3 和 CAMLG 共定位于宿主细胞的 PVM 中。 PVM 插入的 GRA3 与 ER CAMLG 的结构结合表明，在弓形虫寄生期间，ER 的受体配体被募集到 PVM。
A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.