Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms:: gene transcription pattern, ligand binding properties and structural characterisation
Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms:: gene transcription pattern, ligand binding properties and structural characterisation
复制标题
DOI：
10.1016/s0166-6851(02)00253-0
复制
发表时间：
2003-01-01
影响因子：
1.5
通讯作者：
Hotez, PJ
中科院分区：
文献类型：
--
作者：
Basavaraju, S;Zhan, B;Hotez, PJ
针对成年犬钩虫排泄分泌（ES）产物的抗体用于免疫筛选cDNA表达文库，导致分离编码推定钩虫脂肪酸和视黄醇结合蛋白的cDNA。Ac-far-1和Ac-far-2 cDNA编码开放阅读框，对应于具有91%氨基酸同一性的相似的20 kDa蛋白。Ac-FAR-1和Ac-FAR-2与寄生线虫的其他法尔斯表现出明显的相似性，最接近秀丽隐杆线虫的两种FAR蛋白（Ce-FAR-1和Ce-FAR-2）。通过RT-PCR检测，Ac-far-1基因在A.犬的然而，通过免疫印迹仅在成虫钩虫ES产品和成虫提取物中检测到相应的蛋白质。使用基于荧光的结合测定，发现细菌重组Ac-FAR-1结合脂肪酸和视黄醇（维生素A），解离常数在微摩尔区域。圆二色光谱表明Ac-FAR-1具有高水平的α-螺旋，类似于盘尾丝虫Ov-FAR-1。这是第一次演示的功能性FAR分泌的成年钩虫，并提供了进一步的证据表明，FAR蛋白分泌的寄生线虫是至关重要的寄生。(C)2002 Elsevier Science B. V.保留所有权利。
Antibody against adult Ancylostoma caninum excretory-secretory (ES) products was used to immunoscreen a cDNA expression library leading to the isolation of cDNAs encoding putative hookworm fatty-acid and retinol-binding proteins. Ac-far-1 and Ac-far-2 cDNAs encode open reading frames corresponding to similar to20 kDa proteins with 91 percent amino acid identity. Ac-FAR-1 and Ac-FAR-2 exhibit clear similarities to other FARs of parasitic nematodes, most closely to two of the FAR proteins of Caenorhabditis elegans (Ce-FAR-1 and Ce-FAR-2). By reverse transcriptase polymerase chain reaction (RT-PCR) assay, Ac-far-1 mRNA was detected in both adult and third-stage larvae of A. caninum. However, the respective proteins were detectable by immunoblot only in adult hookworm ES products and adult extracts. Using fluorescence-based binding assays, bacterial recombinant Ac-FAR-1 was found to bind fatty acids and retinol (Vitamin A) with dissociation constants in the micromolar region. Circular dichroism spectra indicated that Ac-FAR-1 possesses a high level of alpha-helix, similar to Ov-FAR-1 from Onchocerca volvulus. This is the first demonstration of a functional FAR secreted by adult hookworms and provides further evidence that FAR proteins secreted by parasitic nematodes are crucial to parasitism. (C) 2002 Elsevier Science B.V. All rights reserved.