Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Mod Rheumatol. 2012 Sep;22(5):685-94. doi: 10.1007/s10165-011-0582-9. Epub 2012 Jan 24.
The anti-Wa antibody found in systemic sclerosis patients reacts with a transfer RNA (tRNA)-associated 48-kDa protein and immunoprecipitates several tRNAs. We investigated the Wa antigen and its binding to tRNA species.
We performed molecular cloning of the Wa antigen and made its recombinant protein. To investigate Wa antigen distribution in the cell, we performed an indirect immunofluorescence study. To determine the Wa-bound tRNA species, we performed a reverse transcription (RT)-polymerase chain reaction (PCR) using the RNAs immunoprecipitated by anti-Wa antibody as templates, and synthetic primers of mammalian tRNA sequences. To clarify the tissue expression of Wa antigen, we performed quantitative and semi-quantitative PCR of the cDNA.
We demonstrated that the Wa antigen was identical to NEFA (DNA binding/EF-hand/acidic amino acid rich region), otherwise known as nucleobindin-2. A full-length and an alternative splice variant cDNA lacking exon 11 were isolated by cloning NEFA cDNA. Anti-Wa-positive sera stained both the nucleus and cytoplasm of HEp-2 cells. RT-PCR suggested that Wa binds at least six tRNA species. In human tissues, NEFA is expressed predominantly in exocrine glands.
We have demonstrated that the Wa antigen is NEFA or nucleobindin-2, which binds specific tRNA species, and is distributed in specific human tissues.
系统性硬化症患者体内的抗 Wa 抗体与一种与转移 RNA（tRNA）相关的 48kDa 蛋白发生反应，并免疫沉淀多种 tRNA。我们研究了 Wa 抗原及其与 tRNA 种类的结合。
我们对 Wa 抗原进行了分子克隆并制备了其重组蛋白。为了研究 Wa 抗原在细胞中的分布，我们进行了间接免疫荧光研究。为了确定 Wa 结合的 tRNA 种类，我们使用抗 Wa 抗体免疫沉淀的 RNA 作为模板，使用哺乳动物 tRNA 序列的合成引物进行逆转录（RT）-聚合酶链反应（PCR）。为了阐明 Wa 抗原的组织表达，我们对 cDNA 进行了定量和半定量 PCR。
我们证明 Wa 抗原与 NEFA（DNA 结合/EF 手/酸性氨基酸丰富区）相同，也称为核结合蛋白-2。通过克隆 NEFA cDNA，分离出全长和缺少外显子 11 的替代剪接变体 cDNA。抗 Wa 阳性血清可同时染色 HEp-2 细胞的核和细胞质。RT-PCR 表明 Wa 至少结合六种 tRNA 种类。在人体组织中，NEFA 主要在外分泌腺中表达。
我们已经证明 Wa 抗原是 NEFA 或核结合蛋白-2，它结合特定的 tRNA 种类，并分布在特定的人体组织中。
