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Analysis of the autoantigen for IEN type of IgA pemphigus and sublamina densa type of liner IgA Dermatosis

IEN型IgA天疱疮和密板下型线状IgA皮肤病自身抗原分析

基本信息

批准号：
23791299
负责人：
TUCHISAKA Atunari
金额：
$ 2.66万
依托单位：
Kurume University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23791299/
关键词：
皮膚診断学皮膚正科学免疫沈降皮膚生化学分子生物学

项目摘要

In this study, to identify unknown autoantigen for intraepidermal neutrophilic IgA dermatosis type IgA pemphigus (IEN), we performed a new immunoprecipitation (IP), in which biotinylated KU-8 cell extract was precipitated by peptide M agarose, that has specific binding ability to human IgA. By the IP combined with mass spectrometry, we identified transmembrane secretory component (TSC) as a candidate. TSC was attractive as an antigen for IEN, because TSC delivers IgA in the mucosae. Polyclonal antibody (pAb) to TSC reacted with the epidermis in immunofluorescence (IF). We isolated human TSC cDNAs by PCR using KU-8 cell mRNA, and produced full-length eukaryotic TSC recombinant protein(RP) in COS-7 cells. Surprisingly, the full length TSC RP was recognized by both IEN IgA and normal IgA in both IP and IF studies, indicating that TSC has general binding ability to IgA. To detect antigen-specific binding by IEN IgA, we prepared several TSC cDNA constructs lacking N- or C-terminal gion. All TSC pAb, IEN IgA and normal IgA reacted with TSC RPs without C-terminus, like full length TSC. In contrast, TSC RP withoutN-terminus was recognized by TSC pAb but not by normal IgA, indicating that this RP lacks IgA-binding domain. However, IEN IgA also lost binding ability to this RP, suggestingthat IEN IgA did not react with TSC via antigen-antibody reactivity. Nevertheless, this study should be important, because this is the first study to show the presence of TSC in keratinocytes and to indicate the important role of TSC in immunity not only in the mucosae but also in the skin. Therefore, the TSC cDNA clones should be useful tools for the future studies of TSC functions in the epidermis. This study also indicates that TSCmay hamper detection of antigens for IgA autoantibodies

本研究采用一种新的免疫沉淀法，将生物素化的KU-8细胞提取物经M-琼脂糖沉淀，获得与人IgA具有特异性结合能力的免疫沉淀物，以鉴定表皮内中性粒细胞病型IgA天疱疮的未知自身抗原。通过IP结合质谱分析，我们确定了跨膜分泌成分(TSC)为候选成分。TSC作为一种抗原对Ien很有吸引力，因为TSC在粘膜中递送IgA。抗TSC的多克隆抗体(Pab)与表皮呈免疫荧光反应。本研究以KU-8细胞为模板，采用聚合酶链式反应技术分离人TSC基因，并在COS-7细胞中获得全长真核重组蛋白(RP)。令人惊讶的是，在IP和IF研究中，全长TSC RP都能被Ien IgA和正常IgA识别，表明TSC具有与IgA的一般结合能力。为了检测Ien-IgA与抗原的特异性结合，我们制备了几种缺失N-端或C-端区的TSC基因。所有TSC抗体、Ien-IgA和正常IgA均与无C-末端的TSC RPS反应，如全长TSC。相反，无N末端的TSC RP可被TSC Pab识别，但不能被正常的IgA识别，表明该RP缺乏IgA结合域。然而，Ien IgA也失去了与这个RP的结合能力，这表明Ien IgA不是通过抗原-抗体反应与TSC反应的。然而，这项研究应该是重要的，因为这是第一个显示角质形成细胞中存在TSC的研究，并表明TSC在免疫中的重要作用，不仅在粘膜中，而且在皮肤中。因此，TSC基因克隆有望成为进一步研究TSC在表皮中功能的有用工具。这项研究还表明，TSC可能会阻碍对IgA自身抗体抗原的检测