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

在这项研究中，以确定未知的自身抗原的表皮内嗜酸性伊加皮肤病型伊加天疱疮（IEN），我们进行了一种新的免疫沉淀（IP），其中生物素化的KU-8细胞提取物沉淀的肽M琼脂糖，具有特异性结合能力的人伊加。通过IP结合质谱，我们确定跨膜分泌成分（TSC）作为候选。TSC是有吸引力的作为抗原的肠上皮内瘤变，因为TSC在粘膜中提供伊加。抗TSC多克隆抗体（pAb）与表皮细胞发生免疫荧光反应。本研究利用KU-8细胞的mRNA，通过PCR方法分离出人TSC cDNA，并在COS-7细胞中表达了全长真核TSC重组蛋白（RP）。令人惊讶的是，在IP和IF研究中，全长TSC RP被伊加和正常伊加识别，表明TSC具有与伊加的一般结合能力。为了检测伊加的抗原特异性结合，我们制备了几种缺乏N-或C-末端gion的TSC cDNA构建体。所有TSC pAb、IEN伊加和正常伊加均与无C末端的TSC RP反应，与全长TSC一样。而不含N端的TSCRP能被TSCpAb识别，而不能被正常伊加识别，表明该RP缺乏IgA结合结构域。然而，IEN伊加也失去了与该RP的结合能力，这表明IEN伊加不通过抗原抗体反应性与TSC反应。尽管如此，这项研究应该是重要的，因为这是第一项研究，以显示角质形成细胞中的TSC的存在，并指出TSC的重要作用，不仅在粘膜，而且在皮肤的免疫。因此，TSC的cDNA克隆应该是有用的工具，为今后的研究TSC在表皮中的功能。本研究还表明，TSC可能会妨碍伊加自身抗体抗原的检测

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