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

在本研究中，为了鉴定表皮内嗜中性粒细胞IgA皮肤病型IgA天疱疮（IEN）的未知自身抗原，我们进行了一种新的免疫沉淀（IP），其中生物素化的KU-8细胞提取物用肽M琼脂糖沉淀，具有特异性结合人IgA的能力。通过IP结合质谱，我们确定了跨膜分泌成分（TSC）作为候选成分。TSC作为IEN的抗原具有吸引力，因为TSC在粘膜中传递IgA。抗TSC的多克隆抗体（pAb）在免疫荧光（IF）中与表皮反应。利用KU-8细胞mRNA，采用PCR方法分离人TSC cdna，并在COS-7细胞中制备了全长真核TSC重组蛋白（RP）。令人惊讶的是，在IP和IF研究中，全长TSC RP都被IEN IgA和正常IgA识别，这表明TSC与IgA具有普遍的结合能力。为了检测ieniga的抗原特异性结合，我们制备了几个缺乏N或c端区的TSC cDNA构建体。所有的TSC pAb、IEN IgA和正常IgA都能与不含c末端的TSC RPs发生反应，与全长TSC类似。相比之下，没有n -末端的TSC RP可以被TSC pAb识别，而不能被正常的IgA识别，这表明该RP缺乏IgA结合域。然而，IEN IgA也失去了与该RP的结合能力，这表明IEN IgA不通过抗原抗体反应性与TSC发生反应。尽管如此，这项研究应该是重要的，因为这是第一次表明TSC存在于角质形成细胞中，并表明TSC不仅在粘膜而且在皮肤中在免疫中起重要作用。因此，TSC cDNA克隆将为进一步研究TSC在表皮中的功能提供有用的工具。本研究还表明，tscc可能阻碍IgA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