| Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2012: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2011: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Research Abstract |
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
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