Anti-p200 pemphigoid has been characterized by autoantibodies to an unidentified 200-kDa protein (p200) of the dermal؊epidermal junction. The objective of this study was to identify p200. We performed 2D gel electrophoresis of dermal extracts and immunoblotting with patients' sera, followed by MS analysis of a unique protein band. The protein band corresponded to laminin ␥1. Anti-laminin ␥1 mAb reacted with the anti-p200 immunoprecipitates by immunoblotting. Sera from 32 patients with anti-p200 pemphigoid showed 90% reactivity to the recombinant products of laminin ␥1. None of the healthy control sera reacted with laminin ␥1. By immunoblotting, reactivity of a patient's serum with p200 was competitively inhibited by adding anti-laminin ␥1 C-terminus mAb. Purified anti-p200 IgG also inhibited the reactivity of this mAb to dermal laminin ␥1. Most laminin ␥1-positive sera showed reactivity with recombinant laminin ␥1 C-terminal E8 fragment. Reactivity of patients' sera and purified IgG to dermal laminin ␥1 was higher than reactivity to blood vessel laminin ␥1 under reducing conditions. These results suggest that laminin ␥1 is the autoantigen for patients with anti-p200 pemphigoid. The autoantibodies may specifically recognize dermal laminin ␥1 with unique posttranslational modifications. The epitope is localized to the 246 C-terminal amino acids within the coiled-coil domain. The 9 C-terminal residues are known to be critically involved in laminin recognition by integrins.autoimmune disease ͉ basement membrane ͉ bullous pemphigoid ͉ proteomics

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs. basement membrane ͉ body map ͉ niche ͉ cell adhesion ͉ glycosaminoglycan