Significance of the extracellular cleavage of BP180/type XVII collagen for skin biology and disease

半桥粒介导复杂的复层上皮细胞与基底膜的黏附。大疱性类天疱疮抗原180(BP180)/XVI型胶原是一种半桥粒跨膜蛋白，是锚定细丝的主要成分。对BP180的自身免疫反应和该蛋白的遗传缺陷都会导致表皮下起泡，强调其对基底膜区完整性的重要性。BP180的胞外部分120 kDa可通过蛋白水解性切割从细胞表面脱落。我们最近发现，这种切割是由膜相关的基质金属蛋白酶介导的，并定位于BP180膜近端的NC16A结构域。该位点与A6B4整合素的A6亚单位相关联，代表了BP180最具免疫原性的部分。本提案的目的是研究卵裂过程的生理和病理意义。这种切割将通过酶促消除培养角质形成细胞的胞外胶原部分来复制，并将使用BP230、a6b4整合素和层粘连蛋白5的特异性单抗来研究其对半染色体组分相互作用的影响。通过定点突变，切割位点附近的NC16A结构域将引入氨基酸变化。缺乏BP180的患者的角质形成细胞(GABEB)将被导入这些突变形式的BP180，以确定是否可以取消切割。突变的BP180还将用于研究切割与细胞迁移和分化的相关性。最后，将研究切割过程对患者自身抗体对BP180的NC16A结构域反应性的影响。这些研究将提高我们对基底膜带锚定机制的理解。

Hemidesmosomes mediate adhesion of complex and stratified epithelia to basement membranes. Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a hemidesmosomal transmembrane protein and a major component of anchoring filaments. Both an autoimmune response to BP180 and genetic defects of the protein result in subepidermal blistering emphasizing its importance for the integrity of the basement membrane zone. A 120 kDa extracellular portion of BP180 can be shed from the cell surface as a result of proteolytic cleavage. We recently found the cleavage to be mediated by a membrane-associated matrix metalloproteinase and to localize within the membraneproximal NC16A domain of BP180. This site associates with the a6 subunit of a6b4 integrin and represents the most immunogenic portion of BP180. The aim of the present proposal is to examine the physiological and pathological significance of the cleavage process. The cleavage will be reproduced by enzymatic elimination of the extracellular collagenous portion in cultured keratinocytes and its effect on the interaction between hemidesmosomal components will be studied using specific monoclonal antibodies to BP230, a6b4 integrin, and laminin 5. By sitedirectedmutagenesis, amino acid changes will be introduced within the NC16A domain near the cleavage site. Keratinocytes from patients lacking BP180 (GABEB) will be transfected with these mutant forms of BP180 to determine if the cleavage can be abolished. The mutant BP180 will also be used to study the relevance of the cleavage for cell migration and differentiation. Finally, the effect of the cleavage process on the reactivity of patients' autoantibodies to the NC16A domain of BP180 will be investigated. These studies should improve our understanding of anchoring mechanisms of basement membrane zones.