ADHESIVE AND PROTEOLYTIC FUNCTION OF MDC-L

淋巴细胞对组织的持续监视对于控制和调节全身免疫应答是必要的。 然而，淋巴细胞和其他白细胞的异常运输导致炎症。 在分子水平上了解淋巴细胞渗出可能会带来炎症性疾病的新疗法。 淋巴细胞外渗和迁移到选定的微环境需要粘附分子和金属蛋白酶的协同努力。 MDCs（metalloprotease disintegrin cysteine-rich）是近年来在哺乳动物组织中发现的一类细胞表面蛋白。 该蛋白质家族的成员具有预测具有粘附和蛋白水解功能的结构域。 我们假设MDC蛋白家族成员在白细胞外渗中起作用。 为了研究这一点，首先检查淋巴细胞的MDC蛋白的表达。 我们已经分离出这个家族的一个新成员，MDC-L，它似乎是由淋巴细胞特异性表达的跨膜和分泌形式。 本课题的目的是研究MDC-L的表达和功能。 免疫组化和流式细胞术方法将被用来检查MDC-L表达淋巴细胞的表型和MDC-L形式的微环境中发现。整合素识别MDC-L去整合素域将使用组合的生化方法进行研究。 一旦确定了识别MDC-L的整联蛋白的身份，将使用定点诱变、单克隆抗体（mAb）和合成肽方法来确定该整联蛋白识别MDC-L的分子方面。 所产生的抑制整联蛋白结合的mAb和肽将用于研究MDC-L在体外淋巴细胞迁移试验中作为粘附分子的作用。 将测定金属蛋白酶结构域的底物特异性。 将评估本项目过程中产生的抗MDC-L mAb和现有金属蛋白酶抑制剂的MDC-L抑制活性。 这些蛋白酶抑制剂将用于评估MDC-L金属蛋白酶在体外淋巴细胞迁移试验中的功能。与许多基质金属蛋白酶一样，MDC-L可以作为酶原合成，其在去除前结构域后被激活。 将通过突变MDC-L构建体的重组表达和金属蛋白酶活性测定来研究金属蛋白酶的调节。 从该项目中获得的结果可能确定一类新的潜在治疗靶点，如MDC-L，用于干预淋巴细胞外渗。

Continual surveillance of tissues by lymphocytes is necessary to control and meditate systemic immune responses. However, aberrant trafficking of lymphocytes, and other leukocytes, results in inflammation. Understanding lymphocyte extravasation at the molecular level may lead to new therapies for inflammatory diseases. Lymphocyte extravasation and migration to selected microenvironments requires the concerted efforts of adhesion molecules and metalloproteases. MDCs (metalloprotease disintegrin cysteine-rich) are a family of cell surface proteins recently identified in several mammalian tissues. Members of this protein family possess domains that are predicted to have both adhesive and proteolytic functions. We hypothesize that members of the MDC protein family function in leukocyte extravasation. To investigate this, lymphocytes were initially examined for the expression of MDC proteins. We have isolated a novel member of this family, MDC-L, that appears to be specifically expressed by lymphocytes as both a transmembrane and secreted form. The aims of this project are to examine the expression and function of MDC-L. Both immunohistochemical and flow cytometric approaches will be used to examine the phenotype of MDC-L expressing lymphocytes and the microenvironments each MDC-L form are found. Integrin recognition of the MDC-L disintegrin domain will be investigated using a combination of biochemical methods. Once the identity of the integrin that recognizes MDC-L is determined, a site directed mutagenesis, monoclonal antibody (mAb), and synthetic peptide approach will be used to determine the molecular aspect of MDC-L recognition by that integrin. The mAbs and peptides produced that inhibit integrin binding will be used to investigate the role of MDC-L as an adhesion molecule in in vitro lymphocyte transmigration assays. The substrate specificity of the metalloprotease domain will be determined. Anti-MDC-L mAbs produced during the course of this project and existing inhibitors of metalloproteases will be assessed for MDC-L inhibitory activity. These protease inhibitors will be used to assess the function of the MDC-L metalloprotease in in vitro lymphocyte transmigration assays. Like many matrix metalloproteases, MDC-L may be synthesized as a zymogen that is activated upon removal of the prodomain. The regulation of the metalloprotease will be investigated by recombinant expression of mutant MDC-L constructs and assaying for metalloprotease activity. The results obtained from this project may identify a new class of potential therapeutic targets, such as MDC-L, for intervention of lymphocyte extravasation.