CEMENTUM DERIVED ATTACHMENT PROTEIN

本应用程序目标是克隆和表征一种 从这两种物质中分离出的可能的新细胞附着蛋白 牙骨质和人牙骨质肿瘤。这种牙骨质附着蛋白(CAP) 促进成纤维细胞而不是上皮细胞的附着，其 活性受到多肽的抑制。CaP与纤维连接蛋白结合， 羟基磷灰石，但不是胶原，它可能同时使用α1和 α5-整合素亚基作为其细胞表面受体。多克隆和 抗CAP的单抗可抑制细胞附着，并证明 CAP仅在牙骨质和人牙骨质肿瘤中发现 申请者和他的同事进行培养。在具体目标1中，a 已准备好的培养人牙骨质肿瘤文库将被筛选 利用现有的抗体和CAP的cDNA将被克隆，鉴定 并在体外表达。在具体目标2中，CAP的生物合成将 被研究。申请人发现CAP被合成为43 kDa 根据以下条件加工成56、49、39和26 kDa形式的前体 使用新陈代谢标记的蛋白质分析的初步研究 牙骨质肿瘤细胞。潜在的糖基化、磷酸化和 CAP的硫化部位将用标准方法进行研究。生物合成 标记和免疫沉淀实验，以及当 CAP cDNAs的问世，将用于探索CAP的调控 生长因子调控牙骨质瘤细胞中CAP的表达 其他细胞外基质分子(转化生长因子-β、血小板衍生生长因子、维生素D、甲状旁腺素和 干扰素-伽马)。CAP与细胞表面受体的结合 牙龈和牙周韧带成纤维细胞和骨细胞 用生物素标记CAP和放射性碘标记的方法测定 链霉亲和素。在特定的目标3和4中，将使用免疫组织化学 测定CAP在口腔和其他组织中的分布 正常和病理状态。

The objective of this application is to clone and characterize a putative new cell attachment protein that has been isolated from both cementum and human cementum tumor.This cementum attachment protein (CAP) promotes the attachment of fibroblasts but not epithelial cells, and its activity is inhibited by peptides. CAP binds to fibronectin and hydroxyapatite, but not collagen and it may use both the alpha 1- and alpha 5-integrin subunits as its cell surface receptor. Polyclonal and monoclonal antibodies to CAP inhibit cell attachment and demonstrate that CAP is found exclusively in cementum and in a human cementum tumor adapted to culture by the applicant and his colleagues. In Specific Aim 1, a cultured human cementum tumor library already prepared will be screened with existing antibodies and the CAP cDNA will be cloned, characterized and expressed in vitro. In Specific Aim 2, the biosynthesis of CAP will be studied. The applicant has found that CAP is synthesized as a 43 kDa precursor that is processed to 56, 49, 39,and 26 kDa forms based on preliminary studies that employ Western analysis of metabolically labeled cementum tumor cells. Potential glycosylation, phosphorylation, and sulfation sites of CAP will be studied by standard methods. Biosynthetic labeling and immunoprecipitation experiments, and Northern analysis when the CAP cDNA become available, will be used to explore the regulation of CAP expression in cementum tumor cells by growth factors known to regulate other extracellular matrix molecules (TGF-beta, PDGF, vitamin D, PTH and interferon-gamma). The binding of CAP to cell surface receptors in gingival and periodontal ligament fibroblasts and bone cells will be assayed by the use of biotin-conjugated CAP and radioiodine-labeled streptavidin. In Specific Aims 3 and 4, immunohistochemistry will be used to determine the distribution of CAP in oral and other tissues under both normal and pathological conditions.