CEMENTUM DERIVED ATTACHMENT PROTEIN

牙骨质衍生的附着蛋白

• 批准号: 2131378
• 负责人: A. Sampath NARAYANAN
• 金额: $ 17.97万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2131378
• 关键词: binding proteins; cementum; complementary DNA; fibroblasts; genetic library; glycosylation; growth factor; human tissue; immunocytochemistry; immunoprecipitation; laboratory mouse; laboratory rabbit; molecular cloning; monoclonal antibody; northern blottings; osteocytes; phosphorylation; posttranslational modifications; protein biosynthesis; protein sequence; protein structure function; receptor binding; sulfation; tissue /cell culture; western blottings

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.

该应用程序的目的是克隆和表征 推定的新细胞附着蛋白已从两者中分离出来 牙骨质和人牙骨质肿瘤。这种牙骨质附着蛋白（CAP） 促进成纤维细胞的附着，但不促进上皮细胞的附着，其 活性受到肽的抑制。 CAP 与纤连蛋白结合 羟基磷灰石，但不是胶原蛋白，它可以同时使用 α1- 和 α5-整合素亚基作为其细胞表面受体。 多克隆和 CAP 单克隆抗体抑制细胞附着并证明 CAP 只存在于牙骨质和适应的人牙骨质肿瘤中 申请人及其同事的文化。 在具体目标 1 中， 已制备好的培养人牙骨质肿瘤文库将进行筛选 与现有抗体和 CAP cDNA 将被克隆、表征 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 前体被加工成 56、49、39 和 26 kDa 形式 采用西方分析代谢标记的初步研究 牙骨质肿瘤细胞。 潜在的糖基化、磷酸化和 CAP 的硫酸化位点将通过标准方法进行研究。 生物合成 标记和免疫沉淀实验，以及 Northern 分析 CAP cDNA 可用，将用于探索 牙骨质肿瘤细胞中 CAP 的表达受已知调节的生长因子的影响 其他细胞外基质分子（TGF-β、PDGF、维生素 D、PTH 和 干扰素-γ）。 CAP与细胞表面受体的结合 牙龈和牙周膜成纤维细胞和骨细胞 使用生物素结合的 CAP 和放射性碘标记进行测定 链霉亲和素。 在具体目标 3 和 4 中，将使用免疫组织化学 确定两种情况下 CAP 在口腔和其他组织中的分布 正常和病理条件。