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.
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