Hemidesmosomes, cell-matrix connector devices, are used by oral epithelial to attach to the connective tissue of the gingiva as well as the tooth surface. In the cytoplasm of an oral epithelial cell hemidesmosomes are also sites of cytoskeleton anchorage to the cell surface. During the course of periodontal disease, there is, of necessity, modulation in hemidesmosome integrity as epithelial cells migrate along the tooth to form the """"""""long junctional epithelium"""""""". Before we can study the role of hemidesmosomes in the pathogenesis of periodontal disease we must have a basic understanding of the molecular components involved in both the assembly and structural maintenance of this complex morphological entity. To this end, in Project 1 of this program project application, we plan to focus our efforts on functional analyses of an adhesive extracellular glycoprotein component of hemidesmosomes called laminin-5 and its impact on oral epithelial cells.
In Aim I we address the identification of domains of the laminin-5 heterotrimer involved in nucleation of hemidesmosome assembly. This will involve preparation of recombinant laminin-5 proteins as well as laminin-5 peptides.
In Aim 2, these recombinant proteins and peptides will be assayed for their ability a) to induce rapid adhesion of oral epithelial cells and b) to nucleate assembly of hemidesmosomes. As alternative strategies for identification of functional important domains of laminin-5, in Aim 2 we will use peptides to compete the adhesive properties of intact laminin-5.
In Aim 3, we wish to identify the substrate binding site(s) of laminin-5. To this end, proteolytic fragments of laminin-5 will be assayed for a) their ability to adhere to surfaces and/or b) to compete with native laminin-5 for substrate binding. Functionally active fragments will be further characterized by microsequencing.
In Aim 4, the impact of cell binding to laminin- 5 on hemidesmosome components will be assayed biochemically. We are particularly interested in the possibility that laminin-5 plays a role in matrix-cell signaling via phosphorylation events. Finally, in Aim 5, we wish to identify membrane interactions of laminin-5. This linkage is necessary for the stabilization of the oral epithelium on the tooth surface.
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