The acquired enamel pellicle is a thin film of protein intimately associated with the tooth surface. There is ample evidence to indicate that the formation of the enamel pellicle is a process of biological specificity in which salivary proteins and fragments of these proteins are selectively adsorbed onto the hydroxyapatite surface. This structure plays an important role in mineral/solute interactions which maintain and protect the integrity of surface enamel and also dictates the nature of initial colonization of the tooth surface by specific oral microorganisms. We have made significant progress in our studies using both an in vitro hydroxyapatite model and in the development of techniques allowing the direct investigation of pellicle formed in vivo. During the course of this work we have isolated and characterized new families of pellicle precursor proteins. In addition microtechniques have been developed which now make it possible to directly isolate and characterize minute quantities of proteins/peptides actually present in the pellicle formed in the oral cavity. A multifaceted strategy employing protein microchemistry, immunology and recombinant DNA technology has been incorporated in this proposal in order to determine the structure and function of pellicle components.
The specific aims are: 1. To identify, isolate and characterize individual proteins and peptides in the in vivo acquired enamel pellicle using three different approaches: a) direct isolation and characterization by gel permeation and reversed phase HPLC followed by automated Edman degradation, b) characterization of individual components purified by affinity chromatography employing monoclonal antibodies followed by direct sequencing, and c) determination of the nucleotide sequences of new pellicle precursor protein cDNAs isolated from parotid and submandibular gland libraries. 2. To relate the structural properties of pellicle components to their functional role on tooth surfaces by: a) Analysis of the composition of in vivo pellicles from individual subjects and from different oral sites, b) investigation of the mechanism of pellicle formation by analysis of fragments from purified pellicle precursor proteins proteolytically cleaved in solution or after adsorption to hydroxyapatite, c) determination of physical parameters of pellicle components to gain insights into pellicle formation, d) measurement of calcium phosphate precipitation inhibition to identify pellicle components which inhibit primary and secondary calcification, and e) measurement of the nucleation capacity of pellicle components to identify components which promote calcification and may be important in calculus formation.
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