Oral Fluid Proteolytic Effects on Salivary Protein Structure and Function

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DESCRIPTION (provided by applicant): All biological processes occurring on oral surfaces in health and disease are dictated by the characteristics of the oral fluid environment. The major contributors to whole saliva are exocrine secretions derived from parotid, submandibular and sublingual glands. The predominant salivary proteins in these secretions should be readily identifiable in whole saliva. Surprisingly however, the protein profiles of glandular secretions differ significantly from that of whole saliva. Structure/function studies of salivary proteins isolated from glandular secretions have provided much insight into their potential roles in the maintenance of oral health. The drastic discrepancy between whole saliva and glandular secretions, however, has hardly been addressed in terms of its potential functional implications. One of the major differences between glandular secretions and whole saliva is the pronounced proteolytic activity of whole saliva. This activity appears to be in large part responsible for the apparent loss of intact proteins from this body fluid. Proline-rich proteins (PRPs), statherin and histatins have been well characterized in terms of their properties relating to mineral homeostasis and antimicrobial activity. While PRPs, statherin and histatins have been considered to be functionally among the most relevant salivary proteins, they also belong to those salivary proteins that are highly susceptible to proteolysis upon release into the oral cavity. Our hypothesis is that structural changes in proteins which occur between the release of secretion from glandular excretory ducts and the mixing of these proteins with the liquid and cellular constituents present in the oral cavity will have important functional, and hence physiological, consequences. This proposal focuses on the impact of salivary proteolysis on the structure and function of PRP-1, statherin, histatin-1 and histatin-3 by: 1. Studying the rate and mode of salivary protein degradation using as enzyme sources both liquid and cellular fractions of whole saliva. The degradation products will be subjected to chromatographic separation by RPHPLC and the individual peptides will be identified and characterized by mass spectrometry. 2. Determining the effect of whole saliva proteolysis on protein functions that are directly related to oral health. To achieve this, protein degradation mixtures and individual fragments will be evaluated in assays assessing their capacity to inhibit primary and secondary calcium phosphate precipitation as well as antifungal and antibacterial activities. Project Narrative: To sustain oral health, proper saliva function is a prerequisite. The current proposal will investigate how salivary proteolytic enzymes affect the structure and function of four prominent and biologically important glandular salivary proteins.