Oral Fluid Proteolytic Effects on Salivary Protein Structure and Function

描述（由申请人提供）：健康和疾病中口腔表面发生的所有生物过程都是由口腔液体环境的特征决定的。全唾液的主要贡献者是来自腮腺、下颌骨和舌下腺的外分泌。这些分泌物中的主要唾液蛋白在整个唾液中应该很容易识别。然而，令人惊讶的是，腺体分泌物的蛋白质谱与整个唾液的蛋白质谱有很大的不同。从腺分泌物中分离的唾液蛋白的结构/功能研究为其在维持口腔健康中的潜在作用提供了许多见解。然而，整个唾液和腺体分泌物之间的巨大差异，在其潜在的功能含义方面几乎没有得到解决。腺体分泌物和全唾液之间的主要区别之一是全唾液的明显的蛋白水解活性。这种活性似乎在很大程度上导致了体液中完整蛋白质的明显损失。富含脯氨酸的蛋白质（PRPs）、甾醇蛋白（statherin）和组他汀类药物（histatin）在与矿物质稳态和抗菌活性相关的特性方面已经得到了很好的表征。虽然PRPs、他汀蛋白和组他汀蛋白在功能上被认为是最相关的唾液蛋白，但它们也属于那些在释放到口腔后极易被蛋白水解的唾液蛋白。我们的假设是，在从腺体排泄管释放分泌物和这些蛋白质与口腔中存在的液体和细胞成分混合之间发生的蛋白质结构变化将具有重要的功能和生理后果。本文拟从以下方面研究唾液蛋白水解对PRP-1、statin、histatin-1和histatin-3结构和功能的影响：以全唾液的液体和细胞组分为酶源，研究唾液蛋白降解的速率和方式。降解产物将通过高效液相色谱法进行色谱分离，单个肽将通过质谱法进行鉴定和表征。2. 确定全唾液蛋白水解对与口腔健康直接相关的蛋白质功能的影响。为了实现这一目标，蛋白质降解混合物和单个片段将在测试中评估其抑制初级和次级磷酸钙沉淀的能力以及抗真菌和抗菌活性。项目简介：维持口腔健康，唾液功能正常是先决条件。目前的建议将研究唾液蛋白水解酶如何影响四种突出的和生物学上重要的腺唾液蛋白的结构和功能。

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.