Oral Fluid Proteolytic Effects on Salivary Protein Structure and Function

描述(申请人提供)：健康和疾病中发生在口腔表面的所有生物过程都由口腔液体环境的特征决定。唾液的主要来源是腮腺、颌下腺和舌下腺的外分泌。这些分泌物中的主要唾液蛋白应该很容易在整个唾液中识别出来。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显著不同。从腺体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更多的洞察力。然而，整个唾液和腺体分泌物之间的巨大差异，几乎没有从其潜在的功能影响的角度来解决。腺体分泌物和全唾液的主要区别之一是全唾液具有明显的蛋白分解活性。这种活动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)、他汀类和组抑素在矿物质动态平衡和抗菌活性方面有很好的特性。虽然PRPS、他汀类和组抑素被认为是功能上最相关的唾液蛋白之一，但它们也属于唾液蛋白，它们在释放到口腔中后极易发生蛋白分解。我们的假设是，从腺体排泄管释放分泌物到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。本研究主要从以下几个方面研究唾液蛋白降解对PRP-1、Statherin、组蛋白-1和组蛋白-3结构和功能的影响：1.以全唾液和唾液细胞为酶源，研究唾液蛋白降解的速率和方式。降解产物用反相高效液相色谱分离，单肽用质谱仪进行鉴定和表征。2.确定全唾液蛋白分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，将在评估它们抑制初级和次级磷酸钙沉淀以及抗真菌和抗菌活性的能力的分析中对蛋白质降解混合物和单个片段进行评估。项目简介：要维持口腔健康，适当的唾液功能是先决条件。目前的提案将研究唾液蛋白水解酶如何影响四种突出的、具有生物重要性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.