Oral Fluid Proteolytic Effects on Salivary Protein Structure and Function

描述（由申请人提供）：在健康和疾病状态下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。全唾液的主要贡献者是来自腮腺、下颌下腺和舌下腺的外分泌物。这些分泌物中的主要唾液蛋白在整个唾液中应该是容易识别的。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。从唾液腺分泌物中分离的唾液蛋白的结构/功能研究提供了很多关于它们在维持口腔健康中的潜在作用的见解。然而，整个唾液和腺分泌物之间的巨大差异，几乎没有解决其潜在的功能影响。腺分泌物和全唾液之间的主要区别之一是全唾液的显著蛋白水解活性。这种活动似乎在很大程度上是造成这种体液中完整蛋白质明显丢失的原因。富含脯氨酸的蛋白质（PRP），statherin和histatin已被很好地表征其性质有关的矿物质稳态和抗微生物活性。虽然PRP、唾液酸甘油酶和组胺素被认为是功能上最相关的唾液蛋白，但它们也属于在释放到口腔中时对蛋白水解高度敏感的那些唾液蛋白。我们的假设是，发生在腺排泄管分泌物释放和这些蛋白质与口腔中存在的液体和细胞成分混合之间的蛋白质结构变化将具有重要的功能，因此也具有重要的生理后果。该提案通过以下方式关注唾液蛋白水解对PRP-1、成肌生成素、组胺素-1和组胺素-3的结构和功能的影响：1.以全唾液的液体组分和细胞组分作为酶源，研究唾液蛋白降解的速率和模式。将通过RPHPLC对降解产物进行色谱分离，并通过质谱法对单个肽进行鉴别和表征。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。为了实现这一点，将在试验中评价蛋白质降解混合物和单个片段，以评估其抑制一级和二级磷酸钙沉淀的能力以及抗真菌和抗细菌活性。项目叙述：为了维持口腔健康，适当的唾液功能是先决条件。目前的建议将调查唾液蛋白水解酶如何影响四个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.