Functional Glycomics of Human Saliva

描述(由申请人提供)：我们建议对收集作为导管分泌物的人腮腺和颌下/舌下唾液进行功能糖组分析。人们对唾液蛋白质组有很多了解。相比之下，唾液中的甘露糖受到的关注要少得多。这是一个重大的遗漏，因为唾液中含有非常高比例的糖蛋白，具有非常不寻常的碳水化合物结构。这些低聚糖具有大量具有复杂分支图案的糖，这些糖装饰着血型决定因素(例如，ABO和Lewis家族)和硫酸盐取代基。我们认为，这种复杂性编码了大量的生物信息。例如，我们之前的研究表明，这些多糖在细胞黏附中扮演着许多有趣的角色。在此背景下，我们建议检验唾液多糖调节基本功能的假设，从而对健康和疾病产生重大影响。为了验证这一理论，我们将利用我们团队在基于质谱学的碳水化合物结构分析和人类唾液的“组型”研究中积累的专业知识。我们还开发了一套分析方法，用于研究唾液低聚糖与细菌和/或免疫细胞的粘附性相互作用的功能。因此，我们提出了两个具体目标。目标1的目标是对人类腮腺和下颌下唾液进行血糖分析。具体地说，我们将使用几种分离策略来根据唾液成分的多糖结构来分离它们。我们的方法包括通过固定化凝集素和抗体捕获，识别寡糖结构的特定方面，包括连接位置和异构体/异构体配置。我们最近的工作证明了这些方法对于显著扩展血糖分析的深度的实用性。然后，我们将使用各种基于质谱学的平台，包括新开发的离子迁移率分离方法，来阐明分级多糖的结构。我们还将对它们的肽结合部位进行测序，这将使我们能够更深入地研究唾液蛋白质组。在目标2中，我们将通过唾液多糖与细菌或免疫细胞之间的粘附性相互作用来建立结构-功能关系，从而增加我们对口腔生态学机制的理解。我们将采用的方法包括，作为重要变量，模拟口腔环境的剪应力水平。总而言之，这些实验的数据将提供组成唾液糖的寡糖结构的全面概述，以及它们在协调细胞黏附方面的功能。我们认为，这一重要信息可能会有几个有趣的临床应用。例如，糖链图谱，也就是所谓的“糖型”，正在成为个体患龋齿风险的有用预测指标。此外，竞争性抑制剂可用于破坏与疾病相关的生物体的黏附，相反，支持有益物种黏附的寡糖序列可用于改善口腔生态。

DESCRIPTION (provided by applicant): We propose a functional glycomics analysis of human parotid and submandibular/sublingual salivas collected as the ductal secretions. A great deal is known about the salivary proteome. In contrast, the salivary glycome has received considerably less attention. This is a significant omission because saliva contains an unusually high proportion of glycoproteins with very unusual carbohydrate structures. These oligosaccharides have large numbers of sugars with complex branching patterns that are decorated with blood group determinants (e.g., ABO and Lewis families) and sulfate substituents. We think that this complexity encodes a great deal of biological information. For example, our previous studies suggest that these glycans play many interesting roles in cell adhesion. In this context, we propose testing the hypothesis that salivary glycans mediate essential functions, thereby exerting major influences on health and disease. To test this theory, we will use the accumulated expertise of our group in mass spectrometry-based carbohydrate structural analyses and in "omic-type" studies of human saliva. We have also developed a suite of assays for studying the functions of salivary oligosaccharides in terms of their adhesive interactions with bacteria and/or immune cells. Accordingly, we propose two specific aims. The goal of Aim 1 is a glycomic analysis of human parotid and submandibular salivas. Specifically, we will use several separation strategies to fractionate salivary components based on their glycan structures. Our approach includes capture by immobilized lectins and antibodies, which recognize specific aspects of oligosaccharide structure including linkage positions and anomeric/isomeric configurations. Our recent work demonstrates the utility of these approaches for significantly expanding the depth of glycomic analyses. Then we will use a variety of mass spectrometry-based platforms, including newly developed ion mobility separation approaches, to elucidate the structures of the fractionated glycans. We will also sequence their peptide attachment sites, which will allow us to delve deeper into the salivary proteome. In Aim 2, we will establish structure-function relationships in terms of adhesive interactions between salivary glycans and bacteria or immune cells, thereby increasing our understanding of the mechanisms that specify the oral ecology. The approaches we will employ include, as important variables, levels of shear stress that mimic the environment in the oral cavity. In summary, the data from these experiments will provide a comprehensive overview of the oligosaccharide structures that comprise the salivary glycome and their functions in terms of coordinating cell adhesion. We think that this important information could have several interesting clinical applications. For example, glycan profiles, so-called "glycotypes," are emerging as useful predictors of an individual's risk of developing caries. Additionally, competitive inhibitors could be used to disrupt the adhesion of disease-related organisms and, conversely, oligosaccharide sequences that support adhesion of beneficial species could be used to improve the oral ecology.