Functional Glycomics of Human Saliva

描述（由申请人提供）：我们建议对作为导管分泌物收集的人腮腺和下颌下/舌下唾液进行功能糖组学分析。人们对唾液蛋白质组了解很多。相比之下，唾液糖组受到的关注要少得多。这是一个重大的遗漏，因为唾液含有异常高比例的糖蛋白，并且具有非常不寻常的碳水化合物结构。这些寡糖含有大量具有复杂分支模式的糖，并用血型决定簇（例如 ABO 和 Lewis 家族）和硫酸盐取代基修饰。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聚糖在细胞粘附中发挥着许多有趣的作用。在这种情况下，我们建议测试唾液聚糖介导基本功能，从而对健康和疾病产生重大影响的假设。为了检验这一理论，我们将利用我们小组在基于质谱的碳水化合物结构分析和人类唾液的“组学型”研究中积累的专业知识。我们还开发了一套检测方法，用于研究唾液寡糖在与细菌和/或免疫细胞的粘附相互作用方面的功能。因此，我们提出两个具体目标。目标 1 的目标是对人腮腺和下颌下唾液进行糖组分析。具体来说，我们将使用几种分离策略根据聚糖结构来分离唾液成分。我们的方法包括通过固定化凝集素和抗体进行捕获，这些凝集素和抗体可识别寡糖结构的特定方面，包括连接位置和异头/异构体构型。我们最近的工作证明了这些方法在显着扩展糖组分析深度方面的实用性。然后我们将使用各种基于质谱的平台，包括新开发的离子淌度分离方法，来阐明分级聚糖的结构。我们还将对它们的肽附着位点进行测序，这将使我们能够更深入地研究唾液蛋白质组。在目标 2 中，我们将根据唾液聚糖与细菌或免疫细胞之间的粘附相互作用建立结构-功能关系，从而加深我们对特定口腔生态机制的理解。我们将采用的方法包括模拟口腔环境的剪切应力水平作为重要变量。总之，这些实验的数据将提供对构成唾液糖组的寡糖结构及其在协调细胞粘附方面的功能的全面概述。我们认为这一重要信息可能有几个有趣的临床应用。例如，聚糖谱，即所谓的“糖型”，正在成为个体患龋齿风险的有用预测因子。此外，竞争性抑制剂可用于破坏疾病相关生物体的粘附，相反，支持有益物种粘附的寡糖序列可用于改善口腔生态。 公共健康相关性：拟议工作的目标是使用基于质谱的方法来表征构成人类唾液糖组的寡糖结构，并评估其在介导细菌和/或免疫细胞粘附方面的功能。这些结构-功能相关性将为我们提供对建立健康个体口腔生态的分子机制的重要见解。总的来说，所得数据将形成衡量疾病相关变化的基线，这是这项工作的长期目标。

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. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to use mass spectrometry-based methods to characterize the oligosaccharide structures that comprise the human salivary glycome and to assess their functions in terms of mediating bacterial and/or immune cell adhesion. These structure-function correlations will provide us important insights into the molecular mechanisms that establish the oral ecology of healthy individuals. In the aggregate, the resulting data will form a baseline against which disease-related changes can be measured, the long-term goal of this work.