Functional Glycomics of Human Saliva

描述（由申请人提供）：我们提出了一个功能性糖组学分析的人腮腺和下颌下/舌下唾液收集的导管分泌物。人们对唾液蛋白质组有很多了解。相比之下，唾液糖组受到的关注要少得多。这是一个重要的遗漏，因为唾液中含有非常高比例的糖蛋白，这些糖蛋白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，这些糖被血型决定簇修饰（例如，ABO和刘易斯家族）和硫酸酯取代基。我们认为这种复杂性编码了大量的生物信息。例如，我们以前的研究表明，这些聚糖在细胞粘附中起着许多有趣的作用。在这种情况下，我们建议测试的假设，唾液聚糖介导的基本功能，从而发挥重大影响的健康和疾病。为了验证这一理论，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“组型”研究方面积累的专业知识。我们还开发了一套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的测定。因此，我们提出两个具体目标。目的1的目标是人腮腺和下颌下唾液的糖组学分析。具体而言，我们将使用几种分离策略，以基于其聚糖结构来分离唾液组分。我们的方法包括捕获固定的凝集素和抗体，识别寡糖结构的具体方面，包括连接位置和异头/异构体配置。我们最近的工作表明，这些方法的效用显着扩大糖组学分析的深度。然后，我们将使用各种基于质谱的平台，包括新开发的离子迁移率分离方法，以阐明分级聚糖的结构。我们还将测序它们的肽附着位点，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，从而增加我们对指定口腔生态学的机制的理解。我们将采用的方法包括，作为重要的变量，水平的剪切应力，模仿在口腔中的环境。总之，来自这些实验的数据将提供组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，所谓的“糖型”，正在成为个体患龋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。

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.