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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE021041-04
Application #
8628831
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Burgoon, Penny W
Project Start
2011-04-01
Project End
2016-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
4
Fiscal Year
2014
Total Cost
$386,250
Indirect Cost
$136,250
Name
University of California San Francisco
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143