Glycoprofiling of the Human Salivary Proteome.

Glycosylation is important for a number of biological processes and is perhaps the most abundant and complicated of the known post-translational modifications found on proteins. This work combines two-dimensional polyacrylamide gel electrophoresis (2-DE) and lectin blotting to map the salivary glycome, and mass spectrometry to identity the proteins that are associated with the glycome map. A panel of 15 lectins that recognize six sugar-specific categories was used to visualize the type and extent of glycosylation in saliva from two healthy male individuals. Lectin blots were compared to 2-D gels stained either with Sypro Ruby (protein stain) or Pro-Q Emerald 488 (glycoprotein stain). Each lectin shows a distinct pattern, even those belonging to the same sugar-specific category. In addition, the glycosylation profiles generated from the lectin blots show that most of the salivary proteins are glycosylated and that the pattern is more widespread than is demonstrated by the glycoprotein stained gel. Finally, the co-reactivity between two lectins was measured to determine the glycan structures that are most and least often associated with one another along with the population variation of the lectin reactivity for 66 individuals.

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

A novel caries risk test.

A diagnostic test is particularly beneficial if it reveals the level of susceptibility prior to onset of a disease process. In the case of childhood caries, such a diagnostic test affords the opportunity for preventive measures to be implemented before caries begins. Salivary glycoproteins contain a wealth of individually specific oligosaccharide motifs. Depending on microbial compatibilities and individual genotypes, the glycoproteins that form the pellicle coating of teeth may provide attachment sites that foster colonization leading to cariogenesis. Alternatively, certain oligosaccharides, when present in nonpellicle glycoproteins, can interact with planktonic bacteria and lower their ability to interact with the tooth surface. We have found that in young adults the ratio of the two classes of oligosaccharides present in resting saliva exhibits a strong correlation with caries history (DFT: number of decayed and filled teeth). Oligosaccharide moieties associated with the test are quantitated in dried spots of whole saliva on nitrocellulose using commercially available biotinylated lectins with a variety of reporters. A combination of multiple linear regression and neural net analyses were used to develop the algorithms that describe the relationship between oligosaccharide patterns and DFT. During test development several different groups of adults and children have been studied. The correlation algorithms routinely exceed an R(2) (coefficient of determination) of 0.96. When the test is applied to the saliva of children, it yields a projection of their future caries history. Modifying the test result metric to reflect the groups of teeth with caries in young adults, the test identifies those teeth at risk for future caries in children. This test outcome can then be accompanied with suggested specific preventive measures for each tooth group-based risk level.

A novel saliva test for caries risk assessment.

A new saliva test for caries risk assessment introduced in this study integrates a variety of host factors to predict for children, individual risk levels that are tooth-group specific. These various host factors correlate with caries history, DFT (decayed and filled teeth) or DFS (decayed and filled surfaces) in young adults. The test is based on the pattern of genetically determined oligosaccharides present on salivary glycoproteins. The mechanism behind the test is believed to be centered on the specific oligosaccharides that either facilitate bacterial attachment and colonization at the surface of teeth or protect against colonization by promoting agglutination and removal of free bacteria. It is the ratio of the two classes of oligosaccharides that is very strongly correlated with the numerical range of DFS or DFT observed in a young adult population.