S-layer, surface-accessible, and concanavalin A binding proteins of Methanosarcina acetivorans and Methanosarcina mazei.

The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often posttranslationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus, minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed nonspecifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically localized. This approach provides an alternative strategy to study surface proteins in the archaea.

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.

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.

Differentially expressed protein markers in human submandibular and sublingual secretions.

Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.