Paul C. Denny

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.

Comparison of Whole Saliva Flow Rates and Mucin Concentrations in Healthy Caucasian Young and Aged Adults

Unstimulated and chewing-stimulated whole saliva samples were obtained from 42 healthy Caucasians; 21 were between 18 and 35 years of age, and 21 between 65 and 83 years of age. The unstimulated salivary flow rate was significantly lower in the aged group, but the stimulated flow rate was significantly higher in the aged than in the young group. Both groups showed significantly increased flow during salivary stimulation. MG1 and MG2 concentrations in unstimulated and stimulated saliva samples were significantly lower in the aged group. There were no significant correlations between salivary flow rates and MG1 and MG2 concentrations.

Age-related Changes in Mucins from Human Whole Saliva

The predominant mucins in human whole saliva, MG1 and MG2, serve to protect and to lubricate the oral cavity. In this study, both unstimulated and stimulated whole salivas were collected from two groups of subjects: young (18-35 years of age) and aged (65-83 years of age). The subjects were in apparent good health. Saliva samples from each subject were analyzed by SDS-PAGE. The gels were stained with Stains-all, and both MG1 and MG2 were quantitated by video-image densitometry. The protocol gave reproducible values for each mucin. The stimulated and unstimulated salivas from aged subjects showed significant reductions in concentrations of both MG1 and MG2, as quantitated in mucin dye-binding units. Possible associations of these reductions with the aging process are discussed.

Dynamics of parenchymal cell division, differentiation, and apoptosis in the young adult female mouse submandibular gland

The submandibular salivary gland of the young adult female mouse has two secretory cell types, acinar and granular duct, which are separated by intercalated ducts. Based on the occurrence of autologous cell division in these cells, they have been traditionally classified as expanding populations. However, differentiation from stem or progenitor cells in the intercalated ducts, usually associated with renewing populations, has also been detected.

Determination of sialic acid using 2-thiobarbituric acid in the absence of hazardous sodium arsenite

Sialic acid (SA) is a component of many glycoproteins and glycolipids. Unusual levels of SA have been observed in a variety of cancerous tissues. Recent evidence indicates that serum SA can be an indicator of tumour burden and prognosis [l-4]. The commonly used thiobarbituric acid (TBA) calorimetric [5,6] and fluorometric [7] assays for SA employ sodium arsenite as a reducing agent. The United States Occupational Safety and Health Administration [8] and the State of California Occupational Safety and Health Act [9] have classified all inorganic arsenic salts as carcinogens, and the latter has established rigid standards for their use. This ban on the use of sodium arsenite without special facilities had led us to find an alternative reducing agent for the TBA assay of SA.

Glycoprofiling of the Human Salivary Proteome

IntroductionGlycosylation is an important component for a number of biological processes and is perhaps the most abundant and complicated of the known post-translational modifications found on proteins.MethodsThis work combines two-dimensional (2-D) polyacrylamide gel electrophoresis 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).ResultsEach 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 salivary proteins are glycosylated and that the profiles are more widespread than is demonstrated by the glycoprotein-stained gel. Finally, the coreactivity between lectins was measured to determine what types of glycan structures are associated with one another and also the population variation of the lectin reactivity for 66 individuals were reported.ConclusionsThis starting 2-D gel glycosylation reference map shows that the scientifically accepted, individual oligosaccharide variability is not limited to a few large glycoproteins such as MUC5B, but are found on most members of the salivary proteome. Finally, in order to see the full range of oligosaccharide distribution, multiple reagents or lectins are needed.

Purification and biochemical characterization of a mouse submandibular sialomucin

A sialomucin from the mouse submandibular gland was isolated and purified by a protocol involving Sephacryl S-200 chromatography, acidic dialysis, and preparative, poly(acrylamide)-gel electrophoresis. The mucus glycoprotein was judged to be free from contaminants by analytical and sodium dodecyl sulfate-poly(acrylamide)-gel electrophoresis, isoelectric focusing, immunoelectrophoresis, and immunodiffusion when made visible by Stains-all, periodic acid-Schiff reagent, and Coomassie Blue. The carbohydrate portion constituted 81% of the weight of the mucus glycoprotein, and was composed of 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, sialic acid, D-galactose, and D-mannose. Neither L-fucose nor sulfate was detected. The aliphatic amino acids constituted 60% of the protein core. The sialomucin has an apparent mol. wt. of 140,000 by sodium dodecyl sulfate-gel electrophoresis, and a pI of 2.77-3.63 by isoelectric focusing.

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.

Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

SummaryIn this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.

Mouse submandibular gland mucin: embryo-specific mRNA and protein species

Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein.