Glycoprofiling of the Human Salivary Proteome.
ABSTRACT 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.
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ABSTRACT: Interactions between salivary glycoproteins and many oral bacteria have been shown to depend on O-linked glycans on salivary glycoproteins. Basic proline-rich proteins form the largest group of proteins within human parotid saliva. In the present study human parotid salivary glycoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional electrophoresis, electroblotted onto nitrocellulose and probed with two biotin-labelled lectins from Maclura pomifera (MPA) and Arachis hypogaea (PNA) which are specific for O-linked (galactose beta 1,3 N-Acetylgalactosamine) glycans. Lectin binding was detected with avidin-biotin complex and enhanced chemiluminescence. Two-dimensional electrophoresis in combination with lectin binding indicated that only basic parotid salivary glycoproteins bind the lectin MPA. Following removal of terminal sialic acid residues by sialidase digestion the same glycoproteins were detected by the lectin PNA. Glycosidase digestion with endo-alpha-N-acetylgalactosaminidase (O-glycanase) in conjunction with sialidase eliminated MPA binding. Taken together these results indicate that many basic parotid salivary glycoproteins contain O-glycans, all of which are sialylated.Oral Microbiology and Immunology 11/1999; 14(5):309-15. · 2.81 Impact Factor
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ABSTRACT: The ability of wheat germ agglutinin to form precipitates with a series of synthetic carbohydrate-protein conjugates and with carcinoembryonic antigen and its Smith degradation products was investigated. The precipitation reaction between wheat germ agglutinin and p-azophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside-bovine serum albumin was selected to examine the capacity of a large number of sugar haptens to inhibit this system. Our results indicate that the wheat germ agglutinin binding site is complementary to a sequence of three beta-(1 leads to 4)-linked N-acetyl-D-glucosamine units (N,N'N"-triacetyl chitotriose). The internal carbohydrate portion of carcinoembryonic antigen probably contains two such units and wheat germ agglutinin precipitates with untreated as well as sequentially Smith degraded carcinoembryonic antigen. Compared with other reports certain discrepancies in the relative binding affinities of per N-acetylated chitodextrins and N-acetyl-D-glucosamine were found. These differences are discussed in terms of the methods used and the proposed subsite hypothesis of Allen, A.K., Neuberger, A. and Sharon, N. (1973) Biochem. J. 131, 155-162.Biochimica et Biophysica Acta 10/1975; 405(1):53-61. · 4.66 Impact Factor
Article: Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by two-dimensional gel electrophoresis.[show abstract] [hide abstract]
ABSTRACT: The secretions of the salivary parotid and submandibular-sublingual (SMSL) glands constitute the main part of whole human saliva (WS) in which proline-rich proteins (PRPs) and mucins represent dominant groups. Although proteome analysis had been performed on WS, no identification of PRPs or mucins by 2-DE and MS was achieved in WS and no comprehensive analysis of both glandular secretions is available so far. The aim of this study was to compare the protein map of WS to parotid and SMSL secretions for the display of PRPs and mucins. WS and glandular secretions were subjected to 2-DE and spots were analyzed by MALDI-MS. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic PRPs as well as the proline-rich glycoprotein (PRG) could now be mapped in WS. Acidic PRPs were found equally in parotid and SMSL secretions, whereas basic PRPs and PRG were found primarily in parotid secretion. Salivary mucin MUC7 was identified in SMSL secretion. Thus, the more abundant proteins of WS can be explained mainly by mixed contributions of parotid and SMSL secretions with only few components remaining that may be derived from local sources in the oral cavity.PROTEOMICS 04/2006; 6(5):1631-9. · 4.51 Impact Factor