The long-term goal of this proposal is the design and enhancement of the lubricative function of statherin with a concomitant decrease of its bacterial adhesion properties. Specifically, 15N-enriched statherin will be expressed in an optimized E. coli expression system using pGEX-2T as the cloning vehicle. The solution structure of the purified 15N-enriched statherin will be determined using three-dimensional nuclear magnetic resonance spectroscopy. Fluorescence and FTIR spectroscopy will be used to test whether statherin undergoes any conformational change upon binding to hydroxyapatite. The information gained from these biophysical studies will be used with molecular modeling to deduce the three-dimensional structure of statherin at the enamel interface. Using the model at the interface, specific residues and secondary structural motifs will be selected for substitution using site-directed mutagenesis. The hypothesis is that statherin requires a highly polar N-terminal region to anchor itself at the interface and the C-terminal segment for both lubricity, as well as bacterial attachment. The lubrication property of statherin is dependent upon its ability to provide an amphipathic film upon binding to the enamel surface. This hypothesis will be tested by specifically preparing several analogs of statherin in which the hydrophobicity of the C-terminal region will be increased by individually replacing the tyrosine residues with more hydrophobic residues such as Phe, Val, Ile and Leu. All these analogs will be produced by site-directed mutagenesis. The efficacy of the analogs to enhance lubrication and their ability to decrease the attachment of Actinomyces viscosus to analog-coated hydroxyapatite will be examined. Information from the behavior of these analogs will allow the design and production of an analog with optimum lubrication but minimal bacterial adhesion properties.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Specialized Center (P50)
Project #
3P50DE008240-10S2
Application #
6104747
Study Section
Project Start
1997-12-01
Project End
1999-11-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
10
Fiscal Year
1998
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Ohkusa, Toshifumi; Yoshida, Tsutomu; Sato, Nobuhiro et al. (2009) Commensal bacteria can enter colonic epithelial cells and induce proinflammatory cytokine secretion: a possible pathogenic mechanism of ulcerative colitis. J Med Microbiol 58:535-45
Sojar, Hakimuddin T; Genco, Robert J (2005) Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae. FEMS Immunol Med Microbiol 45:25-30
Satyanarayana, J; Gururaja, T L; Narasimhamurthy, S et al. (2001) Synthesis and conformational features of human salivary mucin C-terminal derived peptide epitope carrying Thomsen-Friedenreich antigen: implications for its role in self-association. Biopolymers 58:500-10
Narasimhamurthy, S; Naganagowda, G A; Janagani, S et al. (2000) Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7). J Biomol Struct Dyn 18:145-54
Gururaja, T L; Levine, J H; Tran, D T et al. (1999) Candidacidal activity prompted by N-terminus histatin-like domain of human salivary mucin (MUC7)1. Biochim Biophys Acta 1431:107-19
Tseng, C C; Miyamoto, M; Ramalingam, K et al. (1999) The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase. Arch Oral Biol 44:119-27
Naganagowda, G A; Gururaja, T L; Satyanarayana, J et al. (1999) NMR analysis of human salivary mucin (MUC7) derived O-linked model glycopeptides: comparison of structural features and carbohydrate-peptide interactions. J Pept Res 54:290-310
Sojar, H T; Han, Y; Hamada, N et al. (1999) Role of the amino-terminal region of Porphyromonas gingivalis fimbriae in adherence to epithelial cells. Infect Immun 67:6173-6
Mettath, S; Munson, B R; Pandey, R K (1999) DNA interaction and photocleavage properties of porphyrins containing cationic substituents at the peripheral position. Bioconjug Chem 10:94-102
Satyanarayana, J; Gururaja, T L; Naganagowda, G A et al. (1998) A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1H NMR assignments and their application in solid-phase glycopeptide synthesis. J Pept Res 52:165-79

Showing the most recent 10 out of 162 publications