Salivary amylase provides an excellent example of the multifunctionality exhibited by salivary proteins. The multifunctional nature of amylase includes: 1) starch hydrolysis; 2) binding to hydroxyapatite (enamel); and 3) binding to bacteria (e.g. viridans streptococci) in solution and when bound to hydroxyapatite. For salivary amylase, its binding to bacteria in solution may result in bacterial clearance (protective) while its presence in the enamel pellicle may facilitate dental plaque formation (harmful). Its binding to viridans streptococci both in solution as well as when bound to the hydroxyapatite surface is dependent upon the maintenance of its native conformation. The goals of this proposal are to elucidate the structure-function relationships of amylase in the context of its role(s) in oral physiology. Characterization of these relationships at the molecular level will improve the understanding of basic mechanisms responsible for the early colonization of streptococci in the oral cavity. The underlying hypothesis of this proposal is that the multifunctionality of this enzyme can play a significant role in dental caries development. In particular, we feel that the structural domains of salivary amylase are critical in the caries process. In this grant period, we propose to generate distinct mutants with biochemical and physiological defects targeted against each of the three functions of salivary amylase. The mutants will be generated using a facile baculovirus expression system and the biological activities of the mutants will be screened with specific assays for bacterial binding, starch hydrolysis or hydroxyapatite binding. The structure of these mutants will be determined using protein crystallography for understanding the effect of mutation on the function. These first generation mutants will provide clues regarding how augmenting or weakening of one function affects the other two. We will obtain these clues from the in vitro biological assays and structure analysis of mutants which exhibit significantly altered activity. Based upon these results, additional mutants representing second and third generations will be constructed. Such mutants will permit the design of strategies to manipulate human salivary amylase-bacterial interactions that favor the host and thus reduce the potential for caries.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE012585-04
Application #
6516504
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Program Officer
Mangan, Dennis F
Project Start
1999-04-01
Project End
2003-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
4
Fiscal Year
2002
Total Cost
$180,707
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Dentistry
Type
Schools of Dentistry
DUNS #
781265475
City
Newark
State
NJ
Country
United States
Zip Code
07101
Gyémánt, Gyöngyi; Zajácz, Agnes; Bécsi, Bálint et al. (2009) Evidence for pentagalloyl glucose binding to human salivary alpha-amylase through aromatic amino acid residues. Biochim Biophys Acta 1794:291-6
Ragunath, Chandran; Manuel, Suba G A; Venkataraman, Venkat et al. (2008) Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding. J Mol Biol 384:1232-48
Ramasubbu, N; Thomas, L M; Ragunath, C et al. (2005) Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans. J Mol Biol 349:475-86
Kaplan, Jeffrey B; Ragunath, Chandran; Velliyagounder, Kabilan et al. (2004) Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 48:2633-6
Ramasubbu, Narayanan; Ragunath, Chandran; Mishra, Prasunkumar J et al. (2004) Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity. Eur J Biochem 271:2517-29
Ramasubbu, Narayanan; Sundar, Krishnan; Ragunath, Chandran et al. (2004) Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity. Arch Biochem Biophys 421:115-24
Kaplan, Jeffrey B; Velliyagounder, Kabilan; Ragunath, Chandran et al. (2004) Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms. J Bacteriol 186:8213-20
Remenyik, Judit; Ragunath, Chandran; Ramasubbu, Narayanan et al. (2003) Introducing transglycosylation activity into human salivary alpha-amylase (HSA). Org Lett 5:4895-8
Kandra, Lili; Gyemant, Gyongyi; Remenyik, Judit et al. (2003) Subsite mapping of human salivary alpha-amylase and the mutant Y151M. FEBS Lett 544:194-8
Kaplan, Jeffrey B; Ragunath, Chandran; Ramasubbu, Narayanan et al. (2003) Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity. J Bacteriol 185:4693-8

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