This R29 application is designed to investigate the correlation between proteolytic processing of the enamel extracellular matrix protein amelogenin and growth of apatite crystals, using an in vitro system. The applicant identified a serine protease, designated ameloprotease-I, as a cleavage product of one of the larger non-amelogenin proteins. This protease was shown by the applicant to degrade amelogenin proteins in vitro. The hypotheses to be examined are: that the activity of ameloprotease-I correlates with formation and maturation of enamel apatite crystals; that ameloprotease-I is activated in the extracellular matrix through proteolytic cleavage of its precursors; and that adsorption and dissociation of both enzyme and substrate onto apatite crystals are involved in proteolytic processing.
Four specific aims are proposed: (I) To examine autocatalytic action of the precursor 89 kDa enamelin in release of the 32 kDA protease in the absence and presence of apatite crystals; (II) To investigate effects of calcium, phosphate, carbonate and fluoride on activity of ameloprotease-I; (III) To determine cleavage sites within amelogenin after proteolysis +/-apatite crystals, and to monitor adsorption of enzyme and substrate to the apatite; and (IV) To investigate cleavage of amelogenin by other serine proteases +/- apatite crystals, to determine site of enzyme accessibility on the protein surface.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29DE012350-04
Application #
6379818
Study Section
Special Emphasis Panel (ZRG4-OBM-2 (02))
Program Officer
Small, Rochelle K
Project Start
1998-04-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
4
Fiscal Year
2001
Total Cost
$114,450
Indirect Cost
Name
University of Southern California
Department
Dentistry
Type
Schools of Dentistry
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Bouropoulos, N; Moradian-Oldak, J (2004) Induction of apatite by the cooperative effect of amelogenin and the 32-kDa enamelin. J Dent Res 83:278-82
Iijima, M; Moradian-Oldak, J (2004) Interactions of amelogenins with octacalcium phosphate crystal faces are dose dependent. Calcif Tissue Int 74:522-31
Fong, Hanson; White, Shane N; Paine, Michael L et al. (2003) Enamel structure properties controlled by engineered proteins in transgenic mice. J Bone Miner Res 18:2052-9
Wen, H B; Moradian-Oldak, J (2003) Modification of calcium-phosphate coatings on titanium by recombinant amelogenin. J Biomed Mater Res A 64:483-90
Bouropoulos, N; Moradian-Oldak, J (2003) Analysis of hydroxyapatite surface coverage by amelogenin nanospheres following the Langmuir model for protein adsorption. Calcif Tissue Int 72:599-603
Moradian-Oldak, J; Iijima, M; Bouropoulos, N et al. (2003) Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology. Connect Tissue Res 44 Suppl 1:58-64
Iijima, M; Moriwaki, Y; Wen, H B et al. (2002) Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow. J Dent Res 81:69-73
Wang, L; Moradian-Oldak, J (2002) Assessment of enamelysin (MMP-20) selectivity to three peptide bonds on amelogenin sequence. J Dent Res 81:664-7
Moradian-Oldak, J; Gharakhanian, N; Jimenez, I (2002) Limited proteolysis of amelogenin: toward understanding the proteolytic processes in enamel extracellular matrix. Connect Tissue Res 43:450-5
Moradian-Oldak, Janet; Bouropoulos, Nikolaos; Wang, Lingli et al. (2002) Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal. Matrix Biol 21:197-205

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