Proteinases are present in forming enamel and are known to cleave both amelogenins and enamelins. Although there is general agreement that the degradation of enamel proteins is an important step in enamel formation, determining the actual function of the proteinases is difficult because of the exceedingly low concentrations of the enzymes in the tissue. Thus, it is very difficult to directly isolate these enzymes using biochemical procedures. In the proposed studies, molecular biology strategies will be used to address this problem. Proteinases will be identified based on their expression in the enamel organ (Aim 1). This will be done by performing RT-PCR on mRNA isolated from the enamel organ. Degenerate primers designed for cDNA which codes for conserved regions of the serine or metalloproteinase gene families will be used in PCR to generate the corresponding cDNAs. Results from in situ hybridization experiments will determine if the ameloblasts produce an identified proteinase (Aim 2). To confirm that a proteinase is actually exported into the forming enamel, the proteinase will be cloned from a cDNA library and antisera will be generated for immunolocalization studies. This antisera will also be used in conjunction with Northern analysis to assess the regulation of individual proteinases at different stages of tooth development (Aim 3). Recombinant proteinases will be produced in a mammalian expression system and substrate specificity will be determined using recombinant amelogenin or extracted enamel proteins (Aim 4). Taken together, these studies will provide key information regarding the functional role of proteinases during amelogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29DE012098-03
Application #
2872160
Study Section
Oral Biology and Medicine Subcommittee 1 (OBM)
Project Start
1997-03-01
Project End
2002-01-31
Budget Start
1999-02-01
Budget End
2000-01-31
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Forsyth Institute
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02142
Turk, Benjamin E; Lee, Daniel H; Yamakoshi, Yasuo et al. (2006) MMP-20 is predominately a tooth-specific enzyme with a deep catalytic pocket that hydrolyzes type V collagen. Biochemistry 45:3863-74
Caron, C; Xue, J; Sun, X et al. (2001) Gelatinase A (MMP-2) in developing tooth tissues and amelogenin hydrolysis. J Dent Res 80:1660-4
Caterina, J; Shi, J; Krakora, S et al. (1999) Isolation, characterization, and chromosomal location of the mouse enamelysin gene. Genomics 62:308-11
Bartlett, J D; Simmer, J P (1999) Proteinases in developing dental enamel. Crit Rev Oral Biol Med 10:425-41
Begue-Kirn, C; Krebsbach, P H; Bartlett, J D et al. (1998) Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci 106:963-70
Bartlett, J D; Ryu, O H; Xue, J et al. (1998) Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin. Connect Tissue Res 39:101-9;discussion 141-9
Fukae, M; Tanabe, T; Uchida, T et al. (1998) Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis. J Dent Res 77:1580-8
Caron, C; Xue, J; Bartlett, J D (1998) Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues. Matrix Biol 17:501-11