Dental enamel is a highly mineralized hard tissue which is unique both in its composition and in its formation. Although the formation of dental enamel is a carefully controlled process little is known about the mechanism(s) by which this tissue is formed. Enamel initially forms as ameloblasts secrete proteins onto dentine. This proteinaceous matrix is composed mostly of amelogenins (hydrophobic, proline rich proteins), which are removed as the enamel matures. A gradual loss of amelogenin and increasing mineralization continues throughout maturation. The resulting fully mineralized tissue contains only a small amount of non-amelogenin protein. The processing of amelogenin protein appears to be an important mechanism for the control of apatite crystal growth in the developing enamel. The initially secreted higher molecular weight amelogenins strongly adsorb to apatite crystals and inhibit further crystal growth. The adsorption affinity towards apatite crystals decreases when lower molecular weight forms of amelogenins are tested in vitro. These observations suggest that mineralization may be controlled by a gradual degradation of amelogenins by proteolytic enzymes. Any disruption of this process, such as in enamel fluorosis where the removal of amelogenin protein is delayed, results in a more porous, less well-mineralized enamel matrix. The specific hypothesis to be tested is that metalloproteinases are responsible for hydrolysis of amelogenins in the developing enamel matrix.
The specific aims are: 1) to purify a 21.5 kDa metalloproteinase from enamel; 2) to characterize this enzyme by determination of amino acid composition, sequence, accurate molecular weight, identification of postranslational modifications, determination of optimal substrate and assay conditions, and enzyme specificity; 3) to purify a well characterized form of bovine amelogenin from secretory stage enamel; 4) to determine the specific proteolytic cleavage sites of amelogenin by the 21.5 kDa proteinase. Purification will be accomplished by ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography and reversed phase HPLC. Accurate molecular weights will be determined by electrospray mass spectroscopy, amino acid composition and sequence analysis will be determined. Purification of amelogenin will be accomplished by preparative isoelectric focusing, G-100 column chromatography and revered phase HPLC. Specific cleavage sites will be determined by separation of the digest by reversed phase HPLC and subsequent sequence analysis. These studies to identify and characterize a major metalloproteinase in developing enamel will lead to a better understanding of the mechanisms by which enamel mineralization occurs. They may also serve to further our understanding of defects which occur in enamel such as fluorosis.
| Den Besten, P K; Punzi, J S; Li, W (1998) Purification and sequencing of a 21 kDa and 25 kDa bovine enamel metalloproteinase. Eur J Oral Sci 106 Suppl 1:345-9 |
