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The control of hydroxyapatite crystal initiation and growth during enamel development is thought to be mediated by the proteins of the extracellular matrix. However, the precise nature of these critical mineral−protein interactions remains obscure. In this study, fluid tapping mode atomic force microscopy was used to image, for the first time, the binding of extracellular proteins found in enamel matrix (amelogenin and albumin) to developing enamel crystals. Both albumin and amelogenin were found to be associated with the crystal surfaces under conditions close to physiological; however, the binding of the two proteins was distinctly different. Albumin appeared to bind as a monomer, whereas amelogenin was bound as aggregates resembling previously described “nanosphere” structures. Both proteins were arrayed on the crystal surface in a distinctive banding pattern, perpendicular to the c-axis. This pattern was coincident with recently identified positively charged domains on the crystal surface, suggesting ...

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.