Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions

The matrix-mediated enamel biomineralization involves secretion of the enam el specific amelogenin proteins that through self-assembly into nanosphere structures provide the framework within which the initial enamel crystallit es are formed. During enamel mineralization, amelogenin proteins are proces sed by tooth-specific proteinases. The aim of this study was to explore the factors that affect the activity of enamel proteases to process amelogenin s. Two factors including amelogenin self-assembly and enzyme specificity ar e considered. We applied a limited proteolysis approach, combined with mass spectrometry, in order to determine the surface accessibility of conserved domains of amelogenin assemblies. A series of commercially available prote inases as well as a recombinant enamelysin were used, and their proteolytic actions on recombinant amelogenin were examined under controlled and limit ed conditions. The N-terminal region of the recombinant mouse amelogenin rM 179 was found to be more accessible to cryptic digest than the C-terminal r egion. The endoproteinase Glu-C cleaved amelogenin at both the N-terminal ( E-18/V) and C-terminal (E-178/V) sires. Chymotrypsin cleaved amelogenin at both the carboxy- (F-151/S) and amino-terminal (W-25/Y) regions. Interestin gly, the peptide bond F/S-152 was also recognized by the action of enamelys in on recombinant mouse amelogenin whereas thermolysin cleaved the S-152/M- 153 peptide bond in addition to T-63/L-64 and I-159/L-160 and M-29/I-30 bon ds. It was then concluded that regions at both the carboxy - and amino-term inal were exposed on the surface of amelogenin nanospheres when the N-termi nal 17 amino acid residues were proposed to be protected from proteolysis, presumably as the result of their involvement in direct protein-protein int eraction. Cleavage around the FSM locus occurred by recombinant enamelysin under limited conditions, in both mouse (F-151/S-152) and pig amelogenins ( S-148/M). Our in vitro observations on the limited proteolysis of amelogeni n bl enamelysin suggest that enamelysin cleaved amelogenin at the C-termina l region showing a preference of the enzyme to cleave the S/M and F/S bonds . The present limited proteolysis studies provided insight into the mechani sms of amelogenin degradation during amelogenesis. (C) 2001 John Wiley & So ns, Inc.