Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions.
The matrix-mediated enamel biomineralization involves secretion of the enamel specific amelogenin proteins that through self-assembly into nanosphere structures provide the framework within which the initial enamel crystallites are formed. During enamel mineralization, amelogenin proteins are processed by tooth-specific proteinases. The aim of this study was to explore the factors that affect the activity of enamel proteases to process amelogenins. Two factors including amelogenin self-assembly and enzyme specificity are 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 proteinases as well as a recombinant enamelysin were used, and their proteolytic actions on recombinant amelogenin were examined under controlled and limited conditions. The N-terminal region of the recombinant mouse amelogenin rM179 was found to be more accessible to tryptic digest than the C-terminal region. The endoproteinase Glu-C cleaved amelogenin at both the N-terminal (E18/V) and C-terminal (E178/V) sites. Chymotrypsin cleaved amelogenin at both the carboxy- (F151/S) and amino-terminal (W25/Y) regions. Interestingly, the peptide bond F/S152 was also recognized by the action of enamelysin on recombinant mouse amelogenin whereas thermolysin cleaved the S152/M153 peptide bond in addition to T63/L64 and I159/L160 and M29/I30 bonds. It was then concluded that regions at both the carboxy- and amino-terminal were exposed on the surface of amelogenin nanospheres when the N-terminal 17 amino acid residues were proposed to be protected from proteolysis, presumably as the result of their involvement in direct protein-protein interaction. Cleavage around the FSM locus occurred by recombinant enamelysin under limited conditions, in both mouse (F151/S152) and pig amelogenins (S148/M). Our in vitro observations on the limited proteolysis of amelogenin by enamelysin suggest that enamelysin cleaved amelogenin at the C-terminal 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 mechanisms of amelogenin degradation during amelogenesis.
Moradian-Oldak
J
J
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, 2250 Alcazar Street, Los Angeles, CA 90033, USA. joldak@hsc.usc.edu
Jimenez
I
I
Maltby
D
D
Fincham
A G
AG
D053523:Amelogenin; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013552:Swine; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D053523:Amelogenin; C507200:Amelx protein, mouse; D003746:Dental Enamel Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; C507111:MMP20 protein, human; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; C507113:Mmp20 protein, mouse; D013820:Thermolysin