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Controlled proteolysis of amelogenins reveals exposure of both carboxy‐ and amino‐terminal regions
Author(s) -
MoradianOldak J.,
Jimenez I.,
Maltby D.,
Fincham A. G.
Publication year - 2001
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/1097-0282(200106)58:7<606::aid-bip1034>3.0.co;2-8
Subject(s) - amelogenin , proteolysis , chemistry , enamel paint , recombinant dna , biochemistry , cleavage (geology) , proteases , chymotrypsin , trypsin , biophysics , enzyme , biology , gene , dentistry , medicine , paleontology , fracture (geology)
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 (E 18 /V) and C‐terminal (E 178 /V) sites. Chymotrypsin cleaved amelogenin at both the carboxy‐ (F 151 /S) and amino‐terminal (W 25 /Y) regions. Interestingly, the peptide bond F/S 152 was also recognized by the action of enamelysin 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 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 (F 151 /S 152 ) and pig amelogenins (S 148 /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. © 2001 John Wiley & Sons, Inc. Biopolymers 58: 606–616, 2001