Purification and characterization of porcine LRAP, TRAP, Klk4, and Mmp-20. (A) The leucine-rich amelogenin protein (LRAP) and the tyrosine-rich amelogenin peptide (TRAP) were purified until they represented single chromatographic peaks on a C18 RP-HPLC column and a single band on SDS-PAGE. The numbers under the chromatograms indicate time (in min). (B) Mass spectrometry of LRAP gave a value of 6618.77 Da (calculated mass, 6617.50), and TRAP gave a value of 5404.28 (calculated mass, 5404.08), which closely matched the calculated masses of the single phosphosphorylated proteins. (C) The final purification of Klk4 was on a C18 column, where Klk4 was collected from a well-defined chromatographic peak and appeared as a doublet above 30 kDa on SDS-PAGE stained with Coomassie Brilliant Blue (CBB), on a gelatin zymogram (Z), and on a Western blot (W). The final purification of Mmp-20 was on a heparin affinity column. Mmp-20 could not be detected by SDS-PAGE, but was evident on a casein zymogram (Z) and on a Western blot stained with an anti-peptide antibody raised against its C-terminal hemopexin-like domain. The Mmp-20 catalytic domain appears as lower bands on the zymogram, but is not detected by the antibody raised against the hemopexin-like domain.(D) Standard aliquots of Mmp-20 and (E) Klk4 were used to digest recombinant pig amelogenin (rP172), LRAP, and TRAP for 0, 1, 2, 4, 8, 12, and 24 hrs. The protein without enzyme was run as a control (C). Both enzymes digested rP172, but produced different patterns of digestion products. Mmp-20 showed little activity against LRAP, but was able to produce a slightly smaller product by 24 hrs. Klk4 degraded LRAP by 8 hrs and generated multiple products. Mmp-20 showed no appreciable activity against TRAP, while Klk4 degraded TRAP.

Sites cleaved by Klk4 and Mmp-20 in the 5 fluorescent peptides. Arrows indicate sites cleaved after 24 hrs. Arrowheads indicate sites in Amel2 cleaved by Mmp-20 in a 48-hour digest (after Gln⁶⁰, Ser⁶¹, His⁶², and Ala⁶³). The His⁶² and Ala⁶³ sites are used in a minor processing pathway that generates extended TRAP and the 11-kDa amelogenin (Appendix).

Digestion of fluorescent peptides Amel1 (Tyr³⁹-Ile⁵⁰), Amel2 (Gln⁵⁷-Ile⁷⁰), Amel3 (Gln¹⁰¹-Gln¹¹³), Amel4 (Leu¹⁴³-Asp¹⁵⁵), and Amel5 (Leu¹⁵³-Lys¹⁶⁶). The numbers under the chromatograms indicate time (in min). The first row shows the UV (blue) and fluorescent (red) chromatograms for each uncut peptide. The second and third rows show each peptide after a 24-hour digestion with a standard aliquot of Klk4 or Mmp-20, respectively. Red arrows point to peaks containing a fluorescent (Abz-containing) cleavage product. Yellow arrows point to cleavage products that do not fluoresce (lack the Abz). Arrowheads point to minor fluorescent (Abz-containing) cleavage products that were not characterized in these 24-hour digests, but became more prominent in 48-hour digests (data not shown). Column 1 (Amel1) shows that Mmp-20 catalyzed the TRAP cleavage (after Trp⁴⁵), but Klk4 did not. Column 2 (Amel2) shows that Klk4 can cleave after His⁶². Mmp-20 cleaved Amel2 after His⁶⁸ near the end of the peptide. This cleavage has not been detected in enamel extracts (in vivo) and might be an artifact of the use of a peptide substrate. Mmp-20 generated multiple cleavages of Amel2 after 48 hrs (Appendix). Neither enzyme showed much activity for Amel3, which contained minor cleavage sites previously shown to be susceptible to cleavage by recombinant Mmp-20 (Ryu et al., 1999). Column 4 (Amel4) shows that Mmp-20, but not Klk4, catalyzes the cleavage after Ser¹⁴⁸ that generates the 20-kDa amelogenin. Column 5 (Amel5) shows that Mmp-20, but not Klk4, catalyzes the cleavage after Pro¹⁶² that generates the 23-kDa amelogenin.

Summary of the digestion of porcine LRAP, TRAP, and the major amelogenin isoform (P173) by Mmp-20 and Klk4. (A) The porcine LRAP sequence. Downward pointing arrows mark the two processing sites (after Pro⁴⁰ and Pro⁴⁵) catalyzed by Mmp-20. Upward pointing arrows are degradation sites catalyzed by Klk4, as determined by LC-MSMS analysis of a 24-hour digest of LRAP. (B) Diagram showing the processing of LRAP by Mmp-20. (C) The porcine TRAP sequence. Mmp-20 has very low catalytic activity against TRAP. Upward pointing arrows are degradation sites catalyzed by Klk4, as determined by LC-MSMS analysis of a 24-hour digest of TRAP. (D) Porcine 173-amino-acid major amelogenin (P173) sequence. Downward arrows show the major Mmp-20 processing sites; arrowheads show the minor Mmp-20 processing sites. (E) Diagram showing the major and minor processing sites that generate the most abundant amelogenin cleavage products in porcine secretory-stage enamel. The secreted protein (P173) is usually processed in two steps. The initial cleavage is after Ser¹⁴⁸ and generates P148, the most abundant amelogenin cleavage product in the matrix. The second cleavage is after Trp⁴⁵ and generates TRAP and the 13-kDa amelogenins. A less-used processing pathway involves an initial cleavage after Pro¹⁶², generating the 23-kDa amelogenin, which is cleaved again to generate P148. P148 is also cleaved in a less-often-used alternative pathway that generates extended TRAP and the 11-kDa amelogenin.