MMP-20 and KLK4 Digestion of LRAP and TRAP

The Tyrosine Rich Amelogenin Peptide (TRAP) and Leucine-Rich Amelogenin Peptide (LRAP) are abundant amelogenin cleavage products in the enamel layer of developing teeth. TRAP is derived from the major amelogenin isoform (P173). LRAP is derived from the second most abundant amelogenin isoform (P56). Although these amelogenin peptides accumulate during the secretory stage, they disappear from the enamel layer early in the maturation stage.

Objectives: To test the hypotheses that 1) TRAP and LRAP are generated by MMP-20 cleavages and accumulate during the secretory stage because they are resistant to further digestion by MMP-20; and 2) TRAP and LRAP disappear from the matrix because they are degraded by KLK4. Methods: We isolated TRAP from developing porcine molars and had the LRAP parent protein (P56) synthesized commercially. Both proteins were verified following purification by mass spectrometry. Native KLK4 and MMP-20 were isolated from develop porcine molars. TRAP and P56 were incubated with MMP-20 and KLK4 and the digestion products were analyzed by SDS-PAGE and LC-MSMS. Results: MMP-20 was not able to digest TRAP and digested P56 to produce LRAP, but LRAP was not further degraded by MMP-20. KLK4 degraded TRAP and LRAP and the exact cleavage sites were determined by mass spectrometry. Conclusions: How MMP-20 cleaves amelogenin determines the nature of the cleavage products that accumulate during the secretory stage. KLK4 degrades accumulated amelogenin protein, facilitating their removal from the matrix during the early maturation stage. This study is supported by NIDCR grants DE015846 and DE016376.