Fig. 2. Porcine amelogenin isoforms and cleavage products, and proteolytic processing pathway.

Based on the dissociative extraction method, ten amelogenin isoforms and their proteolytic cleavage products are observed on SDS-PAGE gel (N : extracts by Sorensen buffer (pH 7.4), AL : extracts by carbonate buffer (pH 10.8)). Of these amelogenins, P173 amelogenin isoform (b : P173) is a major amelogenin and its cleavage products (c : P162, d : P148, f : P46–148, g : P63/64–148, h : P62/63, i : P45 (TRAP)) are abundant during the secretory stage of enamel. The intact LRAP is found in the most outer layer enamel, but its cleavage product (j : LRAP-45) is usually detected in the mixture of outer and inner layer of enamel sample. P157 amelogenin isoform lacking exon 3 has not been identified, but its cleavage product (e : P132) has been characterized. P190 amelogenin isoform containing exon 4 encoded segment (a : P190) is the least abundant amelogenin. The major secreted amelogenin (P173) is usually processed in two steps (Fig. 2B). 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 tyrosine-rich amelogenin polypeptide (TRAP or P45) having an apparent molecular weight of 6-kDa and the 13-kDa amelogenins (P46–148). A minor processing pathway involves an initial cleavage after Pro¹⁶², generating the 23-kDa amelogenin (P162), which is cleaved again to generate P148. P148 is also cleaved in a less-often-used alternative pathway that generates extended TRAP having an apparent molecular weight of 7-kDa (P62/P63) and the 11-kDa amelogenin (P63/64–148). All these processing are caused by Mmp20 and Mmp20 showed little to no activity against TRAP LRAP (P56) is cleaved after Pro⁴⁵ or Pro⁴⁰ by Mmp20 to generate P45 (LRAP-45) and P40 (LRAP-40), and Mmp20 can not degrade any sites of LRAP other than them. The generated TRAP LRAP45 and LRAP-40 are fragmentated by Klk4 during the transition and maturation stage.