Porcine Amelogenin : Alternative Splicing, Proteolytic Processing, Protein - Protein Interactions, and Possible Functions

Abstract

Amelogenin is the major secretory product of ameloblasts and is critical for proper tooth enamel formation. Amelogenin isoforms and their cleavage products comprise over 80% of total secretory stage enamel protein. We have isolated and characterized four secreted amelogenin isoforms from developing porcine enamel : P190 (27-kDa), P173 (25-kDa), P132 (18-kDa) and P56 (6.5-kDa ; leucine rich amelogenin polypeptide or LRAP). P190 and P132 are low abundance amelogenins that contain a novel exon 4-encoded segment of lack the exon 3-encoded segment, respectively. P173 is the most abundant (major) amelogenin isoform. Cleavage of P173 by matrix metalloproteinase 20 (Mmp20) occurs at specific sites that generates a set of N-terminal cleavage products : P162 (23-kDa), P148 (20-kDa), P62/P63 (11-kDa), and Trp(45) (6-kDa, tyrosine rich amelogenin polypeptide or TRAP). P148 is the most abundant protein in developing enamel and influences the conversion of amorphous calcium phosphate into hydroxyapatite in vitro. Mmp20 cleaves LRAP, the second abundant amelogenin isoform after Pro(45) and Pro(40). Processing by Mmp20 allows amelogenin cleavage products to serve separate functions. Over time, Mmp20 catalyzes additional cleavages that facilitate the progressive replacement of amelogenin by mineral, so enamel crystals thicken and widen with depth. Besides proteolytic processing, amelogenin protein-protein interactions are critical for function. Far-Western analyses demonstrate that the larger amelogenins (P173, P162, and P148) are only able to interact with larger amelogenins. No amelogenin-amelogenin interactions are observed for the smaller amelogenin cleavage products, TRAP or LRAP Amelogenin doesn't interact with the 32-kDa glycosylated enamelin cleavage product, unless it it partially deglycosylated.

Fig. 1. Structure of porcine amelogenin genes

Boxes indicate the 7 amelogenin exons. Unfilled boxes correspond to the amelogenin-coding region ; patterned boxes correspond to exonic non-coding regions. The DNA sequences in the X and Y copies of the porcine amelogenin genes are identical in the coding regions for the secreted protein (solid line), but differ in the coding regions for the signal peptides (dashed line).

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.

Fig. 3. Amelogenin-amelogenin and amelogenin—enamelin protein-protein interaction by Far-Western blots

Coomassie Blue stained polyacrylamide gels are shown at the top of column 1. Replicas of these gels were transblotted to nitrocellulose and affinity stained using biotin-labed amelogenins or enamelins probes, the identity of which is provided at the top of each blot. Positive signals are indicated by arrows. Key to lanes : 1) P173, 2) P162, 3) P148, 4) P46–148, 5) P63, 6) P45 (TRAP), 7) LRAP-45, 8) P148–173, 9) intact 32-kDa enamelin, and 10) pig albumin.

Fig. 4. TEM micrographs of calcium phosphate mineral products formed in the presence of amelogenins examined at selected times (15 min, 45 min, 1 hour-4 hours, and 1 day), and the comparison with or without amelogenin phosphorylation examined after 1 day. (Courtesy Dr. H Margolis.)

As shown in the control (A), rP172 (B) and P148 (C) at 15 min, ACP was initially formed in the control and in the presence of rP172 and P148, based on the observed (insets) SAED patterns. In the presence of rP147 (D), both spherical particles and small plate-like crystals were observed, consistent with a somewhat diffuse SAED pattern. Subsequent changes in mineral particle shape and organization with time are described in the text. As described, in the presence of rP172 (B) ACP particles were observed (inset) to align and form needle-like particles at 45 min. After 1 day, based on SAED analyses, randomly arranged plate-like apatitic crystals were found in the control (A, inset, showing circular distribution of apatitic reflections), whereas aligned bundles of needle-like apatitic crystals were formed in the presence of rP172 (B, insets, showing a higher magnification of a bundle of aligned crystals and an arc-like diffraction pattern in the direction of the 002 reflection). In contrast, ACP was observed in the presence of P148 (C, inset), even after 1 day. In the presence of rP147 (D), crystal formation took place over time outside of observed protein masses. Randomly arranged plate-like HA crystals were observed at 1 day that were similar in these respects to those seen in the control, consistent with the SAED patterns obtained (D, 1 day, inset, showing circular distribution of apatitic reflections). Plate-like apatitic crystals that were slightly but significantly smaller than those found in the control were observed when mineralization was carried out in the presence of the non-phosphorylated rP147 (E), whereas bundles of significantly larger plate-like crystals were observed when mineralization was carried out using the partially dephosphorylated P148, dephoso-P148 (G). In sharp contrast, assemblies of spherical ACP particles were observed in the presence of P148 (F).