The importance of a potential phosphorylation site in enamelin on enamel formation

Enamelin (ENAM) has three putative phosphoserines (pSers) phosphorylated by a Golgi-associated secretory pathway kinase (FAM20C) based on their distinctive Ser-x-Glu (S-x-E) motifs. Fam20C-knockout mice show severe enamel defects similar to those in the Enam-knockout mice, implying an important role of the pSers in ENAM. To determine the role of pSer55 in ENAM, we characterized ENAMRgsc514 mice, in which Ser55 cannot be phosphorylated by FAM20C due to an E57>G57 mutation in the S-x-E motif. The enamel microstructure of 4-week-old mice was examined by scanning electron microscopy. The teeth of 6-day-old mice were characterized by histology and immunohistochemistry. The protein lysates of the first lower molars of 4-day-old mice were analyzed by Western immunoblotting using antibodies against ENAM, ameloblastin and amelogenin. ENAMRgsc514 heterozygotes showed a disorganized enamel microstructure, while the homozygotes had no enamel on the dentin surface. The N-terminal fragments of ENAM in the heterozygotes were detained in the ameloblasts and localized in the mineralization front of enamel matrix, while those in the WT mice were secreted out of ameloblasts and distributed evenly in the outer 1/2 of enamel matrix. Surprisingly, the ~15  kDa C-terminal fragments of ameloblastin were not detected in the molar lysates of the homozygotes. These results suggest that the phosphorylation of Ser55 may be an essential posttranslational modification of ENAM and is required for the interaction with other enamel matrix molecules such as ameloblastin in mediating the structural organization of enamel matrix and protein-mineral interactions during enamel formation.


INTRODUCTION
Dental enamel is the most highly mineralized hard tissue in the body and is unique in its composition and process of formation. In humans and many other mammals, the enamel formation is artificially classified into presecretion, secretion, and maturation stages. At the secretory stage, the polarized ameloblasts secrete enamel matrix proteins (EMPs) on the dentin surface, including amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), so on. The EMPs are then proteolytically processed by metalloproteinase-20 (MMP20) at the secretory stage and eventually degraded completely by kallikreinrelated peptidase 4(KLK4) at maturation stages. 1 Evolutionary analyses have classified AMEL, AMBN, and ENAM into a family named 'secretory calcium-binding phosphoproteins' (SCPP), which have one or more Golgi casein kinase phosphorylation sites recognized by their distinctive Ser-x-Glu/phospho-Ser (S-x-E/pS) motifs. 2 Family with sequence similarity 20-member C (FAM20C) is a newly discovered Golgi-associated secretory pathway kinase, and is believed to be the genuine casein kinase phosphory-lating the SCPP proteins. [3][4][5] Fam20C-knockout mice exhibited hypophosphatemic rickets 6 and severe enamel defects that are very similar to those in the Enam-and Ambn-knockout mice, [6][7][8] suggesting that phosphoserines (pSers) in EMPs may have important functional roles.
The consensus sequences of S-x-E motifs in ENAM are highly conserved in vertebrate animals. [9][10] Three putative pSer sites have been identified in the ENAM of pigs (Ser 53 , Ser 191 , and Ser 216 ), mice (Ser 55 , Ser 196 , and Ser 219 ) and humans (Ser 54 , Ser 191 , and Ser 216 ). In a previous study, Masuya et al reported two lines of chemically induced Enam-mutant mice, ENAM Rgsc395 (M100395) and ENAM Rgsc514 (M100514), in which the first S-x-E motifs mutate into I 55 -x-E 57 and S 55 -x-G 57 , respectively. 11 The ENAM Rgsc395 mutation eliminates pSer 55 from the S-x-E motif, while the ENAM Rgsc514 mutation eliminates the potential phosphorylation by changing the motif into an unrecognizable one for FAM20C kinase. The breakage of enamel surfaces in ENAM Rgsc395 and ENAM Rgsc514 heterozygotes suggests that the phosphorylation of Ser 55 might be an important posttranslational modification for ENAM. However, the study by Masuya et al. did not characterize the enamel phenotypes in ENAM Rgsc514 homozygotes, and the biochemical consequence of the potential phosphorylation loss is unclear. In this study, we characterized the enamel defects and the biochemical changes in both ENAM Rgsc514 heterozygotes and homozygotes.

Animals and genotyping
All animal procedures were approved by the Institutional Animal Care and Use Committee of Texas A&M College of Dentistry (Dallas, TX, USA), and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The ENAM Rgsc514 (M100514) heterozygous mice were purchased from RIKEN BRC (Ibaraki, Japan). ENAM Rgsc514 mice have a chemically induced A1745G mutation in the Enam gene, resulting in a Glu 57 -to-Gly (E 57 to G) substitution in the first S-x-E motif of the ENAM protein. A genotyping PCR was performed on tail lysates using primers 5′-TTACGCCTGTGTTGGGTCTT-3′ and 5′-TGGTT TGGCACTAGCTCCTT-3′. The genotypes were determined by restriction digestion on the 715 bp PCR products with EarI (New England Biolabs, MA, USA). The reverse complementary chains of the WT allele were cleaved into 575 bp and a 140 bp fragments, while those of the mutant allele were not cleaved because the mutation had eliminated the EarI restriction site. The WT and heterozygotes showed three fragments (715 bp, 575 bp, and 140 bp), comparing to a single 715 bp fragment in the homozygotes.

Backscattered scanning electron microscopy
Mandibles collected from the 4-week-old ENAM Rgsc514 heterozygous, homozygous, and WT mice were fixed in 4% paraformaldehyde at 4°C overnight and dehydrated through ethanol gradient (70%-100%), followed by embedding in methyl methacrylate. The embedded mandibles were cross-sectioned at the first molar level using a slow-speed diamond saw. The surface of the section was ground smooth using 1200 grade silicon carbide abrasive paper and given a final polish using 0.5 μm diamond paste. The polished enamel surfaces were coated with carbon and examined using field emission scanning electron microscopy (Philips XL30, FEI Company, OR, USA). 12

RESULTS
The ENAM Rgsc514 mice showed severe enamel defects The incisors of the ENAM Rgsc514 heterozygotes and homozygotes had a chalky white color and rugged surface compared with the brown color and smooth surface in the WT mice (Figure 1a-1c). Scanning electron microscopy (SEM) revealed that the well-defined rod and interrod structure of the ENAM Rgsc514 heterozygotes enamel was less distinct and less mineralized compared with WT mice. Notably, the ENAM Rgsc514 homozygotes had no enamel on the dentin surface (Figure 1d-1l).
Histological analyses revealed that the ENAM Rgsc514 heterozygotes formed a thinner and disturbed enamel matrix compared to that in the WT mice (Figure 2a-2f). The ameloblasts of the ENAM Rgsc514 homozygotes lost their polarized shape and detached from the dentin surface; a bubble-like space between the ameloblasts and the dentin surface was filled with an amorphous substance (Figure 2g-2i). The odontoblasts and dentin of the ENAM Rgsc514 mice did not show apparent abnormalities.
The ENAM Rgsc514 mice showed an altered ENAM distribution pattern In 6-day-old ENAM Rgsc514 heterozygotes, the N-terminal fragments of ENAM were localized at the mineralization front of the enamel matrix and retained in the cell body of the ameloblasts. In contrast, these fragments were evenly distributed in the outer half of the enamel matrix and were nearly undetectable in the ameloblasts in the WT mice (Figure 3a-3f). The distribution pattern of the ENAM C-terminal fragments did not show apparent differences between the WT and ENAM Rgsc514 heterozygotes. However, these fragments were not detected in the enamel matrix of the ENAM Rgsc514 homozygotes (Figure 3g-3l). Western immunoblotting revealed that both theE-NAM Rgsc514 heterozygotes and homozygotes had less ENAM N-fragments than those in the WT mice, while their cleavage patterns did not show apparent differences among these animals (Figure 3m). We were not able to detect ENAM C-terminal fragments with Western blot using the anti-ENAM-C antibodies.
The ENAM Rgsc514 homozygotes showed an altered AMBN cleavage pattern The distribution pattern of AMBN N-or C-terminal fragments in the teeth did not show apparent differences between the ENAM Rgsc514 and WT mice at postnatal 6 days (Figure 4a-4l). However, the   (Figure 4m). In addition, the ENAM Rgsc514 mice appeared to have more AMBN expression in the teeth than did the WT mice (Figure 4m).
The distribution and cleavage patterns of AMEL did not show apparent differences between the ENAM Rgsc514 and WT mice, while

DISCUSSION
FAM20C has been identified as the Golgi-associated secretory pathway kinase that phosphorylates the serine residues in the S-x-E motifs of ENAM, AMBN, and AMEL. 4,5 Fam20C-knockout mice showed severe enamel defects similar to those in the Enam-knockout mice, [6][7][8] suggesting that the phosphoserines may have an important role in ENAM function. Mammalian ENAMs have three highly conserved pSer residues believed to be phosphorylated by FAM20C. 5,10 One of these pSers is located at the N terminus of ENAM, while the other two reside in the 32 kDa "functional" fragments. A previous study has demonstrated that the last pSer in the 32 kDa fragment may have an important role in amelogenesis, as a substitutive mutation of the serine residue (p.S216L) led to hypoplastic AI in humans. 17 However, it remains unclear whether the AI was associated with the phosphorylation loss or the amino acid change at this residue, as serine and leucine are very different amino acids. A similar question arose when we tried to explain the enamel defects in ENAM Rgsc514 mice. Theoretically, the E57G substitution prevents FAM20C catalyzed phosphorylation on Ser 55 . However, the residue substitution may be sufficient to cause AI, as Glu and Gly are very different amino acids. The first scenario suggests that the phosphorylation of Ser 55 is an essential posttranslational modification of ENAM, while the second scenario may suggest that the conserved residues in this area are non-replaceable for normal ENAM functions. Future studies are warranted to discern between these possibilities by substituting S 55 or E 57 with amino acids having similar properties, such as A 55 or D 57 , respectively. Under normal conditions, ENAM is secreted immediately after being synthesized and thus can barely be detected in the cell body of ameloblasts by IHC staining (Figure 3a and 3b). The full-length ENAM is cleaved into intermediate fragments by MMP20 and transported into specific locations in the enamel matrix: the N-terminal fragments are located in the newly formed enamel matrix, while the C-terminal fragments reside in more mature enamel matrix (Figure 3a-3d). [18][19] It is important that each step of ENAM secretion, cleavage, and transportation be done correctly in order to form normal enamel. In the ENAM Rgsc514 mice, the fragmentation of ENAM showed a pattern similar to that in the WT mice (Figure 3m), suggesting that the E57G substitution (or the phosphorylation failure of pSer 55 ) may not significantly affect ENAM cleavage. However, the ENAM Rgsc514 mice showed an abnormal transportation of N-terminal ENAM fragments compared with the WT: the N-fragments were localized at the mineralization front of the newly formed enamel matrix and strongly stained in the cell body of the ameloblasts in the ENAM Rgsc514 heterozygotes (Figure 3e and 3f) compared with the normal distribution in WT mice (Figure 3a and 3b), suggesting that the conserved residues in this region or the phosphorylation of pSer 55 is essential for the normal seceration of ENAM N-terminal fragments. We were not able to evaluate the distribution pattern of the N-fragments in the ENAM Rgsc514 homozygotes, as their ameloblasts were malformed and the enamel matrix was amorphous (Figure 3i and 3j). We did not detect the C-terminal fragments of ENAM in the amorphous enamel matrix of the ENAM Rgsc514 homozygotes ( Figure  3k and 3l). It is unclear if the negative staining suggests an accelerated degradation or an aberrant transportation of the C-fragments. Future studies are warranted to trace ENAM fragments by protein labeling and clarify the role of pSer 55 by substituting it with more similar/ specific residues.
Ameloblastin (AMBN) is the second most abundant protein in the enamel matrix. 20-23 MMP20 initially cleaves AMBN at one of the three (g) Western immunoblotting showed similar fragment patterns of AMEL among the WT and ENAM Rgsc514 mice, although the ENAM Rgsc514 homozygotes seemingly had less aggregated AMEL at~50 kDa. In addition, the ENAM Rgsc514 mice showed fewer amounts of~20 kDa AMEL fragments compared to the WT mice. Scale bars: 200 μm in a, c, and e. 50 μm in b, d, and f. sites (after Gln 130 , Arg 170 , or Ala 222 ), then further cleaves the intermediate fragments at secondary sites near the C-terminus. 22 The intact AMBN and cleaved fragments have different functions in certain compartments of the developing enamel layers. [24][25] In general, the N-terminal cleavage forms 13-,15-, and 17-kDa AMBN fragments that accumulate in the sheath space throughout the enamel layer, 22,26 while the intact AMBN (62-kDa) and its C-terminal cleavage products are located in superficial enamel and are undetectable below a depth of 50 µm. [27][28] In this study, the ENAM Rgsc514 mice did not show apparent differences in the AMBN distribution pattern compared with that in the WT mice (Figure 4a-4l). However, we did not detect the~15 kDa fragments of the C-terminal AMBN in the molar lysates prepared from the ENAM Rgsc514 homozygotes by Western immunoblotting compared to those in the WT and ENAM Rgsc514 heterozygotes (Figure 4m), suggesting that the cleavage of AMBN (which was probably mediated by MMP20) may require the phosphorylation of ENAM. Given that the N-fragments of ENAM (which contain the pSer 55 ) showed an overlapped distribution in the newly formed enamel matrix with the C-fragments of AMBN, and that the EMPs coordinate during matrix assembly, 29 we envisage that pSer 55 at the ENAM N-terminus may be essential to the structural organization of the enamel matrix and the protein-mineral interactions during enamel formation. Future work is warranted to test this hypothesis by examining the cleavage patterns of AMBN in Enam-knockout mice, and the coordination among normal and mutant ENAM, AMBN, and MMP20 during macromolecular assembly, as well as the enzymatic cleavage of AMBN by MMP20 in the presence or absence of phosphorylation in ENAM.
Taken together, we hypothesize that the phosphorylation of Ser 55 is likely an essential PTM of ENAM, which is probably needed for the interaction with other EMPs, such as AMBN, in mediating the structural organization of the enamel matrix and the protein-mineral interactions during enamel formation.