M. Fukae

Selective adsorption of porcine-amelogenins onto hydroxyapatite and their inhibitory activity on hydroxyapatite growth in supersaturated solutions

SummaryThe selective adsorption of amelogenins onto synthetic hydroxyapatite (HA) and their inhibitory activity on the seeded HA crystal growth were investigated using enamel proteins obtained from the outer layer of immature porcine-enamel (soft, cheeselike in consistency) of developing permanent incisors. Special interests were paid to the effect of a postsecretory degradation of the original amelogenin(s) on their adsorption and inhibitory activity. In the adsorption studies, it was apparent that the originally secreted amelogenin (25 K), proline, and histidine-rich protein (2a), as well as the higher molecular weight components (60–90 K), showed a strong adsorption affinity onto the HA. This adsorption of protein 2a was related to its inhibition of the crystal growth of seeded HA in a dilute supersaturated solution. On the other hand, the partially degraded product (20 K) of amelogenins, protein 2b, lost the high adsorption affinity onto the HA, and consequently showed no significant inhibitory activity. The observed selective adsorption of protein 2a onto HA was apparent at pH 6.0 and pH 7.4 even in the presence of dissociative solvents, such as 3 M urea or 2 and 4 M guanidine-HCl; however, this selective behavior was sensitive to changes in pH, and was not displayed at pH values of 7.8 or 10.8. The results suggest that the originally secreted amelogenin 2a may play an active role in amelogenesis, and that enamel mineralization could be regulated by the secretion of amelogenins and their inactivation through partial enzymic degradation, prior to their complete removal.

Studies on the proteins of developing bovine enamel

Abstract Enamel samples at four different stages of development were separated from embryonic bovine teeth by scraping, and demineralized by dialysis against 0.5 M acetic acid for 4 days at 4 °C. These extracts were lyophilized and redissolved for subsequent Sephadex G-100 chromatography, disc electrophoresis and determination of N-terminal amino acids. There was a sharp drop in the major chromatographical component of enamel protein between stages of cheese-like enamel and chalk-like enamel. Methionine and leucine were identified as the main N-terminal amino acids of enamel protein, and their amount in samples of the same weight increased with the maturation of enamel.

Dentin Sialophosphoprotein Is Processed by MMP-2 and MMP-20 in Vitro and in Vivo

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper tooth dentin formation. During dentinogenesis, DSPP is proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps, and failure to make these cleavages is a potential cause of developmental tooth defects. We tested the hypothesis that dentin-resident matrix metalloproteinases catalyze the cleavages that process DSPP. We defined the exact DSPP cleavages that are catalyzed by proteases during crown formation by isolating DSPP-derived proteins from developing porcine molars and characterizing their N-terminal sequences and apparent size on SDS-PAGE and Western blots. The in vivo DSPP cleavage sites were on the N-terminal sides of Thr200, Ser330, Val353, Leu360, Ile362, Ser377, Ser408, and Asp458. The initial DSPP cleavage is between dentin glycoprotein (DGP) and dentin phosphoprotein (DPP), generating dentin sialoprotein (DSP)/DGP and DPP. Gelatin and casein zymograms identified MMP-2, MMP-20, and KLK4 in the dentin extracts. MMP-2 and MMP-20 were purified from over 150 g of porcine dentin powder and incubated with DSP-DGP and DPP. These enzymes show no activity in further cleaving DPP. MMP-20 cleaves DSP-DGP to generate DSP and DGP. MMP-20 also cleaves DSP at multiple sites, releasing N-terminal DSP cleavage products ranging in size from 25 to 38 kDa. MMP-2 makes multiple cleavages near the DSP C terminus, releasing larger forms of DGP, or “extended DGPs.” Exact correspondence between DSPP cleavage sites that occur in vivo and those generated in vitro demonstrates that MMP-2 and MMP-20 process DSPP into smaller subunits in the dentin matrix during odontogenesis.

Enamelysin (Matrix Metalloproteinase-20): Localization in the Developing Tooth and Effects of pH and Calcium on Amelogenin Hydrolysis

The formation of dental enamel is a precisely regulated and dynamic developmental process. The forming enamel starts as a soft, protein-rich tissue and ends as a hard tissue that is is over 95% mineral by weight. Intact amelogenin and its proteolytic cleavage products are the most abundant proteins present within the developing enamel. Proteinases are also present within the enamel matrix and are thought to help regulate enamel development and to expedite the removal of proteins prior to enamel maturation. Recently, a novel matrix metalloproteinase named enamelysin was cloned from the porcine enamel organ. Enamelysin transcripts have previously been observed in the enamel organ and dental papillae of the developing tooth. Here, we show that the sources of the enamelysin transcripts are the ameloblasts of the enamel organ and the odontoblasts of the dental papilla. Furthermore, we show that enamelysin is present within the forming enamel and that it is transported in secretory vesicles prior to its secretion from the ameloblasts. We also characterize the ability of recombinant enamelysin (rMMP-20) to degrade amelogenin under conditions of various pHs and calcium ion concentrations. Enamelysin displayed the greatest activity at neutral pH (7.2) and high calcium ion concentration (10 mM). During the initial stages of enamel formation, the enamel matrix maintains a. neutral pH of between 7.0 and 7.4. Thus, enamelysin may play a role in enamel and dentin formation by cleaving proteins that are also present during these initial developmental stages.

Epitaxial overgrowth of apatite crystals on the thin-ribbon precursor at early stages of porcine enamel mineralization

SummaryThe aim of the present work was to investigate changes in cross-sectional morphologies of enamel crystallites as a function of location in secretory porcine enamel. Enamel tissues were obtained from 5- to 6-month-old slaughtered piglets. For examination by electron microscopy, a portion of the secretory enamel was embedded in resin and ultrathin sections were prepared with a diamond knife. In parallel studies, compositional and structural changes of enamel mineral were assessed by chemical analysis and Fourier transform infrared (FTIR) spectroscopy. For this purpose, two consecutive layers of the outer secretory enamel, each approximately 30 μm thick, were separated from the labial side of permanent incisors. Using high-resolution electron microscopy, early events of enamel crystal growth were characterized as the epitaxial growth of small apatite units on the lateral surfaces of the initially precipitated thin ribbon. These apatite units had regular triangle or trapezoid cross-sections. After fusions of those isolated trapezoids on both lateral sides of the platy template, the resulting enamel crystallites had the well-documented flattened-hexagonal shapes in cross-sections. The initially precipitated thin plate was buried inside the overgrown apatite lamella and then retained as a central dark line. Similar morphological evidence for the epitaxial nucleation and overgrowth of carbonatoapatite on the platy template was obtainedin vitro. Chemical and FTIR analyses of the enamel layer samples showed that the characteristics of the youngest enamel mineral were distinct from those of enamel crystals found in older secretory enamel. The overall results support the concept that initial enamel mineralization comprises two events: the initial precipitation of thin ribbons and the subsequent epitaxial growth of apatite crystals on the two-dimensional octacalcium phosphate-like precursor.

The localization and characterization of proteinases for the initial cleavage of porcine amelogenin

SummaryIn the outermost layer of porcine-developing enamel adjacent to the ameloblasts in the secretory stage, the activities of two proteinases having molecular masses of 76 and 78kDa were detected by enzymography using gelatin as a substrate. On the other hand, high activities of known 30 and 34kDa proteinases were localized in the inner layer of the enamel. The 76kDa proteinase cleaved the carboxylterminal peptide of porcine 25kDa amelogenin to convert it to 20kDa amelogenin. The 78kDa proteinase also acted on the 25kDa amelogenin similarly, but its activity was weak. The results indicate that the 25kDa amelogenin synthesized and secreted by ameloblasts is converted to 20kDa amelogenin by the action of proteinase localized in the outermost layer of the secretory enamel, and then further degraded by the proteinases in the inner layer of the enamel associated with the increase of mineralization.

Primary Structure of the Porcine 89-kDa Enamelin

The primary structure of the 89-kDa enamelin found in porcine secretory enamel at an early stage of development was investigated. The fragments of the enamelin cDNA were amplified by polymerase chain-reaction from the first-strand enamelin cDNA, and were sequenced. The results indicated that the 89-kDa enamelin consisted of 627 amino acid residues and had a molecular mass of 70,448. A hydrophobic domain is located in the region of the 21 st-62nd amino acid residues of the molecule. Acidic domains are located in two regions of the molecule-one in the region of the 135th-238th amino acid residues and the other in the C-terminal region. A basic domain is located in the region of the 239th-360th amino acid residues. The results also indicated that the low-molecular-weight enamelins were fragments derived from a prototype enamelin.

Amelogenin Gene Expression in Porcine Odontoblasts

Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples.

Relative Levels of mRNA Encoding Enamel Proteins in Enamel Organ Epithelia and Odontoblasts

Amelogenin, enamelin, sheathlin (ameloblastin/ amelin), enamelysin (MMP-20), and KLK4 (EMSP-1) are the major structural proteins and proteinases in developing tooth enamel. Recently, odontoblasts were reported to express amelogenin, the most abundant enamel protein. In this study, we hypothesized that odontoblasts express all enamel proteins and proteases, and we measured their relative mRNA levels in enamel organ epithelia and odontoblasts associated with porcine secretory- and maturation-stage enamel by RT-PCR, using a LightCycler instrument. The results showed that amelogenin mRNA in secretory-stage EOE is 320-fold higher than in odontoblasts beneath secretory-stage enamel, and over 20,000-fold higher than in odontoblasts under maturation-stage enamel. Similar results were obtained for enamelin and sheathlin. Enamelysin mRNA levels were equivalent in these two tissues, while KLK4 mRNA was higher in odontoblasts than in secretory-stage EOE. These results support the conclusion that odontoblasts are involved in the formation of the enamel layer adjacent to enamel-dentin junction.

Proteolytic Enzyme in Porcine Immature Enamel

A proteolytic enzyme cleaving the main component of enamel proteins obtained from immature enamel has been purified from a soluble extract of porcine immature enamel. It is optimally active around pH 6 against enamel protein. It is completely inhibited by phenylmethylsulfonyl fluoride and diisopropyl phosphofluoridate, and partially by benzamidine. EDTA does not affect its activity. The enzyme seems to sever initially enamel protein into two segments, one containing lysine, arginine and tyrosine and the other being free from these amino acids.