Biochemistry and molecular biology of amelogenin proteins of developing dental enamel (original) (raw)
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The Structural Biology of the Developing Dental Enamel Matrix
Journal of Structural Biology, 1999
The biomineralization of the dental enamel matrix with a carbonated hydroxyapatite mineral generates one of the most remarkable examples of a vertebrate mineralized tissue. Recent advances in the molecular biology of ameloblast gene products have now revealed the primary structures of the principal proteins involved in this extracellular mineralizing system, amelogenins, tuftelins, ameloblastins, enamelins, and proteinases, but details of their secondary, tertiary, and quaternary structures, their interactions with other matrix and or cell surface proteins, and their functional role in dental enamel matrix mineralization are still largely unknown. This paper reviews our current knowledge of these molecules, the probable molecular structure of the enamel matrix, and the functional role of these extracellular matrix proteins. Recent studies on the major structural role played by the amelogenin proteins are discussed, and some new data on synthetic amelogenin matrices are reviewed. 1999 Academic Press
Comparative protein biochemistry of developing dental enamel matrix from five mammalian species
Calcified Tissue International, 1982
The matrix proteins of the developing dental enamel of five mammalian species were isolated and subjected to chromatographic, electrophoretic, and amino acid analyses. It was found that the principal chromatographic fractions showed similarities of both size and amino acid composition among Species. The major amelogenin protein of the cow, hamster, human, and sheep was of about 30,000 daltons and of the pig enamel matrix about 20,000 daltons. In each species a higher molecular weight fraction, greater than 40,000 daltons, was detected. In the lower molecular weight range an amelogenin polypeptide enriched in leucine, a fraction rich in tyrosine, and a fraction of intermediate size (Bovine matrix "Component-14") were identified in each case. It is suggested that these characteristic proteins arise during the degradation of the matrix which accompanied mineralization.
Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development
Frontiers in physiology, 2017
The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral de...
The Anatomical record, 1987
In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout the...
Crystal Growth in Dental Enamel: the Role of Amelogenins and Albumin
Advances in Dental Research, 1996
Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2013
Background-Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated. Methods-Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS. Results-It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N-and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents. Conclusion-These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization. General significance-This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.
Enamel mineralization in the absence of maturation stage ameloblasts
Archives of Oral Biology, 2009
The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5 kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.
Biosynthesis and secretion of enamel proteins during hamster tooth development
Calcified Tissue International, 1983
The synthesis and secretion of enamel proteins (EPs) in rat incisors was examined using cytochemical and biochemical methods. Radioautography after injection of 3 H-methionine showed that ameloblasts in the presecretory, secretory, and maturation stages of amelogenesis actively synthesized and secreted proteins. Immunocytochemistry with an antibody to mouse amelogenins revealed the presence of EPs in the protein synthetic and secretory organelles of these cells at all three stages. Labeling was also found in elements of the endosomal/lysosomal compartment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins extracted from enamel and enamel organ showed several protein bands. However, transfer to nitrocellulose paper and immunoblotting revealed that most of the proteins recognized by the antibody were situated between approximately 14 and 32 kDa. EPs were further characterized by using lectins to examine their carbohydrate content. Lectin-gold cytochemistry on sections showed the binding of wheat germ agglutinin and Helix pomatia lectin to secretory stage enamel. Lectin blotting indicated that the amelogenins were heterogeneously glycosylated and contained the sugars N-acetylglucosamine/N-acetyl-neuraminic acid and N-acetyl-D-galactosamine. Fluorography at 6 and 10 min and 1 h after injection of 35 S-methionine revealed four labeled bands in the main amelogenin group near 22, 28, 30, and 32 kDa. A short-lived protein of approximately 58 kDa was also observed primarily in cells. The appearance of labeled proteins in enamel was paralleled by their disappearance from cells and the intensity of the radiolabeled protein bands, both in enamel and in cells, decreased towards the maturation stage. These data are consistent with the concept that ameloblasts produce multiple amelogenins throughout amelogenesis.
Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth
Archives of Oral Biology, 1991
The primary structures, molecular genetics and biosynthesis of the amelogenin protein of the developing tooth are established, but knowledge of their subsequent post-secretory processing and its relation to enamel biomineralization is fragmentary. Preparations of tooth matrix proteins were isolated from molars (M1) of mice from birth to 15 days and analysed by SDS-PAGE and immunochemical methods. Amelogenin proteins, isolated and partially purified by HPLC, were characterized by amino acid analysis and SDS-PAGE. At birth a 26 kDa amelogenin was present that during subsequent developmental stages generated a series of 20-25 kDa amelogenins differing in apparent size by approximately 1 kDa. Amino acid analyses showed that all these amelogenins have amino-terminal TRAP sequences; analyses for both glycosylation and phosphorylation were negative. It is suggested that these post-secretory amelogenins are generated by a sequence of specific carboxy-terminal cleavages, and that the observed post-secretory processing of amelogenin is functionally linked to the structure of the enamel matrix and the control of crystallite development.