Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth

Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.     

Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.     

The enamel protein amelogenin binds to the N-acetyl-D-glucosamine-mimicking peptide motif of cytokeratins

Amelogenins bind to GlcNAc of the dentine-enamel matrix proteins (Ravindranath, R. M. H., Moradian-Oldak, J., Fincham, A. G. (1999) J. Biol. Chem. 274, 2464-2471). The hypothesis that amelogenins may interact with the peptides that mimic GlcNAc is tested. GlcNAc-mimicking peptide (SFGSGFGGGY) but not its variants with single amino acid substitution at serine, tyrosine, or phenylalanine residues inhibited hemagglutination of amelogenins and the terminal tyrosine-rich amelogenin polypeptide (TRAP). The binding affinity of SFGSGFGGGY to amelogenins was confirmed by dosimetric binding of amelogenins or TRAP with [(3)H]peptide, specific binding in varying concentrations of the peptide, Scatchard plot analysis, and competitive inhibition with the unlabeled peptide. The ability of the peptide or GlcNAc to stoichiometrically inhibit TRAP binding of [(14)C]GlcNAc or [(3)H]peptide indicated that both the peptide and GlcNAc compete for a single binding site. Using different fragments of amelogenins, we have identified the peptide-binding motif in amelogenin to be the same as the GlcNAc-binding "amelogenin trityrosyl motif peptide." The GlcNAc-mimicking peptide failed to bind to the amelogenin trityrosyl motif peptide when the tyrosyl residues were substituted with phenylalanine or when the third proline was replaced with threonine, as in some cases of human X-linked amelogenesis imperfecta. This study documents that molecular mimicry may play a role in stability and organization of amelogenin during amelogenesis.     

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.     

A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1)

Amelogenesis imperfecta is characterized by the defective formation of tooth enamel. Here we present evidence that the X-linked form of this disorder (AIH1) is caused by a structural alteration in one of the predominant proteins in enamel, amelogenin. Southern blot analysis revealed a deletion extending over 5 kb of the amelogenin gene in males with the hypomineralization form of the AIH1. Carrier females were heterozygous for the molecular defect. The deletion appears to include at least two exons of the amelogenin gene and the extent of the deletion was verified by PCR analysis. The mutation was shown to segregate with the disease among 15 analyzed individuals belonging to the same kindred. Our results link a defect in the amelogenin gene to the abnormal formation of enamel. We thus conclude that the amelogenin protein has a role in biomineralization of tooth enamel.     

A transgenic animal model resembling amelogenesis imperfecta related to ameloblastin overexpression

Genetic diseases that affect tooth enamel are grouped under the classification amelogenesis imperfecta. Human pedigrees and experiments on transgenic and null mice have all demonstrated that mutations to the secreted proteins amelogenin, enamelin, and enamelysin result in visibly, structurally, or mechanically defective enamel. In an attempt to better define a physiologic function for ameloblastin during enamel formation, we have produced transgenic mice that misexpress the ameloblastin gene. These transgenic animals exhibit imperfections in their enamel that is evident at the nanoscale level. Specifically, ameloblastin overexpression influences enamel crystallite habit and enamel rod morphology. These findings suggest enamel crystallite habit and rod morphology are influenced by the temporal and spatial expression of ameloblastin and may implicate the role of the ameloblastin gene locus in the etiology of a number of undiagnosed autosomally dominant cases of amelogenesis imperfecta.     

Partial rescue of the amelogenin null dental enamel phenotype

The amelogenins are the most abundant secreted proteins in developing dental enamel. Enamel from amelogenin (Amelx) null mice is hypoplastic and disorganized, similar to that observed in X-linked forms of the human enamel defect amelogenesis imperfecta resulting from amelogenin gene mutations. Both transgenic strains that express the most abundant amelogenin (TgM180) have relatively normal enamel, but strains of mice that express a mutated amelogenin (TgP70T), which leads to amelogenesis imperfecta in humans, have heterogeneous enamel structures. When Amelx null (KO) mice were mated with transgenic mice that produce M180 (TgM180), the resultant TgM180KO offspring showed evidence of rescue in enamel thickness, mineral density, and volume in molar teeth. Rescue was not observed in the molars from the TgP70TKO mice. It was concluded that a single amelogenin protein was able to significantly rescue the KO phenotype and that one amino acid change abrogated this function during development.     

Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides.

Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the approximately 20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by approximately 70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70-80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, approximately 65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen alpha-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.     

Matrix metalloproteinase inhibition impairs the processing, formation and mineralization of dental tissues during mouse molar development

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.     

Analysis of self-assembly and apatite binding properties of amelogenin proteins lacking the hydrophilic C-terminal.

Amelogenins, the major protein component of the mineralizing enamel extracellular matrix, are critical for normal enamel formation as documented in the linkage studies of a group of inherited disorders, with defective enamel formation, called Amelogenesis imperfecta. Recent cases of Amelogenesis imperfecta include mutations that resulted in truncated amelogenin protein lacking the hydrophilic C-terminal amino acids. Current advances in knowledge on amelogenin structure, nanospheres assembly and their effects on crystal growth have supported the hypothesis that amelogenin nanospheres provide the organized microstructure for the initiation and modulated growth of enamel apatite crystals. In order to evaluate the function of the conserved hydrophilic C-terminal telopeptide during enamel biomineralization, the present study was designed to analyze the self-assembly and apatite binding behavior of amelogenin proteins and their isoforms lacking the hydrophilic C-terminal. We applied dynamic light scattering to investigate the size distribution of amelogenin nanospheres formed by a series of native and recombinant proteins. In addition, the apatite binding properties of these amelogenins were examined using commercially available hydroxyapatite crystals. Amelogenins lacking the carboxy-terminal (native P161 and recombinant rM166) formed larger nanospheres than those formed by their full-length precursors: native P173 and recombinant rM179. These data suggest that after removal of the hydrophilic carboxy-terminal segment further association of the nanospheres takes place through hydrophobic interactions. The affinity of amelogenins lacking the carboxy-terminal regions to apatite crystals was significantly lower than their parent amelogenins. These structure-functional analyses suggest that the hydrophilic carboxy-terminal plays critical functional roles in mineralization of enamel and that the lack of this segment causes abnormal mineralization.     

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.     

Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.     

Ameloblastin peptide encoded by exon 5 interacts with amelogenin N-terminus

Interactions between enamel matrix proteins are important for enamel biomineralization. In recent in situ studies, we showed that the N-terminal proteolytic product of ameloblastin co-localized with amelogenin around the prism boundaries. However, the molecular mechanisms of such interactions are still unclear. Here, in order to determine the interacting domains between amelogenin and ameloblastin, we designed four ameloblastin peptides derived from different regions of the full-length protein (AB1, AB2 and AB3 at N-terminus, and AB6 at C-terminus) and studied their interactions with recombinant amelogenin (rP172), and the tyrosine-rich amelogenin polypeptide (TRAP). A series of amelogenin Trp variants (rP172(W25), rP172(W45) and rP172(W161)) were also used for intrinsic fluorescence spectroscopy. Fluorescence spectra of rP172 titrated with AB3, a peptide encoded by exon 5 of ameloblastin, showed a shift in λ_max in a dose-dependent manner, indicating molecular interactions in the region encoded by exon 5 of ameloblastin. Circular dichroism (CD) spectra of amelogenin titrated with AB3 showed that amelogenin was responsible for forming α-helix in the presence of ameloblastin. Fluorescence spectra of amelogenin Trp variants as well as the spectra of TRAP titrated with AB3 showed that the N-terminus of amelogenin is involved in the interaction between ameloblastin and amelogenin. We suggest that macromolecular co-assembly between amelogenin and ameloblastin may play important roles in enamel biomineralization.     

Dental enamel matrix: Sequences of two amelogenin polypeptides

The amino acid sequences of a leucine-rich amelogenin polypeptide (LRAP) and a tyrosine-rich amelogenin polypeptide (TRAP), isolated from foetal bovine enamel matrix, were determined. Both LRAP and TRAP occurred in two forms; in each case, one of the molecular species appeared to be shortened at the COOH terminus by 2 and 4 residues, respectively. A striking finding was that LRAP and TRAP had identical sequences for the first 33 residues but were almost completely different for the remaining 12 amino acids.     

Amelogenin sequence and enamel biomineralization in Rana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.     

Evidence for regulation of amelogenin gene expression by 1,25‐dihydroxyvitamin D3 in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X‐linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D‐deficient (−D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in −D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in −D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady‐state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in −D rats and up‐regulated by an unique injection of 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth‐specific genes. J. Cell. Biochem. 76:194–205, 1999. © 1999 Wiley‐Liss, Inc.     

Defining a New Candidate Gene for Amelogenesis Imperfecta: From Molecular Genetics to Biochemistry

Amelogenesis imperfecta is a group of genetic conditions that affect the structure and clinical appearance of tooth enamel. The types (hypoplastic, hypocalcified, and hypomature) are correlated with defects in different stages of the process of enamel synthesis. Autosomal dominant, recessive, and X-linked types have been previously described. These disorders are considered clinically and genetically heterogeneous in etiology, involving a variety of genes, such as AMELX, ENAM, DLX3, FAM83H, MMP-20, KLK4, and WDR72. The mutations identified within these causal genes explain less than half of all cases of amelogenesis imperfecta. Most of the candidate and causal genes currently identified encode proteins involved in enamel synthesis. We think it is necessary to refocus the search for candidate genes using biochemical processes. This review provides theoretical evidence that the human SLC4A4 gene (sodium bicarbonate cotransporter) may be a new candidate gene.