Amelogenins, cell adhesion proteins, play a role in the biomineralisation of teeth. They seem to regulate formation of crystallites during the secretory stage of tooth enamel development and are thought to play a major role in the structural organisation and mineralisation of developing enamel. The extracellular matrix of the developing enamel comprises two major classes of protein: the hydrophobic amelogenins and the acidic enamelins. Circular dichroism studies of porcine amelogenin have shown that the protein consists of 3 discrete folding units: the N-terminal region appears to contain beta-strand structures, while the C-terminal region displays characteristics of a random coil conformation. Subsequent studies on the bovine protein have indicated the amelogenin structure to contain a repetitive beta-turn segment and a "beta-spiral" between Gln112 and Leu138, which sequester a (Pro, Leu, Gln) rich region. The beta-spiral offers a probable site for interactions with Ca2+ ions. Muatations in the human amelogenin gene (AMGX) cause X-linked hypoplastic amelogenesis imperfecta, a disease characterised by defective enamel. A 9bp deletion in exon 2 of AMGX results in the loss of codons for Ile5, Leu6, Phe7 and Ala8, and replacement by a new threonine codon, disrupting the 16-residue (Met1-Ala16) amelogenin signal peptide.
Amelogenins, cell adhesion proteins, play a role in the biomineralisation of teeth. They seem to regulate formation of crystallites during the secretory stage of tooth enamel development and are thought to play a major role in the structural organisation and mineralisation of developing enamel. The extracellular matrix of the developing enamel comprises two major classes of protein: the hydrophobic amelogenins and the acidic enamelins [ (PUBMED:8118759) ].
Circular dichroism studies of porcine amelogenin have shown that the protein consists of 3 discrete folding units [ (PUBMED:8454575) ]: the N-terminal region appears to contain beta-strand structures, while the C-terminal region displays characteristics of a random coil conformation. Subsequent studies on the bovine protein have indicated the amelogenin structure to contain a repetitive beta-turn segment and a "beta-spiral" between Gln112 and Leu138, which sequester a (Pro, Leu, Gln) rich region [ (PUBMED:2598664) ]. The beta-spiral offers a probable site for interactions with Ca2+ ions.
Muatations in the human amelogenin gene (AMGX) cause X-linked hypoplastic amelogenesis imperfecta, a disease characterised by defective enamel. A 9bp deletion in exon 2 of AMGX results in the loss of codons for Ile5, Leu6, Phe7 and Ala8, and replacement by a new threonine codon, disrupting the 16-residue (Met1-Ala16) amelogenin signal peptide [ (PUBMED:7782077) ].
Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta.
Matrix Biol. 2001; 19: 755-60
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Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.
Amelogenin signal peptide mutation: correlation between mutations in the amelogenin gene (AMGX) and manifestations of X-linked amelogenesis imperfecta.
Genomics. 1995; 26: 159-62
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Formation of tooth enamel is a poorly understood biological process. In this study we describe a 9-bp deletion in exon 2 of the amelogenin gene (AMGX) causing X-linked hypoplastic amelogenesis imperfecta, a disease characterized by defective enamel. The mutation results in the loss of 3 amino acids and exchange of 1 in the signal peptide of the amelogenin protein. This deletion in the signal peptide probably interferes with translocation of the amelogenin protein during synthesis, resulting in the thin enamel observed in affected members of the family. We compare this mutation to a previously reported mutation in the amelogenin gene that causes a different disease phenotype. The study illustrates that molecular analysis can help explain the various manifestations of a tooth disorder and thereby provide insights into the mechanisms of tooth enamel formation.
Production of a monoclonal antibody against human amelogenin.
Calcif Tissue Int. 1994; 54: 76-80
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The extracellular organic matrix of developing human enamel is composed of two major classes of proteins, the hydrophobic amelogenins and the acidic enamelins. In order to identify, purify, and characterize the amelogenins from this complex mixture of proteins, and to study their ultrastructural localization and their pathways of synthesis, secretion, and degradation, specific and sensitive probes are needed. In the present paper the production of a monoclonal antibody against human amelogenin employing an intrasplenic primary immunization protocol is described. The monoclonal antibody produced is IgM and recognizes major human amelogenin protein bands in Western immunoblot assays. It also recognizes amelogenin protein bands from other species, specifically bovine and porcine. Indirect immunohistochemical studies showed the monoclonal antibody to react specifically with the extracellular matrix of human developing enamel. It did not react with the underlying dentin layer.
Molecular conformation of porcine amelogenin in solution: three folding units at the N-terminal, central, and C-terminal regions.
J Biochem (Tokyo). 1993; 113: 55-60
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Circular dichroism (CD) studies were conducted to gain a better insight into the conformation of amelogenins, which were isolated from developing enamel of piglets. The intact porcine amelogenin and its degraded products were purified chromatographically. The 25-residue peptide corresponding to the segment at the C-terminus was synthesized. CD spectra of these samples were measured at pH 5.0-5.3 in the temperature range between 4 and 90 degrees C. The most remarkable finding was that the CD spectrum of the intact amelogenin was accounted for by the sum of the spectra of the three fragments at the N-terminal, central, and C-terminal regions, supporting the hypothesis that the structure of the whole protein consists of discrete folding units. Furthermore, low-angle laser light scattering analysis provided evidence that the 20 kDa amelogenin, the most abundant extracellular matrix protein in forming enamel tissue, exists in a monomeric form at pH 5.3 and 25 degrees C. It was tentatively concluded that the N-terminal region contains beta-sheet structures, while the spectral characteristics of the C-terminal region are similar to those of a random coil conformation. The conformation of the central region was characterized by a strong negative ellipticity at 203 nm, although its nature remains to be defined.
Secondary structure and limited three-dimensional structure of bovine amelogenin.
Connect Tissue Res. 1989; 22: 131-8
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Secondary structural features of bovine amelogenin, a hydrophobic protein of developing enamel implicated in ename mineralization, are derived using 2D NMR spectroscopy in solution and molecular mechanics-dynamics studies. A beta-turn: beta-sheet model with some "unordered" segments was previously proposed from circular dichroism, Fourier-transform infrared and Raman spectroscopy augmented by Chou-Fasman predictive algorithm. The proposed structure contains a repetitive beta-turn segment, "beta-spiral" between Gln112 and Leu138 residues containing a (Pro, Leu, Gln) rich segment. The beta-spiral structure offers a probable site for interaction of Ca++ ions. Assignment of proton resonances using 2D COSY spectroscopy is presently in progress. Preliminary 2D NOESY spectra have revealed the presence of Tyr residues (TRAP segment) on the surface of amelogenin molecule and clusters of cross peaks reminiscent of beta-turns and sheets which are consistent with the primary structure and proposed secondary structures of amelogenin. The channel-like beta-spiral structure embedded in amelogenin provides a novel mechanism for trapping of Ca++ ions and their passage for a hydrophobic protein sparse in Ser(P) and charged amino acid residues.
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