Amelogenins. Sequence homologies in enamel-matrix proteins from three mammalian species.

Partial amino acid sequences for selected amelogenin polypeptides isolated from the developing enamel of cow, pig and human foetuses are reported. It was found that there was an identity of sequence for the initial 28 residues of the polypeptides analysed, irrespective of their origin or size. A tyrosine-rich polypeptide was shown to be the N-terminal fragment of the principal higher-molecular-weight amelogenins, although a leucine-rich polypeptide of similar size was not identified in any other amelogenin structure. The findings demonstrate a striking degree of sequence conservation for the amelogenin proteins of the extracellular enamel matrix and support the concept of a discrete fragmentation of an initial 30 000 Da amelogenin molecule during the mineralization of the enamel.     

Human and mouse cementum proteins immunologically related to enamel proteins

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.     

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.     

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.     

DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.     

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.     

Heterogeneity of amelogenin mRNA in the bovine tooth germ

The amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. To study the molecular mechanisms underlying the heterogeneity of the amelogenins we isolated cDNA clones encoding these proteins. The clones were definitively identified by hybrid-selected translation experiments and by comparison of the DNA sequence with the protein-derived amino acid sequence. Southern hybridization of bovine genomic DNA indicated that amelogenin is a single copy gene. However, Northern hybridization experiments distinctly showed two major species of mRNA, each of which were sufficiently large enough to encode the highest known molecular weight species of amelogenin proteins. Furthermore, immunoprecipitation of hybrid-selected translation products using isolated amelogenin cDNA showed multiple, translated protein products. These data are supportive of a differential mRNA processing mechanism involved in generating a heterogeneous family of amelogenin matrix proteins from a single gene.     

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.     

The nuclear basic proteins of human testes and ejaculated spermatozoa

Human testicular nuclei were fractionated into two fractions according to their sedimentation in a sucrose density gradient. The nuclear basic proteins isolated from these two fractions were similar and also resembled electrophoretic mobilities and amino acid composition of the liver histones. Only quantitative differences among histone electrophoretic bands were observed. The nuclear basic proteins of ejaculated spermatozoa differed totally from those of the testes. The proteins could be divided into two categories on the basis of their electrophoretic mobilities, molecular weights and amino acid compositions. One group (SpH) was similar to testicular histones; another (HP) group was smaller, with nearly twice the electrophoretic mobility and a much higher arginine content. These proteins (HP) represent a new type of nuclear basic protein found in human tissues.     

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.     

Characterization of cuticular proteins from the migratory locust,Locusta migratoria

Proteins were extracted from the still unhardened (teneral) cuticle of the migratory locust, Locusta migratoria. The proteins are soluble only at extreme pH-values and at low ionic strength, the solubility increases with decreasing temperature. The unhardened cuticle contains approx. 100 different proteins according to two-dimensional polyacrylamide gel electrophoresis. The majority of the proteins are very basic. The basicity and solubility properties of the proteins have necessitated development of modified electrophoretic procedures. The amino acid composition of the bulk protein shows that alanine, proline, glycine, valine and tyrosine constitute two thirds of the total amino acid content and that cysteine, methionine and tryptophan are absent.The proteins have been extracted from various parts of the cuticle and analysed by two-dimensional electrophoresis. Characteristic protein compositions were found for cuticle from the different body parts. Amino acid analyses of these extracts are strikingly similar. The only significant difference is in the glycine-alanine ratio. Cuticles that are destined to become hard are extremely rich in alanine, whereas the flexible parts of the cuticle are enriched in glycine. The results indicate that the proteins of locust cuticle constitute a group of structural proteins different from other known structural proteins.     

The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro

Amelogenin is the most abundant protein in developing dental enamel. It is believed to play an important role in the regulation of the growth and organization of enamel crystals. Amelogenin, unlike many other proteins found in biominerals, is mostly hydrophobic except for a 13 amino acid hydrophilic C-terminal domain. To clarify the role of amelogenin in enamel mineralization, we designed calcium phosphate crystal growth experiments in the presence of recombinant amelogenins with or without the charged C-terminal domain. The shape and organization of the crystals were examined by TEM in bright field and diffraction modes. It was found that both full-length and truncated amelogenin inhibit crystal growth in directions normal to the c-axis. At the same time, crystallites organized into parallel arrays only in the presence of the full-length amelogenin in monomeric form. Pre-assembled amelogenins had no effect on crystals organization. These results imply that the hydrophobic portion of amelogenin plays a role in an inhibition of crystal growth, whereas the C-terminal domain is essential for the alignment of crystals into parallel arrays. Our data also suggest that nascent enamel structure emerges as a result of cooperative interactions between forming crystals and assembling proteins.     

The subunit structure of prealbumin

1. Prealbumin, a thyroxine-binding protein, is known to bind also to retinol-binding protein and is suspected on X-ray-crystallographic evidence of having a quaternary structure. 2. Experiments described in this paper suggest that the molecule is a tetramer of mol.wt. about 56000, which is disaggregated with some difficulty into subunits of mol.wt. about 14000. 3. The number and staining properties of the tryptic peptides indicate that the subunits are identical or closely similar. This conclusion is reinforced by comparing the sum of the amino acid compositions of the tryptic peptides with the amino acid composition of the whole protein. All minor peptides that were isolated could be shown to be derived from major peptides. 4. No evidence has been found, either from electrophoretic experiments or from amino acid sequence determination, for any dissimilarity between the subunits.     

Pig plasma protease inhibitor gene complex: isolation and partial characterization of three inhibitors

1. Pig plasma alpha-protease inhibitors (protease inhibitor-1, PI1; protease inhibitor-2, PI2; postalbumin-1A, PO1A; postalbumin-1B, PO1B), all encoded by one gene complex (gene cluster), were isolated by rivanol-ammonium sulphate fractionation and double-one dimensional IPG-PAGE. The proteins were recovered from the polyacrylamide gel by a combination of electrophoresis and isoelectric focusing. 2. Molecular wt estimated by SDS-PAGE under reducing conditions was 63,000 each for PI1 and PI2 and 60,000 each for PO1A and PO1B. The two main components of a genetic variant of PI2 differed in mol. wt by approx. 1000. 3. PO1A, PO1B and PI2 were shown to be glycoproteins. The major component of both PO1A and PO1B contained about 15% carbohydrate and the two components of PI2 had about 24 per cent and 21 per cent carbohydrate, respectively. 4. Neuraminidase treatment showed that the main component of PO1A had 8 sialic acid residues and fast and slow components of PI2 had respectively 11 and 10 residues. 5. Amino acid compositions of PO1A, PO1B and PI2 were very similar to one another, indicating that the genes for these proteins have evolved by regional duplications of a common ancestral gene. 6. The results (mol. wt, amino acid and carbohydrate compositions) confirm that pig PI2 is homologous to human plasma alpha 1-antichymotrypsin.     

Pig plasma postalbumin-2 (alpha 1B-glycoprotein): isolation, partial characterization and immunological cross-reactivity with other mammalian sera

1. Components of pig plasma postalbumin-2 (PO2) protein, after rivanol-ammonium sulphate fractionation of plasma, were separated from other proteins by an easy and rapid method of horizontal double-one dimensional IPG-PAGE. The protein was recovered from polyacrylamide gel by combination of electrophoresis and isoelectric focusing. 2. The mol. wt of PO2 was estimated to be 68,000, using SDS-PAGE. 3. Amino acid and carbohydrate compositions of PO2 were very similar to those of human plasma alpha 1B-glycoprotein (alpha 1B), confirming that PO2 is the porcine homologue of human alpha 1B. 4. Neuraminidase treatment resulted in a decrease of electrophoretic migration velocity of all four studied components of PO2. 5. Homologous proteins to pig PO2 (alpha 1B) were observed, not only in human plasma but also in plasma of dog, horse and rabbit, by immunoblotting.     

Glycinin composition of several perennial species related to soybean

The 7S and 11S seed storage proteins from four perennials related to soybean (Glycine canescens, G. tomentella, G. tabacina, and G. clandestina) were analyzed by sodium dodecyl sulfate-gel electrophoresis. Each species yielded a unique electrophoretic pattern that varied in the total number of bands and their relative mobilities. In every case, the electrophoretic patterns were substantially different from CX635-1-1-1, the strain of G. max used in this study for comparison. Size heterogeneities among both the 7S and 11S polypeptides of the perennials were evident.

Abundant proteins in the 11S fraction from G. tomentella (CSIRO No. 1133) were separated by chromatography on DEAE-Sephadex and then their apparent molecular weights, amino acid compositions, and NH₂-terminal amino acid sequences were determined. A group of proteins were obtained which resembled the A_1b-polypeptide components of glycinin from G. max. They had the same size (M_r 37,000), identical NH₂-terminal sequences, and similar amino acid compositions to A_1b. A second group of acidic proteins (M_r 50,000) in G. tomentella had NH₂-terminal sequences homologous to the A₅ component (M_r 10,000) of glycinin. The latter group of polypeptides had a substantially higher apparent molecular weight than any acidic polypeptide components of glycinin analyzed previously. A third group of polypeptides purified from G. tomentella were the same size as basic polypeptides of glycinin and had homologus NH₂-terminal sequences. The results indicated that the perennials exhibit variability in their seed proteins at a level not found among the cultivars of G. max and G. soja and may be useful in studies concerning the origin and organization of genes involved in the synthesis of storage proteins in cultivated soybeans.

     

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.     

Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions

The matrix-mediated enamel biomineralization involves secretion of the enamel specific amelogenin proteins that through self-assembly into nanosphere structures provide the framework within which the initial enamel crystallites are formed. During enamel mineralization, amelogenin proteins are processed by tooth-specific proteinases. The aim of this study was to explore the factors that affect the activity of enamel proteases to process amelogenins. Two factors including amelogenin self-assembly and enzyme specificity are considered. We applied a limited proteolysis approach, combined with mass spectrometry, in order to determine the surface accessibility of conserved domains of amelogenin assemblies. A series of commercially available proteinases as well as a recombinant enamelysin were used, and their proteolytic actions on recombinant amelogenin were examined under controlled and limited conditions. The N-terminal region of the recombinant mouse amelogenin rM179 was found to be more accessible to tryptic digest than the C-terminal region. The endoproteinase Glu-C cleaved amelogenin at both the N-terminal (E18/V) and C-terminal (E178/V) sites. Chymotrypsin cleaved amelogenin at both the carboxy- (F151/S) and amino-terminal (W25/Y) regions. Interestingly, the peptide bond F/S152 was also recognized by the action of enamelysin on recombinant mouse amelogenin whereas thermolysin cleaved the S152/M153 peptide bond in addition to T63/L64 and I159/L160 and M29/I30 bonds. It was then concluded that regions at both the carboxy- and amino-terminal were exposed on the surface of amelogenin nanospheres when the N-terminal 17 amino acid residues were proposed to be protected from proteolysis, presumably as the result of their involvement in direct protein-protein interaction. Cleavage around the FSM locus occurred by recombinant enamelysin under limited conditions, in both mouse (F151/S152) and pig amelogenins (S148/M). Our in vitro observations on the limited proteolysis of amelogenin by enamelysin suggest that enamelysin cleaved amelogenin at the C-terminal region showing a preference of the enzyme to cleave the S/M and F/S bonds. The present limited proteolysis studies provided insight into the mechanisms of amelogenin degradation during amelogenesis.