pH triggered self-assembly of native and recombinant amelogenins under physiological pH and temperature in vitro

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. This study examines the effect of temperature and pH on amelogenin self-assembly under physiological pH conditions in vitro, using dynamic light scattering, turbidity measurements, and transmission electron microscopy. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated, along with proteolytic cleavage products (rM166 and native P148) lacking the hydrophilic C-terminus of parent molecules. Results indicated that the self-assembly of full-length amelogenin is primarily triggered by pH in the temperature range from 13 to 37 degrees C and not by temperature. Furthermore, very large assemblies of all proteins studied formed through the rearrangement of similarly sized nanospherical particles, although at different pH values: pH 7.7 (P148), pH 7.5 (rM166), pH 7.2 (rP172), and pH 7.2 (rM179). Structural differences were also observed. The full-length molecules formed apparently tightly connected elongated, high-aspect ratio assemblies comprised of small spheres, while the amelogenin cleavage products appeared as loosely associated spherical particles, suggesting that the hydrophilic C-terminus plays an essential role in higher-order amelogenin assembly. Hence, tightly controlled pH values during secretory amelogenesis may serve to regulate the functions of both full-length and cleaved amelogenins.     

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.     

Amelogenin and enamelysin localization in human dental germs

Odontogenesis is extensively studied in animal models but less understood in human. In early amelogenesis, amelogenin constitutes 90% of enamel organic matrix, which is degraded by enamelysin and replaced by hydroxyapatite crystals. Here, amelogenin and enamelysin distribution changes during amelogenesis were shown by co-localization experiments by confocal microscopy. Early bell stage showed more amelogenin labeling than enamelysin, as free immune-reactive granular patches towards basal membrane between ameloblast and odontoblast. Increased amelogenin expression and secretion towards extracellular matrix formation region was found. Enamelysin distribution was perinuclear in early bell stage. During late bell stage, a decreasing amelogenin labeling in contrast with enamelysin increasing along the cells was found, suggesting specific temporal amelogenin degradation. Enamelysin was located initially around nuclei and later was found in all the ameloblast and stellate reticulum cytoplasm. Amelogenin was observed inside ameloblast, stellate reticulum, and intermediate stratum cells in the enamel as well as in the newly formed dentin extracellular matrix. In contrast, in dentin more amelogenin than enamelysin was found located close to the periphery.     

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta

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.     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

Reprint of "Stereology meets electron tomography: towards quantitative 3D electron microscopy" [J. Struct. Biol. 159 (2007) 443-450

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. The full-length amelogenin uniquely regulates the growth, shape, and arrangement of enamel crystals. Protein hydrolysis will ultimately facilitate a tissue with high mineral content. Protein processing is however highly specific suggesting a functional role of the cleaved amelogenins in enamel maturation. Here we hypothesize that the cooperative self-assembly of the recombinant full-length amelogenin 25 kDa and the 23 kDa proteolytic cleavage product is a function of pH, mixing ratio and incubation time and is associated with the isoelectric point of the protein. Self-assembly of amelogenin into nanospheres which increased in size with increasing pH was observed by atomic force microscopy. Elongated structures of about 100 nm length and 25 nm width formed over several days for amelogenin 25 and 23 kDa predominantly at pH-values of 6.5 and 7.5, respectively. When both proteins 25 and 23 kDa were mixed, self-assembled nanostrings of 200–300 nm length consisting of fused nanospheres were obtained at pH around 7.0 within 24 h. The protein nanostrings formed links over time and a continuous mesh was obtained after 7 days. Electrical conductivity data also showed gradual changes when both amelogenins were mixed in solutions supporting the idea that elongated structures form over extended periods of time. We propose that due to the difference in the isoelectric point, self-assembled nanospheres composed of 23 or 25 kDa amelogenin have opposite ionic charges at pH-values around 7.0 and thus experience ionic attraction that enables cooperative self-assembly.     

Amelogenin-Collagen Interactions Regulate Calcium Phosphate Mineralization in Vitro

Collagen and amelogenin are two major extracellular organic matrix proteins of dentin and enamel, the mineralized tissues comprising a tooth crown. They both are present at the dentin-enamel boundary (DEB), a remarkably robust interface holding dentin and enamel together. It is believed that interactions of dentin and enamel protein assemblies regulate growth and structural organization of mineral crystals at the DEB, leading to a continuum at the molecular level between dentin and enamel organic and mineral phases. To gain insight into the mechanisms of the DEB formation and structural basis of its mechanical resiliency we have studied the interactions between collagen fibrils, amelogenin assemblies, and forming mineral in vitro, using electron microscopy. Our data indicate that collagen fibrils guide assembly of amelogenin into elongated chain or filament-like structures oriented along the long axes of the fibrils. We also show that the interactions between collagen fibrils and amelogenin-calcium phosphate mineral complexes lead to oriented deposition of elongated amorphous mineral particles along the fibril axes, triggering mineralization of the bulk of collagen fibril. The resulting structure was similar to the mineralized collagen fibrils found at the DEB, with arrays of smaller well organized crystals inside the collagen fibrils and bundles of larger crystals on the outside of the fibrils. These data suggest that interactions between collagen and amelogenin might play an important role in the formation of the DEB providing structural continuity between dentin and enamel.     

Amelogenin-cytokeratin 14 interaction in ameloblasts during enamel formation.

The enamel protein amelogenin binds to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif is found in the N-terminal region of CK14, a differentiation marker for ameloblasts. The binding affinity of CK14 and amelogenin was confirmed by dosimetric binding of CK14 to recombinant amelogenin (rM179), and to the tyrosine-rich amelogenin polypeptide. The specific binding site for CK14 was identified in the amelogenin trityrosyl motif peptide (ATMP) of tyrosine-rich amelogenin polypeptide and specific interaction between CK14 and [(3)H]ATMP was confirmed by Scatchard analysis. Blocking rM179 with GlcNAc, GMp, or CK14 with ATMP abrogates the CK14-amelogenin interaction. CK14 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Morphometry of developing teeth distinguished three phases of enamel formation; growth initiation phase (days 0-1), prolific growth phase (days 1-7), and growth cessation phase (post-day 7). Confocal microscopy revealed co-assembly of CK14/amelogenin in the perinuclear region of ameloblasts on day 0, migration of the co-assembled CK14/amelogenin to the apical region of the ameloblasts from day 1, reaching a peak on days 3-5, and a collapse of the co-assembly. Autoradiography with [(3)H]ATMP and [(3)H]GMp corroborated the dissociation of the co-assembly at the ameloblast Tomes' process. It is proposed that CK14 play a chaperon role for nascent amelogenin polypeptide during amelogenesis.     

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions

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.     

Spatiotemporal expression of ameloblastin isoforms during murine tooth development

Ameloblasts synthesize and secrete the enamel matrix proteins (amelogenin, ameloblastin, and enamelin). This investigation examined the profiles of ameloblastin in the ameloblasts and in the enamel matrix during different postnatal (PN) days (days 0-9) of development of mouse molar, using an antibody specific for C-terminal sequence of ameloblastin (Ct; GNKVHQPQVHNAWRF). Ameloblastin is found in three different molecular sizes (37, 55, and 66 kDa) in both ameloblasts and enamel matrix during PN development. In the ameloblasts, the sequence of expression of these fractions varied. The 37-kDa fraction was observed (even before the appearances of mRNA of the proteases, enamelysin and kallikrein-4) on days 0 and 1, persisted until day 3, and was not found thereafter. Other isoforms (55 and 66 kDa) distinctly appeared in ameloblasts after day 1, reached a peak on day 5, and remained thereafter. The Ct-positive granules appeared beaded in the ameloblasts on day 3. In the extracellular matrix, a 37-kDa (but not 66- or 55-kDa) fraction was detected on days 0 and 1 and remained in the matrix throughout the PN days. The larger isoforms (55 and 66 kDa) appeared in the enamel matrix from day 3 onward. On days 0-3, but not later, the 37-kDa isoform co-localizes with amelogenin in Tomes' process and formative enamel, as revealed by laser scan confocal microscopy. Autoradiography confirmed accumulation of 3H-labeled amelogenin trityrosyl motif peptide in the region of Tomes' process and formative enamel from day 0 to 3. These observations suggest that the 37-kDa isoform interacts with amelogenin during early tooth development.     

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.     

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods’ axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.     

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.     

The Evolution of Enamel Microstructure: How Important Is Amelogenin?

The shape, size, and orientation of enamel prisms have heretofore been thought to be controlled solely by the shape of the Tomes' process. It is known, however, that amelogenin proteins play an important role in enamel deposition and maturation and it is possible that they contribute independently to enamel structure. Using a phylogenetic framework, we clarify the role of amelogenin proteins in the formation of enamel microstructure. We found a negative association between evolutionary changes in amelogenin protein sequences and enamel complexity: amelogenin evolution slows as enamel complexity increases. This is probably because selective constraints on amelogenin increase as enamel complexity increases. Monotremes, which have lost their adult dentition, have particularly high rates of amelogenin evolution while rodents, which have very complex enamel, have very low rates. There is a positive correlation between the number of different amelogenin proteins in a given species and the complexity of its enamel microstructure. An increased number of amelogenins may be necessary for the formation of multiple enamel types in the same tooth. Alternative splicing of amelogenin exons, which allows multiple protein products to be produced from the same gene, may be a key innovation in the diversification of enamel microstructure.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Cloning and Expression Analysis of Human Amelogenin in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> Plants by Means of a Transient Expression System

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF–MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.     

Studies of the morphology and structure of the plasma lipid transfer particle from the tobacco hornworm, Manduca sexta

Morphological features of Manduca sexta plasma lipid transfer particle (LTP) have been investigated by electron microscopy. LTP was found to be an asymmetric particle with two major structural features: a roughly spherical head and an elongated, hinged tail. The hinge occurs approximately at the midpoint of the tail section with the two halves forming angles ranging from 30 degrees to 180 degrees. A molecular mass estimate of 1.4 x 10(6) daltons based on the dimensions of LTP suggests that multiple copies (two or three) of each of the three LTP apoproteins exist in the native complex. Limited digestion studies of LTP suggest that apoLTP-III is less susceptible to trypsin cleavage than apoLTP-I or -II, and therefore may be less exposed to the aqueous environment. Digestion for 1 h at a 1:50 trypsin-LTP protein ratio did not alter the flotation properties of LTP or its morphological features; thus, although significant proteolysis occurred, the particle retained its overall structure. Transfer activity, on the other hand, was affected by trypsin digestion with 30 +/- 14% inhibition of LTP activity occurring upon proteolysis at a 1:50 trypsin-LTP protein ratio. Treatment of LTP with phospholipase A2 resulted in the conversion of LTP-associated phosphatidylcholine and phosphatidylethanolamine to their corresponding lyso forms. Phospholipase A2 treatment did not, however, alter the SDS-PAGE profile, transfer activity, flotation pattern, or the microscopic features of LTP. These results suggest that the products of the phospholipase reaction remain associated with the particle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amelogenin supramolecular assembly in nanospheres defined by a complex helix-coil-PPII helix 3D-structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 3(10) helices (～P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule.