Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5

Abstract

Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.

Keywords: Ameloblastin; Amelogenin; Extracellular Matrix Proteins; Intrinsically Disordered Proteins; Protein Purification; Protein Self-assembly; Tooth.

FIGURE 1.

Production and analysis of recombinant human ameloblastin. A, scheme of the production and purification of AMBN. The fusion protein used for AMBN purification consists of AMBN (lacking the N-terminal secretion signal, Sec), which was genetically fused at the N terminus to a double His purification tag (dH), Trx, and a cleavable TEV specific peptide and at the C terminus to the TEV peptide and a single His purification tag (sH), respectively. After expression in E. coli BL21(λDE3) cells, the fusion protein is captured on an Ni-NTA affinity matrix, whereas the contaminating cell extract proteins are washed away from the column. After elution and dialysis, the fusion protein is processed in solution with TEV protease, and the excised AMBN is separated from Trx and purification tags by reversed phase chromatography on a PLRP-S column. The resulting purified recombinant AMBN harbors three extra residues (GAS) at the N terminus and a short nonapeptide (PREENLYFQ) at the C terminus. B, the samples collected throughout the AMBN production process were separated by SDS-PAGE (12.5%) and stained with Coomassie Blue. Lane St, molecular mass standards; lane 1, crude extract from uninduced cells; lane 2, crude extract from cells induced for production of the Trx-AMBN fusion protein; lane 3, clarified crude extract from induced cells; lane 4, Ni-NTA column flowthrough; lane 5, Ni-NTA column wash; lane 6, fraction of eluted Trx-AMBN fusion protein from Ni-NTA column; lane 7, products of Trx-AMBN fusion cleavage by TEV protease; lane 8, AMBN after reversed phase chromatography on a PLRP-S column. Lane W, purified AMBN (lane 8) was separated by SDS-PAGE (12.5%), transferred to a nitrocellulose membrane, and immunodetected by a polyclonal anti-AMBN rabbit serum. *, bands analyzed by MS.

FIGURE 2.

Ameloblastin forms supramolecular structures, similarly as amelogenin. A, the purified full-length AMBN and AMEL proteins were analyzed by size exclusion chromatography on a Superdex 200 10/30 column. The absorbances at 280 nm of eluted proteins are indicated for AMBN (red line, left y axis) or AMEL (black line, right y axis). The molecular masses of the globular protein standards are indicated above the chromatograms in kDa. V₀, void volume. B, SDS-PAGE analysis of the AMBN and AMEL peak fractions collected during size exclusion chromatography. Lane St, molecular mass standards; lane 1, AMBN excluded peak; lane 2, AMBN included peak; lane 3, AMEL excluded peak; lane 4, AMEL included peak. The samples were analyzed on a 12.5% SDS-polyacrylamide gel and stained with Coomassie Blue. C and D, sedimentation velocity measurements were performed to analyze the high molecular mass structures of AMBN (C) and AMEL (D). Experiments were performed with different concentrations of AMBN or AMEL in 20 mm Tris-HCl (pH 7.4), 200 mm NaCl, at 20 °C and 25,000 rpm. Protein peaks were detected by using the absorption optical system at 280 nm. Rel. Abs., relative absorption.

FIGURE 3.

Residues 36–72 are essential for self-association of ameloblastin. A, schematic representation of truncated AMBN variants. The portions deleted in AMBN are indicated by the solid lines separating the colored bars. The numbers following the symbol Δ indicate the first and the last residue numbers of the respective portion deleted in the given protein construct. S_36–72-AMBN-C_term represents a fusion between segment 36–72 of AMBN and AMBN-C_term. B and C, the purified AMBN mutant variants were analyzed by size exclusion chromatography on a Superdex 200 10/30 column. The molecular masses of the protein standards are indicated above chromatograms in kDa. V₀, void volume. D and E, sedimentation velocity measurements were used to characterize the C-terminal domain of AMBN (D) and the AMBNΔ36–72 mutant variant (E). Experiments were performed with different concentrations of AMBN-C_term or AMBNΔ36–72 in 20 mm Tris-HCl (pH 7.4), 200 mm NaCl, at 20 °C and 48,000 rpm. Protein peaks were detected by using the absorption optical system at 280 nm. Rel. abs., relative absorption.

FIGURE 4.

The AMBN-derived peptide involved in ameloblastin self-association consists of two regions with a relatively well defined conformation. Structures are represented by best fit superpositions of the peptide backbone for 20 converged structures with the region including residues Phe-46 to Pro-57 highlighted in red and the region including residues Ser-60 to Pro-66 highlighted in green. A, superposition over the region from Phe-46 to Pro-66. B, superposition over the first structured region (Phe-46 to Pro-57), which includes a short α-helix between residues Ser-48 and Gly-53. C, superposition over the second relatively structured region (Ser-59 to Pro-66). Both N and C termini are disordered.

FIGURE 5.

Transmission electron micrographs of ameloblastin and its mutant variants. Purified AMBN (A–D), AMBN-N_term (E and F), AMBN-C_term (G), AMBNΔ36–72 (H), S_36–72-AMBN-C_term (I and J), and AMEL (K) were applied onto glow discharge-activated carbon coated grids and negatively stained with 2% uranyl acetate. Samples were examined at a magnification of 64,000×. J shows a 2.84-fold digitally magnified detail of the ribbon-like structure of the S_36–72-AMBN-C_term protein filament presented in I. Scale bars, 100 nm (A–I and K), or 25 nm (J).

FIGURE 6.

Atomic force microscopy analysis of ameloblastin and its mutant variants. A, AMBN. A typical cross-section profile of a self-assembled ribbon (1) and a defect in protein monolayer (2) is shown on graph bellow the image. B, AMBNΔ36–72 mutant. A typical cross-section profile of a protein monomer (1) and a defect in protein monolayer (2) is shown on graph bellow the image. C, histogram of thickness of AMBNΔ36–72 monolayer measured at defects. D, height histogram of AMBNΔ36–72 monomers. E, height histogram of the C-terminal domain of AMBN.

FIGURE 7.

Schematic view of the possible arrangements of ameloblastin monomers within supramolecular structures. AMBN monomers with parallel (A) or antiparallel (B) orientation form supramolecular structures through interactions in the N-terminal segment comprising residues 36–72. The C-terminal domain of AMBN is apparently not critical for the self-assembly, and because of its highly charged character, it is likely to be important for protein solubility.