Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties

Abstract

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN-its oligomerization ability-is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca²⁺)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca²⁺ may explain the role of AMBN in biomineralization and more generally in Ca²⁺ homeostasis processes.

Keywords: ameloblastin; biomineralization; calcium binding; intrinsically disordered protein (IDPs); oligomerization.

Figure 1

(A) Circular dichroism (CD) spectra of ameloblastin full-length isoform I (AMBN ISO I), ameloblastin full-length isoform II (AMBN ISO II) and AMBN with deleted exon 5 (AMBN del E5). Sample before (black) and after (red) addition of 10 mM CaCl₂. (B) Analysis of autocorrelation functions (ACF) from dynamic light scattering (DLS) spectra of AMBN ISO I, AMBN ISO II, AMBN del E5, and bovine serum albumin (BSA) as a standard for globular protein. ACF of proteins in the absence or presence of 10mM CaCl₂. (C) First derivative of thermal unfolding curves of AMBN ISO I, AMBN ISO II, and AMBN del E5 in the presence or absence of 10 mM CaCl₂.

Figure 2

(A) Transmission electron micrographs of AMBN ISO I. Samples were examined at a magnification of 200,000×; scale bar = 100 nm. (B) Magnified detail of the AMBN ISO I presented in panel A. (C) Transmission electron micrographs of AMBN ISO II. Samples were examined at a magnification of 200,000×; scale bar = 100 nm. (D) Magnified detail of the AMBN ISO II presented in panel C.

Figure 3

Sedimentation velocity analysis of (A) AMBN ISO I and AMBN ISO II; (B) AMBN del E5; in the absence or presence of 10 mM CaCl₂. (C) Dependence of the effective electrophoretic mobility, m_P,eff, of AMBN ISO I and AMBN ISO II proteins on the concentration of CaCl₂ (c_CaCl2), in the background electrolyte (BGE) composed of 30 mM Tris, 25 mM acetic acid, pH = 7.4. (D) Microscale thermophoresis analysis of the interaction of the AMBN ISO I, AMBN del E5, and AMBN-Cterm with CaCl₂. Titration of CaCl₂ against a constant concentration of AMBN ISO I revealed a standard binding curve, while the titration of AMBN del E5 and AMBN C-term shows no binding.2.4. AMBN Oligomers of Both Isoforms Have a Heterogenous Character.

Figure 4

The schematic view of possible AMBN oligomerization state. AMBN monomers forming oligomers (e.g., hexamer). The blue rectangle illustrates AMBN-Nterm, while the green rectangle shows oligomerization inducing exon 5. Red lines show the possible arrangements of AMBN-Cterm in the absence of Ca²⁺ (A) and in the presence of Ca²⁺ (B) with yellow circles representing Ca²⁺. Black lines represent possible Ca²⁺ binding to AMBN-Cterm. (C) Schematic detail of possible Ca²⁺-binding sites of two C-termini of AMBN. Black rectangles represent sequences with a high abundance of negatively charged amino acids. (D) Detail view of potential Ca²⁺-binding sites at AMBN C-termini is shown in panel C. Amino acid sequences of AMBN-Cterm are in black rectangles. Negatively charged amino acids as a high preferable binding sites are bold white.