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.

Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

“…In addition, some conserved sequences can be traced back even further in the evolution of whole Sarcopterygii and Osteichthyes lineages. Such observations support the theory of the non-canonical roles of EMPs in more general physiological processes over entire vertebrate evolution 22 .Therefore, EMPs are highly likely to sustain yet unknown physiological functions.…”

“…The impact of missing EMPs was demonstrated in the wide phenotyping screening results and the detailed changes in bone tissue, where their expression has also been revealed by other authors 39,40 , causing changes in their physical properties. We suggest that it is connected with imbalanced bone homeostasis 22,39,41 , probably by affecting the function of osteoblasts or osteoclasts. It was proven, that e.g.…”

“…1), and many functional motifs are stable over vertebrate evolution (Supplementary table 1). This implies that there is still a controlling selective pressure over the protein structure protecting some functional motifs from degradation, even though there are neither enamel nor teeth 22,34 . Our current analysis of the conserved sequences shows more regions, where many motifs are stable in Mammalia regardless of the presence of teeth and some are common even for Amniota, Tetrapoda or all evolutionary lineages in our analysis (Fig.…”

“…Already Girondot and Sire 20 tested this possibility, EMPs may have another role than production of enamel matrix, e.g. modulating calcium metabolism demonstrated by its deposition in bone tissues 21,22 . This can be highlighted by the theory of the Cambrian explosion when calcium storage became an important evolutionary advantage 23 .…”

Biomineralization of Enamel and Dentin Mediated by Matrix Proteins

“…Ameloblastin is considered critical for proper enamel formation because a severely hypoplastic enamel layer appears on the teeth of Ambn mutant mice (Fukumoto et al 2004). Its high calcium binding affinity is an indication that ameloblastin might be directly involved in controlling crystal formation (Vetyskova et al 2020).…”