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.

2nd Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague 5 Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technicka 5 166 28 Prague Czech Republic

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 128 40 Prague Czech Republic

Department of Biomaterials Institute of Clinical Dentistry University of Oslo 0317 Oslo Norway

Department of Biotechnology University of Chemistry and Technology Prague Technicka 5 166 28 Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences Prumyslova 595 252 50 Vestec Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nam 2 160 00 Prague Czech Republic

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Proteolytic profiles of two isoforms of human AMBN expressed in E. coli by MMP-20 and KLK-4 proteases

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