Ameloblastin: Evolution, Structure and Function in Enamel Formation.

Ameloblastin;Enamel Formation;Tooth Development;Enamel Protein;Dentistry;Health Sciences;Oral Health Sciences

Chun, Yong-Hee PatriciaMcCauley, Laurie Kay ;

University of Michigan

关键词: Ameloblastin;    Enamel Formation;    Tooth Development;    Enamel Protein;    Dentistry;    Health Sciences;    Oral Health Sciences;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/63831/ychun_2.pdf?sequence=2&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Dental enamel formation is dependent upon the secretion of three enamel proteins: amelogenin, enamelin, and ameloblastin (Ambn). These proteins contribute to a mineralization front on the secretory surface of ameloblasts. At the mineralization front, thin mineral ribbons grow in length, while enamelysin (Mmp-20) proteolytically processes the secreted enamel proteins. The absence of any one of these proteins results in major defects in the enamel layer. Once the enamel ribbons reach their final length, kallikrein 4 (Klk4) is secreted and degrades the residual enamel proteins, allowing the crystallites to widen and grow together. The goal of this work was to investigate the structure and function of Ambn and its evolution. Ambn belongs to the secretory calcium-binding phosphoproteins, which descended from SPARC (secreted protein, acidic and rich in cysteine). All are involved in biomineralization. To better understand the structure of SPARC in mineralizing systems, we isolated the SPARC cDNA and protein from developing pig teeth, and determined its quaternary structure by homology modeling after the human SPARC crystal structure. Porcine Ambn is secreted as a 395 amino acid glycoprotein. Since Ambn is cleaved rapidly following its secretion, the intact protein has not been isolated from in vivo sources. We expressed recombinant porcine Ambn (rpAmbn) in mammalian (HEK293) cells. Recombinant Ambn was isolated by ammonium sulfate precipitation, metal- affinity chromatography, and reversed-phase high performance liquid chromatography. The purified, uncleaved protein migrated at 64 kDa on SDS-PAGE, was glycosylated, and based upon positive Stains-all staining, bound calcium. Ambn processing is essential for enamel mineralization. In vitro Ambn cleavage sites generated by rpMmp-20 and native Klk4 were characterized using rpAmbn and synthetic fluorescent peptides. Mmp-20 cleaved rpAmbn at sites matching those of Ambn cleavage products isolated from immature enamel. In contrast, Klk4 degraded rpAmbn. As enamel crystals cannot be synthesized in vitro, there are no functional assays for Ambn. We developed an in vivo functional assay by generating a transgenic mouse that expresses Ambn from the AmelX promoter. This transgenic mouse was crossed with the Ambn null mouse. The hybrid (Ambn+/-Tg) showed normal enamel thickness and prisms similar to wildtype.