Leah Dafni

Enamel Matrix Proteins and Ameloblast Biology

The paper reviews the changes in ameloblast ultrastructure, concomitant with the changes in its functions across the major stages of amelogenesis. It describes the mechanisms associated with the major events in biosynthesis and degradation of the major enamel proteins (amelogenins and tuftelin/enamelins) and with the presecretory and postsecretory mechanisms leading to the heterogeneity of these extracellular matrix proteins. The gene structure, chromosomal localization, protein, primary structure and possible function, and the involvement of the different proteins in X-linked (amelogenin) and possibly in autosomally linked (tuftelin) amelogenesis imperfecta, the most common hereditary disease of enamel, are also discussed.

Amelogenin expression in long bone and cartilage cells and in bone marrow progenitor cells

The amelogenin protein is considered as the major molecular marker of developing ectodermal enamel. Recent data suggest other roles for amelogenin beyond structural regulation of enamel mineral crystal growth. Here we describe our novel discovery of amelogenin expression in long bone cells, in cartilage cells, in cells of the epiphyseal growth plate, and in bone marrow stromal cells. Anat Rec, 2007.

Regeneration of bone and periodontal ligament induced by recombinant amelogenin after periodontitis.

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth‐supporting tissues, alveolar bone, periodontal‐ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM+), induced in vivo regeneration of all tooth‐supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM+ induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio‐temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM+.

The Human Tuftelin Gene and the Expression of Tuftelin in Mineralizing and Nonmineralizing Tissues

Tuftelin has been suggested to play an important role during the development and mineralization of enamel, but its precise function is still unclear. This article reviews major milestones in the discovery, structural characterization, expression, localization, and conservation of tuftelin in different vertebrate species. It focuses on the structure of the human tuftelin gene, which has recently been deciphered [12]. It describes the exon-intron organization, sizes and structure, the promoter structure, and the newly discovered alternatively spliced human tooth-bud tuftelin mRNA transcripts. It also examines information on the structural motifs in the human-derived tuftelin protein and how they relate to tuftelin from other species. It reviews our recent results on the transcription of tuftelin mRNA and protein expression in several nonmineralizing soft tissues, using reverse-transcription polymerase chain reaction (RT-PCR) followed by DNA cloning and sequencing, indirect immunohistochemistry, immunohistochemistry combined with confocal microscopy, and in situ hybridization. These results and earlier Northern blot results show that tuftelin, in addition to being expressed in the developing and mineralizing tooth, is also expressed in several nonmineralizing soft tissues, suggesting that tuftelin has a universal function and/or a multifunctional role.

Enamelin and Enameloid

Immunological studies have indicated that enamel proteins have common antigenic determinants across a wide range of vertebrates suggesting the structure of some of these enamel proteins to be highly conserved during the 450 million years of vertebrate evolution [1]. These and other biochemical studies [2, 3, 4, 5, 6] have further shown that the acidic glycoprotein enamelins are predominant in certain aquatic vertebrates such as fishes and sharks, whereas terrestrial vertebrates enamelins are detected in lower proportion relative to the predominant amelogenins [7, 8, 9]. This finding, that enameloid mineralization occurs in the presence of enamel ins but does not require the presence of amelogenins, is indicative of the important biological role of these proteins.

Localization, Quantification, and Characterization of Tuftelin in Soft Tissues

Tuftelin was initially found in the developing and mature extracellular enamel. Here we describe our novel discovery of tuftelin cellular distribution (protein and mRNA) in six soft tissues. The expression levels of tuftelin mRNA were significantly higher in mouse kidney and testis, in which oxygen levels are hovering closely to hypoxia under normal conditions. Anat Rec, 2007.

High Expression of Human Amelogenin in E. Coli

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells

Tuftelin is an acidic protein expressed at very early stages of mouse odontogenesis. It was suggested to play a role during epithelial-mesenchymal interactions, and later, when enamel formation commences, to be involved in enamel mineralization. Tuftelin was also detected in several normal soft tissues of different origins and some of their corresponding cancerous tissues. Tuftelin is expressed in low quantities, and undergoes degradation in the enamel extracellular matrix. To investigate the structure and function of tuftelin, the full length recombinant human tuftelin protein was produced. The full length human tuftelin cDNA was cloned using Gateway recombination into the Bac-to-Bac system compatible transfer vector pDest10. This vector adds a hexahistidine tag to the N-terminus of the expressed protein, enabling one-step affinity purification on nickel column. The recombinant human tuftelin protein was transposed into the bacmid and expressed in Spodoptera frugiperda (Sf9) insect cells. The yield of the purified, his-tagged recombinant full length human Tuftelin (rHTuft+) was 5-8 mg/L culture. rHTuft+ was characterized by SDS-PAGE, Western blot, ESI-TOF spectrometry, restriction mapping and MS/MS sequencing. The availability of the purified, full length recombinant human tuftelin protein opened up the possibility to investigate novel functions of tuftelin. Application of rHTuft+ agarose beads onto embryonic mouse mandibular explants caused changes in the surrounding epithelial cells, including morphology, orientation and spatial organization. Further studies using DiI labeling, revealed that rHTuft+, placed on the tooth germ region, brought about recruitment of adjacent embryonic mesenchymal cells. These findings support the hypothesis that tuftelin plays an important role during embryogenesis.

Tuftelin mRNA is expressed in a human ameloblastoma tumor.

RT-PCR, Southern blotting and DNA sequencing have established for the first time that tuftelin mRNA is expressed in human ameloblastoma tumor. The expression of amelogenin mRNA in ameloblastoma was also established, confirming earlier reports by Snead et al. These results corroborate, on a molecular level, the enamel organ epithelial origin of ameloblastoma. In view of the present results, it is interesting that previous studies have indicated that although ameloblastoma, a non-mineralized odontogenic tumor, transcribes amelogenin mRNA, amelogenin (and enamelin) proteins are not expressed in this tissue. However, in mineralizing odontogenic tumors, both these classes of proteins are expressed.