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.

The enamelin (tuftelin) gene.

This paper reviews the primary structure, characteristics and possible function of tuftelin/enamelin protein. It describes the distribution of tuftelin in the ameloblast cell and in the extracellular enamel matrix, employing high resolution protein-A gold immunocytochemistry. The chromosomal localization of the human tuftelin gene and its possible involvement in autosomally linked Amelogenesis Imperfecta, the most common hereditary disease of enamel, is also discussed.

The human tuftelin gene: cloning and characterization.

Tuftelin has been suggested to play an important role during the development and mineralization of enamel. We isolated the full-length human tuftelin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5' RACE and 3' RACE) methods. Sequence analysis of the tuftelin cDNA revealed an open reading frame of 1170 bp encoding a 390 amino acid protein with a molecular mass of 44.3 kDa and an isoelectric point of 5.7. The human tuftelin protein shares 89 and 88% amino acid sequence identity with the bovine and mouse tuftelin, respectively. It contains a coiled-coil region, recently reported to be involved with tuftelin self-assembly and with the interaction of tuftelin with TIP39 (a novel tuftelin interacting protein). Detailed DNA analysis of the cloned genomic DNA revealed that the human tuftelin gene contains 13 exons and is larger than 26 kb. Two alternatively spliced tuftelin mRNA transcripts have now been identified in the human tooth bud, one lacking exon 2, and the other lacking exon 2 and exon 3. Primer extension analysis, corroborated by RT-PCR and DNA sequencing, revealed multiple transcription initiation sites. The cloned 1.6 kb promoter region contained several GC boxes and several transcription factor binding sites such as those for activator protein 1 and stimulatory protein 1. Our blast search of the human and mouse expressed sequence tag data bases, as well as our RT-PCR and DNA sequencing results, and a previous study using Northern blot analysis revealed that tuftelin cDNA sequences are also expressed in normal and cancerous non-mineralizing soft tissues, suggesting that tuftelin has a universal function. We have now identified and characterized different alternatively spliced mouse tuftelin mRNAs in several non-mineralizing tissues. These results provide an important baseline for future understanding of the biological role of tuftelin.

Long term use of inert intrauterine contraceptive devices in 94 women in Israel.

A 22 year retrospective survey was undertaken in an Israeli family practice to determine how long inert (plastic only, unmedicated) intrauterine contraceptive devices could be safely left in place. Ninety four women were identified who had used 100 inert intrauterine contraceptive devices continuously for between five and 19 years. Fifty nine women complained of pain, increased uterine bleeding or increased vaginal discharge, but these were the reasons for removal of the device in only 32 women. There was only one case of pelvic inflammatory disease but this was treated without removal of the intrauterine contraceptive device. Of 14 women who requested to have their device removed after between five and nine years without having had any side effects, 11 women conceived within nine months. The results of the study indicate that inert intrauterine contraceptive devices can be safely left in place until the menopause. As it will take many years before the new type of copper devices can be shown to be as safe for long term use, it seems an appropriate time to reintroduce inert intrauterine contraceptive devices for women.

PIP: A retrospective study of 94 Israeli women who had worn an inert IUD for 5-19 years was made from records in a family practice including 1 Arab village, 2 Jewish coops, and 3 Jewish kibutzim. The IUDs, 74 Lippes Loops and 26 Saf-T-Coils, were inserted at a mean age of 34 years and at mean parities of 3-6. Removals for an IUD-related event, such as bleeding, pain, vaginal discharge, broken IUD or positive cervical smear, were very common in the first 60 months, less so between 60 and 108 months, and even less common up to 168 months. After 168 months only 1 was removed for a medical event at 226 months. Of the 94 first IUDs, 27 were in place between 5 and 19 years. 20 were removed for bleeding or pain between 5 and 9 years, and only 4 between 10 and 19 years. 50 women complained of side effects, but only 32 requested removal. There was 1 spontaneous expulsion at 8 years, 2 intrauterine pregnancies at 60 and 82 months, and 1 case of pelvic inflammatory disease treated without removal and 3 broken IUDS. 2 IUDS were difficult to remove, one requiring curettage. 11 of the 14 women who requested removal were known to have conceived within 9 months. These data, and those cited in the discussion, suggest that the long-term safety of inert IUDs should be re-examined.

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.

Tuftelin: enamel mineralization and amelogenesis imperfecta.

Tuftelin is a novel acidic enamel protein thought to play a major role in enamel mineralization. Its identity and localization has been confirmed by amino acid composition, enzyme-linked immunosorbant assay, Western blots, indirect immunohistochemistry and high resolution protein-A gold immunocytochemistry. The deduced tuftelin protein (pI 5.2) contains 389 amino acids and has a calculated peptide molecular mass of 43,814 Da. Immunological studies suggest conservation of tuftelin structure between species throughout vertebrate evolution. The cDNA sequence encodes for several putative post-translation sites including one N-glycosylation consensus site, seven O-glycosylation sites and seven phosphorylation sites, as well as an EF-hand calcium-binding domain (with mismatch), localized towards the N-terminal region. At the C-terminal region (residues 252-345) tuftelin contains structurally relevant determinants for self assembly. We recently cloned and partially sequenced the human tuftelin gene (four exons have now been sequenced). These sequences include exon 1 and over 1000 bases of the putative promoter region. Employing fluorescent in situ hybridization, we mapped the human tuftelin gene to chromosome 1q 21-31. Localization of the human tuftelin gene to a well-defined cytogenetic region may be important in understanding the aetiology of autosomally inherited amelogenesis imperfecta, the most common enamel hereditary disease.

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.

High expression of human amelogenin in E. coli.

A human cDNA, encoding for the 175-amino-acid 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.

Tuftelin--aspects of protein and gene structure.

The acidic enamel protein tuftelin has now been cDNA cloned, sequenced and characterized in a number of vertebrate species. Recently, the bovine tuftelin gene structure was elucidated. Cloning of the human tuftelin gene and partial sequencing of a number of exons have also been achieved. Immunologically, the protein has been shown to be conserved throughout 550 million years of vertebrate evolution. The gene has been localized to the long arm of the autosomal chromosome 1. The mapping of the human tuftelin gene to a well-defined cytogenetic region could be important in understanding the etiology of autosomally inherited amelogenesis imperfecta, the most common hereditary disease of enamel. The present paper reviews the primary structure, mRNA/cDNA structure, and gene structure of tuftelin. It describes its immunolocalization at the light microscope level and at the ultrastructural level in both the ameloblast cells and in the extracellular enamel matrix. The timing of tuftelin expression and its possible roles in enamel formation are discussed.

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.