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Cloning and characterization of the human ameloblastin gene
Gene 256 (2000) 1–11 www.elsevier.com/locate/gene
Cloning and characterization of the human ameloblastin gene k Satoru Toyosawa a, *, Taku Fujiwara b, Takashi Ooshima b, Seikou Shintani b, Akie Sato c, Yuzo Ogawa a, Shizuo Sobue b, Naokuni Ijuhin a a Department of Oral Pathology, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Pedodontics, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan c Max-Planck-Institut fu¨r Biologie, Abteilung Immungenetik, Corrensstrasse 42, D-72076 Tu¨bingen, Germany Received 31 March 2000; received in revised form 3 August 2000; accepted 14 August 2000 Received by T. Sekiya
Abstract We isolated the full-length human ameloblastin (AMBN ) cDNA clone using reverse transcription-polymerase chain reaction (RT-PCR) methods. Sequence analysis of the AMBN cDNA revealed an open reading frame of 1341 bp encoding a 447-amino acid protein. Comparison with pig, cattle, rat, and mouse AMBN sequences showed a high amino acid sequence similarity and led to the identification of a novel 78 bp (26 amino acids) insert resulting from internal sequence duplication. By DNA analysis of a human genomic clones, the AMBN gene was shown to consist of 13 exons and a novel 78 bp segment, which proved to comprise two small exons. Human ameloblastomas express AMBN transcripts that contain some mutations. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Human ameloblastin gene; Tooth-specific gene; cDNA sequence; Duplication; Ameloblastoma
1. Introduction The three principal hard tissues of a mammalian tooth are enamel, dentin, and cementum (Miles and Poole, 1967). Enamel is a mineralized extracellular matrix secreted by ameloblasts. The enamel matrix consists of amelogenin (90%) and non-amelogenin proteins (10%). One of the non-amelogenin proteins identified with the help of recombinant DNA techniques is ameloblastin (AMBN; Krebsbach et al., 1996). AMBN, also known as amelin and sheathlin (Cerny et al., 1996; Hu et al., 1997), is a group of related, if not identical, proteins encoded in genes that have been cloned and isolated from a number of species. In situ hybridization and immunohistochemistry have shown that AMBN is principally produced by the enamel-forming ameloblasts and deposited in the developing enamel matrix. The Abbreviations: AMBN, ameloblastin; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription PCR. k The nucleotide sequences of the human ameloblastin has been deposited in GenBank under the Accession No. AF219994. * Corresponding author. Tel.: +81-6-6879-2892; fax: +81-6-6879-2895. E-mail address: [email protected] (S. Toyosawa)
AMBN gene, however, is also expressed in Hertwig’s epithelial root sheath ( Fong et al., 1996). Immunohistochemical studies have demonstrated that AMBN is a member of the group of enamel sheath proteins that are present in the enamel prism sheath ( Uchida et al., 1995). The initial cloning studies revealed that the rat AMBN gene encoded an open reading frame of 422 amino acid residues corresponding to a putative 45 kDa protein ( Krebsbach et al., 1996). Homologs of the AMBN gene or its transcripts have also been cloned in the pig (Hu et al., 1997), mouse (GenBank Accession No. U65021), and cattle (GenBank Accession No. AF157019). In humans, using fluorescence in situ hybridization (FISH ), the AMBN gene maps to chromosome 4q21 in a locus that is linked to an autosomal dominant form of amelogenesis imperfecta (MacDougall et al., 1997), but no human sequence has been presented to date. The human ameloblastoma, characterized by persistent local growth in the maxillofacial area, is the most common human odontogenic epithelial tumor ( Regezi and Sciubba, 1999) whose cells resemble ameloblasts and stellate reticulums of the enamel-forming organ. However, these cells do not produce an identifiable extracellular matrix that is unique to enamel, in contrast
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to normal tooth development, during which the inner enamel epithelium differentiates to functional ameloblasts responsible for enamel formation. The major product of ameloblasts, amelogenin, is a structural protein that is believed to regulate the formation of enamel hydroxyapatite crystallites. Expression of amelogenin in ameloblastomas has been demonstrated by immunohistochemical studies (Mori et al., 1991), Northern blot analysis, and in situ hybridization (Snead et al., 1992). By contrast, expression of AMBN could not be detected in ameloblastomas by immunohistochemical methods ( Takata et al., 2000). Here, we describe the cloning and characterization of full-length cDNAs encoding human AMBN and demonstrate that human ameloblastomas express AMBN transcripts.
2. Materials and methods 2.1. Source and isolation of genomic DNA Human tooth germs, the pulp of adult human teeth, three human ameloblastomas, and other human tissues were obtained at the time of surgery and frozen immediately in liquid nitrogen for later use. Five-micrometer sections of three tumor tissues fixed in 4% paraformaldehyde and embedded in paraffin were used for histologic diagnosis. Genomic DNA was isolated by phenol/ chloroform extraction (Ausubel et al., 1994). The procedures were carried out in full compliance with Japanese Public Health Service, Osaka University of Health Guidelines for Studies Involving Human Subjects and were approved by the Institute Review Board. Human tissues were considered infectious, and appropriate biologic containment was executed during tissue manipulations.
2.2. Isolation of RNA, cDNA synthesis, and isolation of AMBN cDNA fragment Total RNA was isolated using the RNA purification kit (Promega, Madison, WI ). The first-strand cDNAs were synthesized with oligo(dT ) by RNaseH− reverse transcriptase (Gibco/BRL, Grand Island, NY ). The pools of cDNA from tooth germs were screened by polymerase chain reaction (PCR) amplification using the primers AMB10 (sense: 5∞-ATTGAAGATTCCACTTTTCAAAATGAA-3∞) and AMB2 (antisense: 5∞-CTCTCAGGGCTCTTGGAAACGCCA-3∞), based on an alignment of pig, cattle, rat, and mouse AMBN sequences. The amplification product of these two primers spans a cDNA segment of approximately 1.3 kb.
2.3. 5∞ and 3∞ rapid amplification of cDNA ends (5∞- and 3∞-RACE) To obtain a full-length clone, a reverse transcription ( RT )-PCR was carried out using a 5∞- and 3∞-RACE System (Gibco BRL). For 5∞-RACE, 1 mg of total RNA prepared from tooth germs was used for the first-strand cDNA synthesis with the human AMBN gene-specific primer AMB15 (antisense: 5∞-ACTGTCTCATTGTCTCAAGG-3∞). An intermediate terminal deoxynucleotidyltransferase-tailing reaction was performed prior to amplification. The first-strand cDNAs were then amplified by PCR with the human AMBN genespecific primer AMB16 (antisense: 5∞-CCAGGAGGCATAGGATCAGTATCAGGT-3∞). For 3∞-RACE, the first-strand cDNAs synthesized with oligo(dT ) primers were amplified by PCR with the human AMBN genespecific primer AMB5 (sense: 5∞-GCTCCTTGCTCTCCCTAAGGATGA-3∞). The clones amplified with the AMB26 (sense: 5∞-ATCTTGGTTGGCATCATCAGGC-3∞) and the AMB27 (antisense: 5∞-ACAAAGCTGGGAAGCTTGTGCA-3∞) primers were used to confirm the AMBN coding sequence. 2.4. PCR amplifications The pool of the cDNA (1 ml ) was amplified by PCR in 50 ml of PCR buffer (1.5 mM MgCl , 200 mM dNTP, 2 10 mM Tris buffer, pH 8.5) in the presence of the sense and antisense primers and 2.5 units of Taq polymerase (Amersham Pharmacia Biotech, Buckinghamshire, UK ). Amplifications were performed in a GeneAmp 2400 thermal cycler (Applied Biosystems, Mihama, Japan) in 35 cycles, each cycle consisting of 30 s denaturation at 94°C, 30 s annealing at the annealing temperature, and 2 min extension at 72°C. The final extension was 10 min at 72°C. The annealing temperatures varied from 55 to 62°C, depending on the primer combination used. To determine the genomic organization of the human AMBN gene, genomic DNA was amplified by long PCR using human AMBN gene-specific primers. The primer pairs were AMB26L (sense: 5∞-CTTAGAACTATCTTGGTTGGCATCATCAGGC-3∞) and AMB38L (antisense: 5∞-AGGTGAATTGGAGGCTGAAGTGCTTCTT-3∞), as well as AMB12L (sense; 5∞-CTTGAAGCCTCAACAGCCAGGACTGAAA-3∞) and AMB27L (antisense: 5∞-GGGACAAAGCTGGGAAGCTTGTGCA-3∞). Long PCR was carried out with the help of the TaKaRa LA PCR Kit ( TaKaRa Biomedical, Kyoto, Japan) and consisted of one cycle at 98°C for 2 min, followed by 12 cycles, each for 30 s at 98°C and 10 min at 68°C. In the next 24 cycles, the reaction time at 68°C was extended by 15 s in every cycle. The cDNAs synthesized from human ameloblastomas were amplified with human AMBN gene-specific prim-
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Fig. 1. Nucleotide and translated amino acid sequences of human AMBN cDNA clones. Amino acid residues are given in the IUPAC-IUB singleletter code. The signal peptide is underlined with a dotted line, and the stop codon is indicated by an asterisk. The two polyadenylation signals are underlined with a single line. The 78 bp, 26-amino-acid inserted sequence is double-underlined. Vertical lines indicate the positions of the exon boundaries. Primer positions and orientations are indicated by an arrowed line above their location in the sequence. This sequence can be accessed through the GenBank database, Accession No. AF219994.
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ers. The primer pairs were AMB 26 and AMB 27, AMB10 and AMB8 (antisense: 5∞-TGGGTTTTCTAGATTGTCTGGGTTGG-3∞), as well as AMB 11 (sense: 5∞-GACCAAGGGAACATGAAACTCAACA-3∞) and AMB 8. 2.5. Cloning and sequencing Ten microliters of the PCR product were purified by 0.8–2% low-melting-point agarose (Gibco BRL) gel electrophoresis. The bands were excised and isolated from the gel using QIAEX II extraction kits (Qiagen, Hilden, Germany). The isolated DNA was ligated to pGEM-T Easy (Promega) or pCR-XL-TOPO (Invitogen, Carlsbad, CA) vectors and then transformed into competent E. coli bacteria. For a DNA-sequencing template, plasmid DNA was prepared using an ABI PRISMTM Minipreps Kit (Applied Biosystems). The dideoxy dye-termination reaction was performed with an ABI PRISM cycle-sequencing kit (Applied Biosystems) in a GeneAmp 2400 thermal cycler. The products were then analyzed using an automated DNA sequencer model 373 (Applied Biosystems). 2.6. Southern blot analysis For Southern blots, 10 mg of genomic DNA were digested with restriction enzymes, and the resulting fragments were separated by electrophoresis on a 0.8% agarose gel (Gibco BRL) and transferred to a hybridization membrane (Hybond-N+, Amersham Pharmacia Biotech) using the VacuGene blotting system (Amersham Pharmacia Biotech). The filters were incubated for 5 h in a prehybridization solution and then hybridized with a 32P-labeled probe encompassing either the full-length cDNA or only the last exon of the AMBN gene. Filters were washed in 2× SSC, 0.1% SDS and then used to expose XAR5 films ( Kodak, Rochester, NY ).
cDNA clones (one amplified by 5∞-RACE, another amplified with AMB10 and AMB2, and a third amplified by 3∞-RACE ). The clones amplified with the AMB26 and AMB27 primers were used to confirm the coding sequence. The complete human AMBN coding sequence was 1341 bp long and contained an open reading frame of 447 amino acid residues beginning with the putative translation start site at 66 bp ( Fig. 1). A 26-residue hydrophobic signal peptide sequence was present at sites 66–143. The stop codon at 1407 bp was followed by a 3∞ untranslated region ( UTR) of 227 bp with two putative polyadenylation signals (AATAAA) at sites 1537 and 1597. A self-diagonal plot was constructed using the MacVector software comparing the human AMBN sequence with itself for regions of homology (Fig. 2). Using a window size of 20 nucleotides and plotting position with a minimum of 50% homology, significant lines parallel to the diagonal suggested that duplications were involved in the formation of the human AMBN coding region, as they were also in the formation of amelogenin gene (Bonass et al., 1994). A 78 bp (26 amino acid) long segment that had not been found in other reported mammalian AMBN cDNAs was identified in the human sequence. To determine the true character of the 78 bp insert and the genomic organization of the human AMBN gene, a long PCR amplification was performed from two human genomic DNAs using the primer pairs AMB26L and AMB38L, as well as AMB12L and AMB27L. These genomic amplicons
2.7. Data analysis Amino acid sequence alignments (human, pig, cattle, rat, and mouse), self-diagonal plots, and constructions of phylogenetic trees were performed using the MacVector 6.53 DNA sequence analysis software ( Teijin System Tech, Tokyo, Japan). The same software was also used to calculate amino acid compositions and isoelectric points.
3. Results and discussion 3.1. Characterization of the human AMBN cDNA The complete coding sequence of the human AMBN cDNA was deduced from three separate overlapping
Fig. 2. Self-diagonal plot of the human AMBN cDNA sequence. The 1341-nucleotide AMBN cDNA sequence is presented on both the vertical and horizontal axes. The central diagnoal line indicates the homology expected from aligning two identical sequences. Several lines parallel to the diagonal at approximately 600 indicate internal homology.
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were subcloned into a vector and their nucleotide sequence determined. The analysis of the sequence revealed that human AMBN gene consists of 13 exons corresponding to the cDNA segment encompassing nucleotides 50–1438 ( Fig. 3). All the determined splice sites in this segment were of type O, occurring between codons, with all donors sites containing ‘GT’ and all acceptors sites containing ‘AG’ consensus splice sequences. The 78 bp insert represents two exons, which are absent in other reported mammalian AMBN sequences due to alternative splicing at the mRNA level. The two extra exons (exons 8 and 9) represented an internal sequence duplication of exon 7. There was no polymorphism in the coding region of the human AMBN gene between the cDNA sequence and sequences of two human genomic DNAs. The coding region of the human AMBN gene, including the part specifying the signal peptide, has a high sequence similarity with other reported mammalian AMBN cDNA sequences (Fig. 4): 66, 61, 61, and 58% with the pig, cattle, rat, and mouse sequences, respectively. The predicted translation product of the human AMBN gene is an acidic protein, pI 4.54, with a calcu-
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lated molecular weight (M ) of 45 300 Da, excluding the r signal peptide. The protein sequence predicted from the human AMBN cDNA sequence contains 15.2% proline, 10.2% leucine, and 9.0% glycine, which are values comparable with those found in other mammalian AMBN sequences. A comparison of the amino acid compositions of human, pig, cattle, rat, and mouse AMBN is shown in Table 1. The functional protein motifs include potential phosphorylation sites for casein kinase II at residue 261, protein kinase C (residues 46), and protein tyrosine kinase (residue 99). These motifs, which are conserved in the pig, cattle, rat, and mouse amino acid sequences, are highlighted in Fig. 4. The hydrophilicity plots of these sequences share the following characteristics (Fig. 5): the hydrophobic leader peptide of approximately 30 amino acid residues is followed by a short hydrophilic segment (about 10 residues), and then by a more variable internal segment of irregularly alternating hydrophilicity and hydrophobicity; the C-terminal region is hydrophilic. A phylogenetic tree based on amino acid sequences of all available AMBN sequences ( Fig. 6) indicates that the sequences group as expected from their taxonomical positions.
Fig. 3. Genomic organization of the human AMBN gene. The genomic structure of the human AMBN cDNA was determined by DNA sequence analysis of genomic subclones generated by PCR. Hatched rectangles indicate that the three exons represent the internal sequence duplication. Intron sequences and exon sequences are indicated in lower-case and upper-case letters, respectively. Abbreviations: TGA, stop codon; UTR, untranslated region; ND, not determined.
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Fig. 5. Hydrophilicity plot of AMBNs from all available sequences prepared using the method of Kyte and Doolittle (1982). The plots share the following characteristics: the hydrophobic leader peptide of approximately 30 amino acid residues is followed by a short hydrophilic segment (about 10 resudues), a short hydrophobic segment (10 residues), strongly hydrophilic segment (residues 80–110), and then a more vaariable internal segment (residues 110–370) of irregularly alternating hydrophilicity and hydrophobicity; the C-terminal regioans are all hydrophilic.
To investigate the possibility that other AMBN-like loci exist in humans, we carried out a Southern blot analysis using either a full-length cDNA probe or a probe encompassing nearly the entire last exon of the
AMBN gene. Using the full-length probe, a single band was found following digestion with both EcoRI and BamHI restriction endonucleases; two bands were found using the HindIII and TaqI enzymes (data not shown).
Fig. 4. Amino acid sequence alignment of human, pig, cattle, rat, and mouse AMBNs. A dash (−) indicates an alignment gap. Potential phosphorylation sites (PKC, protein kinase C; TK, tyrosine kinase; CK2, casein kinase II ) are indicated by boxes. An asterisk under the alignment indicates amino acid residues conserved in all species. Amino acid residues are given in the IUPAC-IUB single-letter code.
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Fig. 6. Neighbor-joining tree of sequences shown in Fig. 3. Genetic distances (numbers on branches) were estimated from the proportion of differences in pairwise comparison following exclusion of gaps.
However, using the single-exon probe, a single band was found with the NotI, PstI, and TaqI enzymes. These results are consistent with the presence of a single AMBN locus in the human genome. 3.2. Expression of the AMBN gene in human ameloblastomas cDNAs were reversely transcribed from three ameloblastomas and tooth germs and PCR amplified with AMBN gene-specific primers. The expected sizes of the normal amplification products with the primer pairs AMB26 and AMB27, AMB10 and AMB8, as well as AMB11 and AMB8, were 1597, 1008, and 748 bp,
respectively. In all three ameloblastoma samples, amplification products corresponding in size to those amplified from human tooth germs could be detected (Fig. 7). As a negative control, cDNA derived from the pulp of adult human teeth was tested with AMBN cDNAspecific primers, but no band was obtained (data not shown). The products amplified from the three ameloblastoma samples with the primer pair AMB26 and AMB27 were then subcloned into a vector and sequenced. The sequence of one ameloblastoma (tumor 1) contained an open reading frame of 1329 bp encoding a 443 amino acid protein, which had three substitutions ( TC: MetThr; AG: GlyGly; AG: HisArg) and one deletion (12 nucleotides) compared with the tooth-germ cDNA (Fig. 8). The AMBN sequence from the other two ameloblastomas (tumors 2 and 3) had substitutions but no deletion (Fig. 8). Since no polymorphism was found in the coding regions of the AMBN gene in normal tissues, substitutions and deletions found in the ameloblastomas sequences were probably the result of tumor-specific mutations, possibly as a result of the abnormal metabolic changes in the tumor cells. The expression of the AMBN gene might become a useful marker of the ameloblast cell-lineage. Compared with amelogenin, AMBN is expressed in broader area of the inner enamel epithelium containing presecretory ameloblast and Hertwig’s epithelial sheath (Fong et al., 1996; Lee et al., 1996).
Fig. 7. RT-PCR analysis of the human ameloblastomas. The cDNAs synthesized from the tooth germs and ameloblastomas, were amplified using AMBN cDNA-specific primer sets. Lanes 1, 2, and 3, tooth germs; lanes 4, 5, and 6, ameloblastomas (tumor 1); lanes 7,8, and 9 ameloblastoma (tumor 2); lanes 10. 11, and 12, ameloblastoma (tumor 3). Lanes 1, 4, 7 and 10 were amplified with primers AMB26 and AMB27; lanes 2, 5, 8, and 11 with primers AMB10 and AMB8; and lanes 3, 6, 9, and 12 with primers AMB11 and AMB8. M indicates the lanes containing a DNA size marker, a 100 bp ladder.
Fig. 8. Sequence comparison of the AMBN homologs of ameloblastoma with the normal AMBN cDNAs. DNA sequence of the products amplified with the AMB26 and AMB27 primers from three ameloblastomas (tumors 1, 2, 3) and tooth germs, were compared using the Multiple Alignment program of the MacVector. Identical nucleotides between the sequences are indicated by an asterisk under the alignment, and an absent nucleotide by a dash. Primer positions and orientations are indicated by an arrowed line above their location in the sequence. The upper-case letters above the sequence are the IUPAC-IUB single-letter code designations of amino acids translated from human AMBN cDNA, while the upper-case letters below the sequence are amino acids translated from the mutations.
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Table 1 Amino acid composition of human, pig, cattle, rat, and mouse ameloblastin Amino acid
Human Residues
Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamie Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Trytophan Tyrosine Valine Totals
Pig Res/1000
35 83 11 26 13 31 20 48 0 0 26 62 29 69 38 90 11 26 6 14 43 102 12 29 22 52 18 43 64 152 30 71 20 48 3 7 9 21 11 26 421 M 45.3 kDa r pI 4.54
Residues
Cattle Res/1000
31 79 14 35 11 28 13 33 0 0 23 58 31 79 46 117 6 15 7 18 33 84 12 30 16 41 15 38 59 149 31 79 18 46 3 8 11 28 15 38 395 M 42.0 kDa r pI 4.98
Residues
Rat Res/1000
29 79 13 36 11 30 14 38 0 0 21 57 31 85 40 109 9 25 8 22 35 96 9 25 14 38 15 41 52 142 20 55 21 57 3 8 8 22 13 36 366 M 39.3 kDa r pI 4.93
Residues
Mouse Res/1000
32 81 11 28 14 35 12 30 0 0 23 58 30 76 41 104 13 33 8 20 35 88 9 23 16 40 15 38 63 159 24 61 20 51 3 8 10 25 17 43 396 M 42.4 kDa r pI 5.11
Residues
Res/1000
34 89 15 39 14 37 11 29 0 0 22 58 30 79 39 102 10 26 9 24 34 89 9 24 15 39 16 42 56 147 23 64 19 50 3 8 7 18 15 39 381 M 40.8 kDa r pI 5.50
3.3. Conclusions
References
The human AMBN gene shows a high sequence similarity with pig, cattle, rat, and mouse genes. The proteins encoded in all these genes share several features such as the presence of potential phosphorylation sites (casein kinase II, protein kinase C, and tyrosine kinase), similar patterns of hydrophilicity, and a high proportion of Pro, Leu, and Gly residues. The human AMBN gene consists of 13 exons and contains a novel 78 bp (26 amino acid) insert representing two short exons produced by internal sequence duplication. There appears to be only a single AMBN locus in humans. The AMBN transcripts are expressed in human ameloblastomas and contain some tumor-specific mutations.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1994. Current Protocols in Molecular Biology. Wiley, New York. Bonass, W.A., Robinson, P.A., Kirkham, J., Shore, R.C., Robinson, C., 1994. Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence. Biochem. Biophys. Res. Commun. 198, 755–763. Ceˇrny´, R., Slaby, I., Hammarstro¨m, L., Wurtz, T., 1996. A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains. J. Bone Miner. Res. 11, 883–891. Fong, C.D., Slaby, I., Hammarstro¨m, C., 1996. Amelin: an enamelrelated protein, transcribed in the cells of epithelial root sheath. J. Bone Miner. Res. 11, 892–898. Hu, C.C., Fukae, M., Uchida, T., Qian, Q., Zhang, C.H., Ryu, O.H., Tanabe, T., Yamakoshi, Y., Murakami, C., Dohi, N., Shimizu, M., Simmer, J.P., 1997. Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins. J. Dent. Res. 76, 648–657. Krebsbach, P.H., Lee, S.K., Matsuki, Y., Kozak, C.A., Yamada, K.M., Yamada, Y., 1996. Full-length sequence localization, chromosomal mapping of ameloblastin: a novel tooth-specific gene. J. Biol. Chem. 271, 4431–4435. Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. Lee, S.K., Krebsbach, P.H., Matsuki, Y., Nanci, A., Yamada, K.M., Yamada, Y., 1996. Ameloblastin expression in rat incisors and human tooth germs. Int. J. Dev. Biol. 40, 1141–1150. MacDougall, M., Dupont, B.R., Simmons, D., Reus, B., Krebsbach, P.H, Karrman, C., Holmgren, G., Leach, R.J., Forsman, K., 1997. Ameloblastin gene (AMBN ) maps within the critical region for
Acknowledgements We thank Professor Jan Klein for comments and suggestions and Ms Jane Kraushaar for editorial assistance. We would like to express our gratitude to all the patients who participated in this study. This work was supported by Grant-in-aid 11671800 from Ministry of Education, Science, Sports and Culture, Japan, and supported by JSPS Research for the Future Program Biological Tissue Engineering Project no. JSPS-RFTF 98I00201.
S. Toyosawa et al. / Gene 256 (2000) 1–11 autosomal dominant amelogenesis imperfecta at chromosome 4q21. Genomics 41, 115–118. Miles, A.E.W., Poole, D.F.G., 1967. The history and general organization of dentitions. In: Miles, A.E.W. ( Ed.), Structural and Chemical Organization of Teeth. Academic Press, London, pp. 3–44. Mori, M., Yamada, K., Kasai, T., Yamada, T., Shimokawa, H., Sasaki, S., 1991. Immunohistochemical expression of amelogenins in odontogenic epithelial tumours and cysts. Virchows Arch. A Pathol. Anat. Histopathol. 418, 319–325. Regezi, J.A., Sciubba, J.J., 1999. Odontogenic tumors. Oral Pathology: Clinical Pathologic Correlations. third ed., W.B. Saunders, Philadelphia, PA.
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Snead, M.L., Luo, W., Hsu, D.D.-J., Melrose, R.J., Lau, E.C., Stenman, G., 1992. Human ameloblastoma tumors express the amelogenin gene. Oral Surg. Oral Med. Oral Pathol. 74, 64–72. Takata, T., Zhao, M., Uchida, T., Kudo, Y., Sato, S., Nikai, H., 2000. Immunohistochemical demonstration of an enamel sheath protein, sheathlin, in odontogenic tumors. Virchows Arch. 436, 324–329. Uchida, T., Fukae, M., Tanabe, T., Yamakoshi, Y., Satoda, T., Murakami, C., Yakahashi, O., Shimizu, M., 1995. Immunochemical and immunocytochemical study of a 15KDa non-amelogenin and related proteins in the porcine immature enamel: Proposal of a new group of enamel proteins ‘sheath proteins’. Biomed. Res. 16, 131–140.
Cloning and characterization of the human ameloblastin gene k Satoru Toyosawa a, *, Taku Fujiwara b, Takashi Ooshima b, Seikou Shintani b, Akie Sato c, Yuzo Ogawa a, Shizuo Sobue b, Naokuni Ijuhin a a Department of Oral Pathology, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan b Department of Pedodontics, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan c Max-Planck-Institut fu¨r Biologie, Abteilung Immungenetik, Corrensstrasse 42, D-72076 Tu¨bingen, Germany Received 31 March 2000; received in revised form 3 August 2000; accepted 14 August 2000 Received by T. Sekiya
Abstract We isolated the full-length human ameloblastin (AMBN ) cDNA clone using reverse transcription-polymerase chain reaction (RT-PCR) methods. Sequence analysis of the AMBN cDNA revealed an open reading frame of 1341 bp encoding a 447-amino acid protein. Comparison with pig, cattle, rat, and mouse AMBN sequences showed a high amino acid sequence similarity and led to the identification of a novel 78 bp (26 amino acids) insert resulting from internal sequence duplication. By DNA analysis of a human genomic clones, the AMBN gene was shown to consist of 13 exons and a novel 78 bp segment, which proved to comprise two small exons. Human ameloblastomas express AMBN transcripts that contain some mutations. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Human ameloblastin gene; Tooth-specific gene; cDNA sequence; Duplication; Ameloblastoma
1. Introduction The three principal hard tissues of a mammalian tooth are enamel, dentin, and cementum (Miles and Poole, 1967). Enamel is a mineralized extracellular matrix secreted by ameloblasts. The enamel matrix consists of amelogenin (90%) and non-amelogenin proteins (10%). One of the non-amelogenin proteins identified with the help of recombinant DNA techniques is ameloblastin (AMBN; Krebsbach et al., 1996). AMBN, also known as amelin and sheathlin (Cerny et al., 1996; Hu et al., 1997), is a group of related, if not identical, proteins encoded in genes that have been cloned and isolated from a number of species. In situ hybridization and immunohistochemistry have shown that AMBN is principally produced by the enamel-forming ameloblasts and deposited in the developing enamel matrix. The Abbreviations: AMBN, ameloblastin; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription PCR. k The nucleotide sequences of the human ameloblastin has been deposited in GenBank under the Accession No. AF219994. * Corresponding author. Tel.: +81-6-6879-2892; fax: +81-6-6879-2895. E-mail address: [email protected] (S. Toyosawa)
AMBN gene, however, is also expressed in Hertwig’s epithelial root sheath ( Fong et al., 1996). Immunohistochemical studies have demonstrated that AMBN is a member of the group of enamel sheath proteins that are present in the enamel prism sheath ( Uchida et al., 1995). The initial cloning studies revealed that the rat AMBN gene encoded an open reading frame of 422 amino acid residues corresponding to a putative 45 kDa protein ( Krebsbach et al., 1996). Homologs of the AMBN gene or its transcripts have also been cloned in the pig (Hu et al., 1997), mouse (GenBank Accession No. U65021), and cattle (GenBank Accession No. AF157019). In humans, using fluorescence in situ hybridization (FISH ), the AMBN gene maps to chromosome 4q21 in a locus that is linked to an autosomal dominant form of amelogenesis imperfecta (MacDougall et al., 1997), but no human sequence has been presented to date. The human ameloblastoma, characterized by persistent local growth in the maxillofacial area, is the most common human odontogenic epithelial tumor ( Regezi and Sciubba, 1999) whose cells resemble ameloblasts and stellate reticulums of the enamel-forming organ. However, these cells do not produce an identifiable extracellular matrix that is unique to enamel, in contrast
0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 37 9 - 6
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to normal tooth development, during which the inner enamel epithelium differentiates to functional ameloblasts responsible for enamel formation. The major product of ameloblasts, amelogenin, is a structural protein that is believed to regulate the formation of enamel hydroxyapatite crystallites. Expression of amelogenin in ameloblastomas has been demonstrated by immunohistochemical studies (Mori et al., 1991), Northern blot analysis, and in situ hybridization (Snead et al., 1992). By contrast, expression of AMBN could not be detected in ameloblastomas by immunohistochemical methods ( Takata et al., 2000). Here, we describe the cloning and characterization of full-length cDNAs encoding human AMBN and demonstrate that human ameloblastomas express AMBN transcripts.
2. Materials and methods 2.1. Source and isolation of genomic DNA Human tooth germs, the pulp of adult human teeth, three human ameloblastomas, and other human tissues were obtained at the time of surgery and frozen immediately in liquid nitrogen for later use. Five-micrometer sections of three tumor tissues fixed in 4% paraformaldehyde and embedded in paraffin were used for histologic diagnosis. Genomic DNA was isolated by phenol/ chloroform extraction (Ausubel et al., 1994). The procedures were carried out in full compliance with Japanese Public Health Service, Osaka University of Health Guidelines for Studies Involving Human Subjects and were approved by the Institute Review Board. Human tissues were considered infectious, and appropriate biologic containment was executed during tissue manipulations.
2.2. Isolation of RNA, cDNA synthesis, and isolation of AMBN cDNA fragment Total RNA was isolated using the RNA purification kit (Promega, Madison, WI ). The first-strand cDNAs were synthesized with oligo(dT ) by RNaseH− reverse transcriptase (Gibco/BRL, Grand Island, NY ). The pools of cDNA from tooth germs were screened by polymerase chain reaction (PCR) amplification using the primers AMB10 (sense: 5∞-ATTGAAGATTCCACTTTTCAAAATGAA-3∞) and AMB2 (antisense: 5∞-CTCTCAGGGCTCTTGGAAACGCCA-3∞), based on an alignment of pig, cattle, rat, and mouse AMBN sequences. The amplification product of these two primers spans a cDNA segment of approximately 1.3 kb.
2.3. 5∞ and 3∞ rapid amplification of cDNA ends (5∞- and 3∞-RACE) To obtain a full-length clone, a reverse transcription ( RT )-PCR was carried out using a 5∞- and 3∞-RACE System (Gibco BRL). For 5∞-RACE, 1 mg of total RNA prepared from tooth germs was used for the first-strand cDNA synthesis with the human AMBN gene-specific primer AMB15 (antisense: 5∞-ACTGTCTCATTGTCTCAAGG-3∞). An intermediate terminal deoxynucleotidyltransferase-tailing reaction was performed prior to amplification. The first-strand cDNAs were then amplified by PCR with the human AMBN genespecific primer AMB16 (antisense: 5∞-CCAGGAGGCATAGGATCAGTATCAGGT-3∞). For 3∞-RACE, the first-strand cDNAs synthesized with oligo(dT ) primers were amplified by PCR with the human AMBN genespecific primer AMB5 (sense: 5∞-GCTCCTTGCTCTCCCTAAGGATGA-3∞). The clones amplified with the AMB26 (sense: 5∞-ATCTTGGTTGGCATCATCAGGC-3∞) and the AMB27 (antisense: 5∞-ACAAAGCTGGGAAGCTTGTGCA-3∞) primers were used to confirm the AMBN coding sequence. 2.4. PCR amplifications The pool of the cDNA (1 ml ) was amplified by PCR in 50 ml of PCR buffer (1.5 mM MgCl , 200 mM dNTP, 2 10 mM Tris buffer, pH 8.5) in the presence of the sense and antisense primers and 2.5 units of Taq polymerase (Amersham Pharmacia Biotech, Buckinghamshire, UK ). Amplifications were performed in a GeneAmp 2400 thermal cycler (Applied Biosystems, Mihama, Japan) in 35 cycles, each cycle consisting of 30 s denaturation at 94°C, 30 s annealing at the annealing temperature, and 2 min extension at 72°C. The final extension was 10 min at 72°C. The annealing temperatures varied from 55 to 62°C, depending on the primer combination used. To determine the genomic organization of the human AMBN gene, genomic DNA was amplified by long PCR using human AMBN gene-specific primers. The primer pairs were AMB26L (sense: 5∞-CTTAGAACTATCTTGGTTGGCATCATCAGGC-3∞) and AMB38L (antisense: 5∞-AGGTGAATTGGAGGCTGAAGTGCTTCTT-3∞), as well as AMB12L (sense; 5∞-CTTGAAGCCTCAACAGCCAGGACTGAAA-3∞) and AMB27L (antisense: 5∞-GGGACAAAGCTGGGAAGCTTGTGCA-3∞). Long PCR was carried out with the help of the TaKaRa LA PCR Kit ( TaKaRa Biomedical, Kyoto, Japan) and consisted of one cycle at 98°C for 2 min, followed by 12 cycles, each for 30 s at 98°C and 10 min at 68°C. In the next 24 cycles, the reaction time at 68°C was extended by 15 s in every cycle. The cDNAs synthesized from human ameloblastomas were amplified with human AMBN gene-specific prim-
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Fig. 1. Nucleotide and translated amino acid sequences of human AMBN cDNA clones. Amino acid residues are given in the IUPAC-IUB singleletter code. The signal peptide is underlined with a dotted line, and the stop codon is indicated by an asterisk. The two polyadenylation signals are underlined with a single line. The 78 bp, 26-amino-acid inserted sequence is double-underlined. Vertical lines indicate the positions of the exon boundaries. Primer positions and orientations are indicated by an arrowed line above their location in the sequence. This sequence can be accessed through the GenBank database, Accession No. AF219994.
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ers. The primer pairs were AMB 26 and AMB 27, AMB10 and AMB8 (antisense: 5∞-TGGGTTTTCTAGATTGTCTGGGTTGG-3∞), as well as AMB 11 (sense: 5∞-GACCAAGGGAACATGAAACTCAACA-3∞) and AMB 8. 2.5. Cloning and sequencing Ten microliters of the PCR product were purified by 0.8–2% low-melting-point agarose (Gibco BRL) gel electrophoresis. The bands were excised and isolated from the gel using QIAEX II extraction kits (Qiagen, Hilden, Germany). The isolated DNA was ligated to pGEM-T Easy (Promega) or pCR-XL-TOPO (Invitogen, Carlsbad, CA) vectors and then transformed into competent E. coli bacteria. For a DNA-sequencing template, plasmid DNA was prepared using an ABI PRISMTM Minipreps Kit (Applied Biosystems). The dideoxy dye-termination reaction was performed with an ABI PRISM cycle-sequencing kit (Applied Biosystems) in a GeneAmp 2400 thermal cycler. The products were then analyzed using an automated DNA sequencer model 373 (Applied Biosystems). 2.6. Southern blot analysis For Southern blots, 10 mg of genomic DNA were digested with restriction enzymes, and the resulting fragments were separated by electrophoresis on a 0.8% agarose gel (Gibco BRL) and transferred to a hybridization membrane (Hybond-N+, Amersham Pharmacia Biotech) using the VacuGene blotting system (Amersham Pharmacia Biotech). The filters were incubated for 5 h in a prehybridization solution and then hybridized with a 32P-labeled probe encompassing either the full-length cDNA or only the last exon of the AMBN gene. Filters were washed in 2× SSC, 0.1% SDS and then used to expose XAR5 films ( Kodak, Rochester, NY ).
cDNA clones (one amplified by 5∞-RACE, another amplified with AMB10 and AMB2, and a third amplified by 3∞-RACE ). The clones amplified with the AMB26 and AMB27 primers were used to confirm the coding sequence. The complete human AMBN coding sequence was 1341 bp long and contained an open reading frame of 447 amino acid residues beginning with the putative translation start site at 66 bp ( Fig. 1). A 26-residue hydrophobic signal peptide sequence was present at sites 66–143. The stop codon at 1407 bp was followed by a 3∞ untranslated region ( UTR) of 227 bp with two putative polyadenylation signals (AATAAA) at sites 1537 and 1597. A self-diagonal plot was constructed using the MacVector software comparing the human AMBN sequence with itself for regions of homology (Fig. 2). Using a window size of 20 nucleotides and plotting position with a minimum of 50% homology, significant lines parallel to the diagonal suggested that duplications were involved in the formation of the human AMBN coding region, as they were also in the formation of amelogenin gene (Bonass et al., 1994). A 78 bp (26 amino acid) long segment that had not been found in other reported mammalian AMBN cDNAs was identified in the human sequence. To determine the true character of the 78 bp insert and the genomic organization of the human AMBN gene, a long PCR amplification was performed from two human genomic DNAs using the primer pairs AMB26L and AMB38L, as well as AMB12L and AMB27L. These genomic amplicons
2.7. Data analysis Amino acid sequence alignments (human, pig, cattle, rat, and mouse), self-diagonal plots, and constructions of phylogenetic trees were performed using the MacVector 6.53 DNA sequence analysis software ( Teijin System Tech, Tokyo, Japan). The same software was also used to calculate amino acid compositions and isoelectric points.
3. Results and discussion 3.1. Characterization of the human AMBN cDNA The complete coding sequence of the human AMBN cDNA was deduced from three separate overlapping
Fig. 2. Self-diagonal plot of the human AMBN cDNA sequence. The 1341-nucleotide AMBN cDNA sequence is presented on both the vertical and horizontal axes. The central diagnoal line indicates the homology expected from aligning two identical sequences. Several lines parallel to the diagonal at approximately 600 indicate internal homology.
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were subcloned into a vector and their nucleotide sequence determined. The analysis of the sequence revealed that human AMBN gene consists of 13 exons corresponding to the cDNA segment encompassing nucleotides 50–1438 ( Fig. 3). All the determined splice sites in this segment were of type O, occurring between codons, with all donors sites containing ‘GT’ and all acceptors sites containing ‘AG’ consensus splice sequences. The 78 bp insert represents two exons, which are absent in other reported mammalian AMBN sequences due to alternative splicing at the mRNA level. The two extra exons (exons 8 and 9) represented an internal sequence duplication of exon 7. There was no polymorphism in the coding region of the human AMBN gene between the cDNA sequence and sequences of two human genomic DNAs. The coding region of the human AMBN gene, including the part specifying the signal peptide, has a high sequence similarity with other reported mammalian AMBN cDNA sequences (Fig. 4): 66, 61, 61, and 58% with the pig, cattle, rat, and mouse sequences, respectively. The predicted translation product of the human AMBN gene is an acidic protein, pI 4.54, with a calcu-
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lated molecular weight (M ) of 45 300 Da, excluding the r signal peptide. The protein sequence predicted from the human AMBN cDNA sequence contains 15.2% proline, 10.2% leucine, and 9.0% glycine, which are values comparable with those found in other mammalian AMBN sequences. A comparison of the amino acid compositions of human, pig, cattle, rat, and mouse AMBN is shown in Table 1. The functional protein motifs include potential phosphorylation sites for casein kinase II at residue 261, protein kinase C (residues 46), and protein tyrosine kinase (residue 99). These motifs, which are conserved in the pig, cattle, rat, and mouse amino acid sequences, are highlighted in Fig. 4. The hydrophilicity plots of these sequences share the following characteristics (Fig. 5): the hydrophobic leader peptide of approximately 30 amino acid residues is followed by a short hydrophilic segment (about 10 residues), and then by a more variable internal segment of irregularly alternating hydrophilicity and hydrophobicity; the C-terminal region is hydrophilic. A phylogenetic tree based on amino acid sequences of all available AMBN sequences ( Fig. 6) indicates that the sequences group as expected from their taxonomical positions.
Fig. 3. Genomic organization of the human AMBN gene. The genomic structure of the human AMBN cDNA was determined by DNA sequence analysis of genomic subclones generated by PCR. Hatched rectangles indicate that the three exons represent the internal sequence duplication. Intron sequences and exon sequences are indicated in lower-case and upper-case letters, respectively. Abbreviations: TGA, stop codon; UTR, untranslated region; ND, not determined.
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Fig. 5. Hydrophilicity plot of AMBNs from all available sequences prepared using the method of Kyte and Doolittle (1982). The plots share the following characteristics: the hydrophobic leader peptide of approximately 30 amino acid residues is followed by a short hydrophilic segment (about 10 resudues), a short hydrophobic segment (10 residues), strongly hydrophilic segment (residues 80–110), and then a more vaariable internal segment (residues 110–370) of irregularly alternating hydrophilicity and hydrophobicity; the C-terminal regioans are all hydrophilic.
To investigate the possibility that other AMBN-like loci exist in humans, we carried out a Southern blot analysis using either a full-length cDNA probe or a probe encompassing nearly the entire last exon of the
AMBN gene. Using the full-length probe, a single band was found following digestion with both EcoRI and BamHI restriction endonucleases; two bands were found using the HindIII and TaqI enzymes (data not shown).
Fig. 4. Amino acid sequence alignment of human, pig, cattle, rat, and mouse AMBNs. A dash (−) indicates an alignment gap. Potential phosphorylation sites (PKC, protein kinase C; TK, tyrosine kinase; CK2, casein kinase II ) are indicated by boxes. An asterisk under the alignment indicates amino acid residues conserved in all species. Amino acid residues are given in the IUPAC-IUB single-letter code.
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Fig. 6. Neighbor-joining tree of sequences shown in Fig. 3. Genetic distances (numbers on branches) were estimated from the proportion of differences in pairwise comparison following exclusion of gaps.
However, using the single-exon probe, a single band was found with the NotI, PstI, and TaqI enzymes. These results are consistent with the presence of a single AMBN locus in the human genome. 3.2. Expression of the AMBN gene in human ameloblastomas cDNAs were reversely transcribed from three ameloblastomas and tooth germs and PCR amplified with AMBN gene-specific primers. The expected sizes of the normal amplification products with the primer pairs AMB26 and AMB27, AMB10 and AMB8, as well as AMB11 and AMB8, were 1597, 1008, and 748 bp,
respectively. In all three ameloblastoma samples, amplification products corresponding in size to those amplified from human tooth germs could be detected (Fig. 7). As a negative control, cDNA derived from the pulp of adult human teeth was tested with AMBN cDNAspecific primers, but no band was obtained (data not shown). The products amplified from the three ameloblastoma samples with the primer pair AMB26 and AMB27 were then subcloned into a vector and sequenced. The sequence of one ameloblastoma (tumor 1) contained an open reading frame of 1329 bp encoding a 443 amino acid protein, which had three substitutions ( TC: MetThr; AG: GlyGly; AG: HisArg) and one deletion (12 nucleotides) compared with the tooth-germ cDNA (Fig. 8). The AMBN sequence from the other two ameloblastomas (tumors 2 and 3) had substitutions but no deletion (Fig. 8). Since no polymorphism was found in the coding regions of the AMBN gene in normal tissues, substitutions and deletions found in the ameloblastomas sequences were probably the result of tumor-specific mutations, possibly as a result of the abnormal metabolic changes in the tumor cells. The expression of the AMBN gene might become a useful marker of the ameloblast cell-lineage. Compared with amelogenin, AMBN is expressed in broader area of the inner enamel epithelium containing presecretory ameloblast and Hertwig’s epithelial sheath (Fong et al., 1996; Lee et al., 1996).
Fig. 7. RT-PCR analysis of the human ameloblastomas. The cDNAs synthesized from the tooth germs and ameloblastomas, were amplified using AMBN cDNA-specific primer sets. Lanes 1, 2, and 3, tooth germs; lanes 4, 5, and 6, ameloblastomas (tumor 1); lanes 7,8, and 9 ameloblastoma (tumor 2); lanes 10. 11, and 12, ameloblastoma (tumor 3). Lanes 1, 4, 7 and 10 were amplified with primers AMB26 and AMB27; lanes 2, 5, 8, and 11 with primers AMB10 and AMB8; and lanes 3, 6, 9, and 12 with primers AMB11 and AMB8. M indicates the lanes containing a DNA size marker, a 100 bp ladder.
Fig. 8. Sequence comparison of the AMBN homologs of ameloblastoma with the normal AMBN cDNAs. DNA sequence of the products amplified with the AMB26 and AMB27 primers from three ameloblastomas (tumors 1, 2, 3) and tooth germs, were compared using the Multiple Alignment program of the MacVector. Identical nucleotides between the sequences are indicated by an asterisk under the alignment, and an absent nucleotide by a dash. Primer positions and orientations are indicated by an arrowed line above their location in the sequence. The upper-case letters above the sequence are the IUPAC-IUB single-letter code designations of amino acids translated from human AMBN cDNA, while the upper-case letters below the sequence are amino acids translated from the mutations.
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Table 1 Amino acid composition of human, pig, cattle, rat, and mouse ameloblastin Amino acid
Human Residues
Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamie Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Trytophan Tyrosine Valine Totals
Pig Res/1000
35 83 11 26 13 31 20 48 0 0 26 62 29 69 38 90 11 26 6 14 43 102 12 29 22 52 18 43 64 152 30 71 20 48 3 7 9 21 11 26 421 M 45.3 kDa r pI 4.54
Residues
Cattle Res/1000
31 79 14 35 11 28 13 33 0 0 23 58 31 79 46 117 6 15 7 18 33 84 12 30 16 41 15 38 59 149 31 79 18 46 3 8 11 28 15 38 395 M 42.0 kDa r pI 4.98
Residues
Rat Res/1000
29 79 13 36 11 30 14 38 0 0 21 57 31 85 40 109 9 25 8 22 35 96 9 25 14 38 15 41 52 142 20 55 21 57 3 8 8 22 13 36 366 M 39.3 kDa r pI 4.93
Residues
Mouse Res/1000
32 81 11 28 14 35 12 30 0 0 23 58 30 76 41 104 13 33 8 20 35 88 9 23 16 40 15 38 63 159 24 61 20 51 3 8 10 25 17 43 396 M 42.4 kDa r pI 5.11
Residues
Res/1000
34 89 15 39 14 37 11 29 0 0 22 58 30 79 39 102 10 26 9 24 34 89 9 24 15 39 16 42 56 147 23 64 19 50 3 8 7 18 15 39 381 M 40.8 kDa r pI 5.50
3.3. Conclusions
References
The human AMBN gene shows a high sequence similarity with pig, cattle, rat, and mouse genes. The proteins encoded in all these genes share several features such as the presence of potential phosphorylation sites (casein kinase II, protein kinase C, and tyrosine kinase), similar patterns of hydrophilicity, and a high proportion of Pro, Leu, and Gly residues. The human AMBN gene consists of 13 exons and contains a novel 78 bp (26 amino acid) insert representing two short exons produced by internal sequence duplication. There appears to be only a single AMBN locus in humans. The AMBN transcripts are expressed in human ameloblastomas and contain some tumor-specific mutations.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1994. Current Protocols in Molecular Biology. Wiley, New York. Bonass, W.A., Robinson, P.A., Kirkham, J., Shore, R.C., Robinson, C., 1994. Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence. Biochem. Biophys. Res. Commun. 198, 755–763. Ceˇrny´, R., Slaby, I., Hammarstro¨m, L., Wurtz, T., 1996. A novel gene expressed in rat ameloblasts codes for proteins with cell binding domains. J. Bone Miner. Res. 11, 883–891. Fong, C.D., Slaby, I., Hammarstro¨m, C., 1996. Amelin: an enamelrelated protein, transcribed in the cells of epithelial root sheath. J. Bone Miner. Res. 11, 892–898. Hu, C.C., Fukae, M., Uchida, T., Qian, Q., Zhang, C.H., Ryu, O.H., Tanabe, T., Yamakoshi, Y., Murakami, C., Dohi, N., Shimizu, M., Simmer, J.P., 1997. Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins. J. Dent. Res. 76, 648–657. Krebsbach, P.H., Lee, S.K., Matsuki, Y., Kozak, C.A., Yamada, K.M., Yamada, Y., 1996. Full-length sequence localization, chromosomal mapping of ameloblastin: a novel tooth-specific gene. J. Biol. Chem. 271, 4431–4435. Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132. Lee, S.K., Krebsbach, P.H., Matsuki, Y., Nanci, A., Yamada, K.M., Yamada, Y., 1996. Ameloblastin expression in rat incisors and human tooth germs. Int. J. Dev. Biol. 40, 1141–1150. MacDougall, M., Dupont, B.R., Simmons, D., Reus, B., Krebsbach, P.H, Karrman, C., Holmgren, G., Leach, R.J., Forsman, K., 1997. Ameloblastin gene (AMBN ) maps within the critical region for
Acknowledgements We thank Professor Jan Klein for comments and suggestions and Ms Jane Kraushaar for editorial assistance. We would like to express our gratitude to all the patients who participated in this study. This work was supported by Grant-in-aid 11671800 from Ministry of Education, Science, Sports and Culture, Japan, and supported by JSPS Research for the Future Program Biological Tissue Engineering Project no. JSPS-RFTF 98I00201.
S. Toyosawa et al. / Gene 256 (2000) 1–11 autosomal dominant amelogenesis imperfecta at chromosome 4q21. Genomics 41, 115–118. Miles, A.E.W., Poole, D.F.G., 1967. The history and general organization of dentitions. In: Miles, A.E.W. ( Ed.), Structural and Chemical Organization of Teeth. Academic Press, London, pp. 3–44. Mori, M., Yamada, K., Kasai, T., Yamada, T., Shimokawa, H., Sasaki, S., 1991. Immunohistochemical expression of amelogenins in odontogenic epithelial tumours and cysts. Virchows Arch. A Pathol. Anat. Histopathol. 418, 319–325. Regezi, J.A., Sciubba, J.J., 1999. Odontogenic tumors. Oral Pathology: Clinical Pathologic Correlations. third ed., W.B. Saunders, Philadelphia, PA.
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Snead, M.L., Luo, W., Hsu, D.D.-J., Melrose, R.J., Lau, E.C., Stenman, G., 1992. Human ameloblastoma tumors express the amelogenin gene. Oral Surg. Oral Med. Oral Pathol. 74, 64–72. Takata, T., Zhao, M., Uchida, T., Kudo, Y., Sato, S., Nikai, H., 2000. Immunohistochemical demonstration of an enamel sheath protein, sheathlin, in odontogenic tumors. Virchows Arch. 436, 324–329. Uchida, T., Fukae, M., Tanabe, T., Yamakoshi, Y., Satoda, T., Murakami, C., Yakahashi, O., Shimizu, M., 1995. Immunochemical and immunocytochemical study of a 15KDa non-amelogenin and related proteins in the porcine immature enamel: Proposal of a new group of enamel proteins ‘sheath proteins’. Biomed. Res. 16, 131–140.