©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Full-length Sequence, Localization, and Chromosomal Mapping of Ameloblastin
A NOVEL TOOTH-SPECIFIC GENE (*)

(Received for publication, September 18, 1995; and in revised form, November 6, 1995)

Paul H. Krebsbach Suk Keun Lee Yutaka Matsuki Christine A. Kozak (1) Kenneth M. Yamada Yoshihiko Yamada (§)

From the Laboratory of Developmental Biology, NIDR and the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-4370

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization.


INTRODUCTION

The mammalian tooth is a specialized structure that develops through a series of reciprocal epithelial-mesenchymal interaction culminating in the formation of three different mineralized tissues: enamel, dentin, and cementum(1, 2, 3) . The tooth has provided a valuable model system for understanding specific gene regulation, morphogenesis, and mineralization. Enamel is an oral epithelial-derived mineralized tissue, while dentin and cementum are formed by cranial neural crest-derived ectomesenchyme(4, 5, 6) . This cranial neural crest-derived ectomesenchyme is derived from the neuroectoderm of rhombomere 2 of the vertebrate hindbrain(7, 8) . As in bone, the premineralized matrix of dentin and cementum contains a collagen-rich matrix that is maintained throughout the mature tissue. However, enamel differs from these other mineralized tissues in two important ways: 1) the epithelial-derived enamel matrix does not contain type I collagen; and 2) the organic matrix of enamel is essentially lost during tissue maturation, resulting in a tissue that is more than 98% mineral in the form of hydroxyapatite(9, 10) .

Amelogenesis, the formation of tooth enamel, is divided into discrete developmental stages(11) . Following a presecretory stage in which the inner enamel epithelium receives developmental cues from the dental papillae, the inner enamel epithelium differentiates into secretory ameloblasts. The enamel matrix, secreted by the ameloblast, is composed of two classes of proteins termed the amelogenins and the enamelins (12) . The amelogenins are hydrophobic proteins rich in proline, histidine, and glutamic acid and comprise about 90% of the enamel matrix. Diversity of the amelogenins found in enamel matrix is due to alternative mRNA splicing(13) . The remaining 10% of enamel matrix is made up of acidic enamelins (14) and other non-amelogenin proteins such as amelogeninase(15, 16) . Once the full thickness of enamel matrix is formed, significant morphological and functional changes occur within the ameloblast as it passes through the transition, maturation, and protective stages of development. Thus, the developing tooth is a unique mineralizing organ and provides an interesting developmental system.

Although a few tooth-specific genes have been identified, the precise mechanisms of tooth morphogenesis, mineralization and the pathophysiology of inherited enamel and dentin defects are not well defined. We have therefore initiated a genome project with an ultimate goal of identifying novel genes involved in tooth formation which may help explain the mechanisms of odontogenesis(17) . We chose to use the continuously erupting rat incisor as a model system, because the complete developmental sequence of odontogenesis can be analyzed in a single tooth. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3-4-week-old rats, sequenced clones, and classified sequences based on their homology to known genes(17) . One clone termed Y224 was the most abundant novel gene expressed and was partially sequenced(17) . Here, we report the full-length sequencing, cell-specific expression, and protein localization pattern of a newly described ameloblast-specific gene which we have designated ameloblastin.


EXPERIMENTAL PROCEDURES

DNA Sequence Analysis

Both strands of the initial Y224 cDNA insert were sequenced by the dideoxynucleotide chain termination method using the Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Corp.) or by automated DNA sequencing (Applied Biosystems, model 370A). Specific oligonucleotide primers were synthesized on an Applied Biosystems model 380B DNA synthesizer.

Recombinant Protein and Antibody Production

A recombinant protein was made by subcloning a fragment of Y224 (17) corresponding to +613 to +1134 base pairs into an expression vector that encodes six histidine residues at the N terminus of the recombinant protein (pQE-30, Qiagen). The recombinant plasmid (pKQE-17) was transformed into competent M15(pREP4)cells and the protein was induced with isopropyl-beta-D-thiogalactopyranoside. The recombinant protein was purified using a nickel-nitrilotriacetic acid-agarose affinity column (Qiagen). Polyclonal antibodies against pKQE-17 were made using standard procedures. New Zealand White rabbits were inoculated with 500 µg of pKQE-17 in an equal volume of Freund's complete adjuvant. The rabbit was boosted three times at 2-week intervals using 250 µg of pKQE-17 and an equal volume of Freund's incomplete adjuvant (Duncroft Inc.). Isolation of IgG from serum were performed using a protein A-agarose column (Affi-Gel MAPS II, Bio-Rad).

Probes Used for in Situ Hybridization

The Y224 clone was linearized by BamHI and XhoI for antisense and sense probe production, respectively. Digoxigenin-UTP-labeled, single-strand antisense and sense RNA probes were prepared by T7 RNA polymerase and T3 RNA polymerase, respectively, using a RNA labeling kit (Boehringer Mannheim).

In Situ Hybridization

For in situ hybridization, 3-4-week-old Sprague-Dawley rats were anesthetized with ketamine hydrochloride (Sigma) and perfused with 4% paraformaldehyde in phosphate-buffered saline (pH 7.0) through the left ventricle. Mandibles, including the incisors, were excised and decalcified in 10% EDTA (pH 8.0) for 10 days and embedded in paraffin. In situ hybridization was performed on 5-µm sections(18) . Hybridization of tissue sections was performed in a hybridization solution (50% formamide, 10 mM Tris-HCl (pH 7.6), 200 µg/ml tRNA, 1 times Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA) at 50 °C for 16 h in a humidified chamber. Slides were sequentially washed in 2 times SSC containing 50% formamide at 55 °C for 30 min and then rinsed at 37 °C for 10 min in TNE (10 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA). Unhybridized transcripts were digested with 20 µg/ml RNase A (Sigma) in TNE at 37 °C for 30 min. The slides were washed again in TNE at 37 °C for 10 min, once with 2 times SSC at 50 °C for 20 min and finally twice with 0.2 times SSC at 50 °C for 20 min. Specific transcripts were detected with anti-digoxigenin-conjugated alkaline phosphatase antibody (Boehringer Mannheim).

Immunohistochemistry Methods

Paraffin sections were deparaffinized and hydrated before inactivation of endogenous peroxidases by 3% hydrogen peroxide for 10 min. The sections were blocked with normal goat serum, and three-layer immunolabeling was performed. For immunolabeling, an antigen affinity-purified mono-specific antibody raised against the pKQE-17 recombinant protein was used. The sections were incubated with a biotinylated secondary antibody followed by streptoavidin-peroxidase. Immunolocalization was visualized by diaminobenzidine (HistoMark, Kirkegaard and Perry Laboratories). As a negative control, the antibody against pKQE-17 was preabsorbed with the fusion protein and was used in the same immunostaining procedure described above.

RNA Isolation and Analysis

Total RNA was extracted from tissues using an acid-guanidinium-phenol-chloroform method(19) . RNA pellets were stored at -80 °C until analyzed. Ten micrograms of total RNA were separated on a 1% agarose, 1 M formaldehyde gel and transferred by capillary action to Nylon membranes (Magna NT, Micron Separations Inc.). A full-length cDNA (Y224) probe was labeled with [alphaP]dCTP by a random-primed oligonucleotide method and hybridized at 42 °C(20) .

Chromosomal Mapping of the Mouse Ameloblastin Gene

The ameloblastin gene was mapped by analysis of two sets of multilocus crosses: (NFS/N or C58/J times Mus musculus musculus) times M. m. musculus(21) and (NFS/N times Mus spretus) times M. spretus or C58/J(22) . DNAs from these crosses have been typed for over 850 loci, including the chromosome 5 loci Kit (kit oncogene), Afp (alpha-fetoprotein), Ibsp (bone sialoprotein), and Pdeb (phosphodiesterase beta) as described previously(23, 24) . Data from these crosses are stored and analyzed using the program LOCUS developed by C. E. Buckler (National Institutes of Health, Bethesda, MD)


RESULTS

We previously constructed a unidirectional cDNA library from the non-calcified tissues of rat incisors and partially sequenced the 5` portion of the coding strands of about 400 randomly selected clones (17) . Sequence homology searches through GenBank(TM) data base and PIR protein data base revealed that the majority of the clones (about 56%) represented previously unidentified genes. To identify novel genes involved in tooth development we have first focused on the most frequently expressed genes from this previously unidentified cDNA population.

Tissue-specific Expression of Y224

Northern blot analysis of total RNA extracted from rat spleen, brain, eye, salivary gland, heart, thymus, lung, kidney, and incisors revealed that mRNA for Y224 was detected only in incisor tissue (Fig. 1A). Ameloblastin mRNA was not detected by Northern blot analysis in other mineralized tissues such as bone and cartilage (data not shown). Two transcripts were identified at approximately 2.0 and 1.6 kb. (^1)An identical restricted pattern of mRNA expression was observed in mouse tissues (Fig. 1B).


Figure 1: Tissue-specific expression of ameloblastin mRNA. RNA extracted from rat (A) or mouse (B) tissues was subjected to Northern blot analysis. A 10-µg sample of total RNA was separated on a 1% agarose-formaldehyde gel and transferred to a nylon membrane by capillary action. Filters were hybridized with a [alphaP]dCTP-labeled cDNA (Y224). The lower panel shows ethidium bromide staining of 18 and 28 S ribosomal RNAs.



Localization of Y224 Using in Situ Hybridization

We examined the expression pattern of clone Y224 by in situ hybridization (Fig. 2). Expression of Y224 mRNA was limited to the ameloblast cell layer (Fig. 2B). Especially high levels of expression were observed in the distal cytoplasm of secretory ameloblasts. The mouse incisor showed identical expression using the rat antisense RNA probe (data not shown). Since in situ hybridization showed high levels of expression of Y224 in both rat and mouse incisors, and because it was localized primarily in cells of the ameloblast lineage, we named its gene product ameloblastin and further characterized the cDNA and studied the localization of its gene product.


Figure 2: Ameloblastin expression in rat incisors (times 400, no counterstain). A, in situ hybridization using the Y224 sense RNA probe (negative control). B, in situ hybridization using the Y224 antisense probe. Ameloblastin is localized to the cytoplasm of secretory ameloblasts (Am). C, immunohistochemical staining of ameloblastin. Ameloblastin is localized to the distal cytoplasm and Tomes' processes of secretory ameloblasts (closed arrow). Extracellular staining is evident at the dentino-enamel junction (open arrow). E represents enamel matrix.



Tissue Distribution of Ameloblastin

To analyze the expression of ameloblastin protein in rat incisors, we raised antibodies against the recombinant protein pKQE-17. The IgG fraction was purified using a protein A-agarose column. As shown in Fig. 2C, anti-ameloblastin antibodies reacted exclusively with ameloblasts in the developing rat incisors. Intense immunostaining was found in the specialized cytoplasmic extension termed the Tomes' process. This distinctive apical, cytoplasmic structure develops during the secretory phase of amelogenesis at the interface between the secreting cells and forming enamel, suggesting that ameloblastin may be involved in the secretion of enamel matrix.

DNA Sequence Analysis and Deduced Protein Characteristics

The cDNA sequence of ameloblastin is illustrated in Fig. 3. The first methionine codon is designated base 1 and is followed by a hydrophobic stretch consistent with a signal sequence and a predicted signal sequence cleavage site(25) . Two additional methionine residues at bases 114 and 129, respectively, are surrounded by amino acids that correlate more closely with the reported translation start site consensus sequence(26) . Presuming the use of the first methionine in the open reading frame as the translation start site, ameloblastin encodes an open reading frame of 422 amino acids corresponding to a protein of 45 kDa. Ameloblastin is an acidic protein (pI = 5.54) rich in proline (15.2%), glycine (9.9%), and leucine (9.9%). The sequence contains a number of potential myristoylation sites (residues 56-61, 250-255, 271-276, 279-284, 345-350). A presumptive polyadenylation signal begins at base 1797 and is followed by a poly(A) tail. Other protein motifs include potential phosphorylation sites for casein kinase II at residue 241, protein kinase C (residues 51, 268, and 276), and protein tyrosine kinase (residue 104). Data base analysis through GenBank(TM) and the PIR protein data bases indicated that ameloblastin does not exhibit sequence similarities to any reported gene except for the Y224 partial sequence that we had reported (17) and thus represents a novel tooth-specific gene.


Figure 3: Nucleotide sequence and deduced amino acid sequence of rat ameloblastin. The 1929-base pair nucleotide sequence of the full-length cDNA is shown, including untranslated 5` and 3` sequences. The numbers on the left refer to nucleotide positions. The numbers on the right refer to amino acid positions. Potential polyadenylation signals are boxed. The presumptive signal sequence is underlined and the closed arrow represents the predicted signal sequence cleavage site(25) .



The Ameloblastin Gene Maps to Mouse Chromosome 5

Southern blotting identified hybridization of Y224 to XbaI fragments of 5.0 and 4.8 kb in M. m. musculus and 4.8 and 4.5 in NFS/N and C58/J. BamI digestion produced Y224 reactive fragments of 21 kb in NFS/N and C58/J and 15 kb in M. spretus. Inheritance of these fragments in the progeny of the two sets of crosses was compared with inheritance of previously typed markers, and the gene for ameloblastin, Ambn, was mapped to chromosome 5 (Fig. 4). This gene was positioned just distal to Kit in a region of conserved linkage with human chromosome 4q, suggesting a human map location for this gene.


Figure 4: Map location of the ameloblastin gene on mouse chromosome 5. To the right of the map are recombination fractions for adjacent loci: the first fraction represents data from the M. m. musculus crosses, and the second fraction is from the M. spretus crosses. In parentheses are recombinational distances and standard errors calculated according to Green(35) , Map locations of the human homologs are indicated to the left.




DISCUSSION

As a result of our initial strategy designed to identify new genes involved in odontogenesis, we obtained partial DNA sequence on over 400 cDNA clones from a rat incisor library(17) . One clone that appeared multiple times during random sequencing, Y224, was further characterized and named ameloblastin. We have completed the sequencing of the cDNA for ameloblastin, characterized its expression in the continuously erupting rat incisor, and mapped its locus within the mouse genome. Four other clones that share partial sequence with ameloblastin were identified and are currently being investigated as potential alternatively spliced transcripts.

The cDNA for ameloblastin encodes an open reading frame of 422 amino acids that corresponds to a predicted 45-kDa protein with no significant homology to any reported gene except Y224. The predicted protein is rich in proline and glycine, but it does not display a primary structure consistent with reported collagens. Thus, ameloblastin is a novel tooth-specific gene. Although ameloblastin does not share sequence homology with other reported genes, it does contain potential phosphorylation sites, including a casein kinase II phosphorylation site that is shared by proteins involved in mineralization such as in osteopontin(27) , bone sialoprotein(28) , bone acidic glycoprotein-75(29) , and dentin phosphoprotein(30) , as well as a tyrosine kinase site and three protein kinase C sites.

Northern blot analysis of multiple rat and mouse tissues showed that ameloblastin expression was restricted to the developing tooth. Two transcripts were observed at 2.0 and 1.6 kb. It is not yet determined if the two transcripts result from alternative mRNA splicing, the use of multiple transcription start sites, or the use of two different polyadenylation sites.

The immunohistochemical analysis of ameloblastin was consistent with the results obtained by in situ hybridization. The finding that ameloblastin became condensed in the Tomes' processes of secretory ameloblasts and in the distal cytoplasm of mature ameloblasts may imply that the production of ameloblastin is closely related to the process of secretion. Diffuse staining was seen in the superficial enamel layer near the mature ameloblasts, a region that is mainly composed of homogenous enamel matrix. Irregular positive immunolocalization of ameloblastin was also observed in the dentino-enamel junction in a pattern similar to that reported for tuftelin(14) . The persistent expression of ameloblastin in the cytoplasm of mature ameloblasts and the dispersed positive reaction of ameloblastin in the superficial enamel layer near post-secretory ameloblasts support the notion that ameloblastin could play a role in enamel mineralization. However, the precise function of ameloblastin during amelogenesis remains to be determined.

Previous studies have demonstrated that the region of the Ambn locus on mouse chromosome 5, and the corresponding region on human chromosome 4 contain a number of genes important in mineralization, including osteopontin, bone sialoprotein, and bone morphogenetic protein 3(31) . Ambn is not, however, clustered with these more distally located genes. Recent studies, however, place the human disorder dentinogenesis imperfecta type II in close linkage with human osteopontin(32, 33) . Since both osteopontin and bone sialoprotein have been excluded as candidates for this disorder(23, 33) , we are pursuing the possibility that a defect in Ambn might be responsible for this phenotype as well as an autosomal dominant form of amelogenesis imperfecta, which has been linked to human chromosome 4q(34) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35097[GenBank].

§
To whom correspondence should be addressed: Laboratory of Developmental Biology, National Institute of Dental Research, Bldg. 30, Rm. 413, 30 Convent Dr., MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-2111; Fax: 301-402-0897; :yamada{at}yoda.nidr.nih.gov.

(^1)
The abbreviation used is: kb, kilobase pair(s).


ACKNOWLEDGEMENTS

-We thank Drs. Suzanne Bernier and Ken Nakata for helpful discussions and critical reading of the manuscript.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.