Eduardo C. Lau

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.

DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.

Isolation and characterization of a mouse amelogenin expressed in Escherichia coli

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Alternative splicing of the mouse amelogenin primary RNA transcript

A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5′ ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5′ untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.

Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth

The primary structures, molecular genetics and biosynthesis of the amelogenin protein of the developing tooth are established, but knowledge of their subsequent post-secretory processing and its relation to enamel biomineralization is fragmentary. Preparations of tooth matrix proteins were isolated from molars (M1) of mice from birth to 15 days and analysed by SDS-PAGE and immunochemical methods. Amelogenin proteins, isolated and partially purified by HPLC, were characterized by amino acid analysis and SDS-PAGE. At birth a 26 kDa amelogenin was present that during subsequent developmental stages generated a series of 20-25 kDa amelogenins differing in apparent size by approximately 1 kDa. Amino acid analyses showed that all these amelogenins have amino-terminal TRAP sequences; analyses for both glycosylation and phosphorylation were negative. It is suggested that these post-secretory amelogenins are generated by a sequence of specific carboxy-terminal cleavages, and that the observed post-secretory processing of amelogenin is functionally linked to the structure of the enamel matrix and the control of crystallite development.

Linkage of amelogenin (Amel) to the distal portion of the mouse X chromosome

Amelogenins are hydrophobic, proline-rich proteins that are the primary biosynthetic products of ameloblasts. These cells are responsible for the formation of tooth enamel, and amelogenins play an important role in the process of biomineralization. A cDNA, corresponding to the mouse 26-kDa amelogenin, has been molecularly cloned and sequenced. Southern blot analysis of genomic DNA from the mouse using this cDNA as a probe indicates that there is only one mouse amelogenin (Amel) gene. This paper describes restriction site variation for the Amel gene that we have identified between C57BL/6 and M. spretus and the segregation of that variation as an X-chromosome gene. The position of the amelogenin locus (Amel) relative to the loci for alpha-galactosidase (Ags), proteolipoprotein (Plp), and the random genomic probe DXWas31 has been determined. Amel is established as: (1) the most distal locus in the genetic map of the mouse X chromosome, (2) lying proximal to the X:Y pairing region, and (3) being restricted to the mouse X chromosome.

Human developing enamel proteins exhibit a sex-linked dimorphism.

SummaryThe amelogenin protein of developing dental enamel is generally accepted to mediate the regulation of the form and size of the hydroxyapatite crystallites during enamel biomineralization (1). A genetic disorder of enamel development (amelogenesis imperfecta) has been linked to theamelogenin geneAMEL (2–3), and loci regulating enamel thickness and tooth size have been mapped to the human sex chromosomes (4). In the human genome there are twoAMEL loci with one copy of the gene on each of the sex chromosomes (AMELX andAMELY), whereas in the mouse only anAMELX locus is present (5). It is presently unknown if humanAMELY is transcriptionally active. These observations prompted us to examine specimens of human developing enamel for sexual dimorphism at the protein level. We report here, for the first time, a diagnosis of differences in human enamel proteins which permits the distinction of specimens according to the sex of the individual.

Human ameloblastoma tumors express the amelogenin gene

Instructive signals are responsible for the regulation of the expression of gene products characteristic of many cell lineages during normal development and potentially during neoplasia. The odontogenic origin of ameloblastomas is based largely on the similarity in histologic appearance between the tumor and the developing tooth organ. A pathognomonic pattern for odontogenic tissue-specific gene expression in ameloblastomas has not been previously shown. In these studies, the gene expression parameters for human ameloblastomas have been characterized with the techniques of messenger RNA phenotyping in combination with Northern and in situ hybridization analysis of messenger RNA. The results of these studies confirm that amelogenin, a gene transcribed solely by differentiated ameloblasts, was expressed by epithelial cells from human ameloblastomas. This observation suggests that the instructive signals required for ameloblast differentiation are shared during normal development and tumorigenesis of odontogenic epithelium.

Autosomal localization of the amelogenin gene in monotremes and marsupials: implications for mammalian sex chromosome evolution.

We have determined by Southern blot analysis that DNA sequences homologous to the AMG gene probe are present in the genomes of both marsupial and monotreme mammals, although adult monotremes lack teeth. In situ hybridization and Southern analysis of cell hybrids demonstrate that AMG homologues are located on autosomes. In the Tammar Wallaby, AMG homologues are located on chromosomes 5q and 1q and in the Platypus, on chromosomes 1 and 2. The autosomal location of the AMG homologues provides additional support for the hypothesis that an autosomal region equivalent to the human Xp was translocated to the X chromosome in the Eutheria after the divergence of the marsupials 150 million years ago. The region containing the AMG gene is therefore likely to have been added 80-150 million years ago to a pseudoautosomal region shared by the ancestral eutherian X and Y chromosome; the X and Y alleles must have begun diverging after this date.

Analysis of Human Enamel Genes: Insights Into Genetic Disorders of Enamel

A number of inherited craniofacial diseases are known to be associated with gene mutations. Inherited genetic disorders of enamel formation called amelogenesis imperfecta (AI) affect the human population with a prevalence of 1 in 14,000 in the United States. Amelogenins, the major proteins in developing enamel matrix of mammalian teeth, have been suggested to participate in normal enamel matrix biomineralization, as well as with abnormal biomineralization such as seen in AI. The complementary DNA for mouse amelogenin gene (AMEL) has been cloned, characterized, and used as a probe to establish the chromosomal locations of AMEL for mouse and man. The human AMEL gene sequences have been located to the distal short arm p22.1----p22.3 region of the X chromosome, and the pericentromeric region of the Y chromosome. An assignment of human AMEL gene to the X chromosome p22 region together with a recent assignment of the X-linked AI disease locus to the Xp22.2 region support the association of the AMEL-X gene with AI. This also leads us to propose that a mutated AMEL-X gene produces altered amelogenin polypeptide, which is defective in its ability to participate in mineralization of enamel matrix, thus giving rise to the X-linked phenotypes of AI.