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Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

ABSTRACT: Amelogenin, the major enamel matrix protein in tooth development, has been demonstrated to play a significant role in tooth enamel formation. Previous studies have identified the alternative splicing of amelogenin in many mammalian vertebrates as one mechanism for amelogenin heterogeneous expression in teeth. While amelogenin and its splicing forms in mammalian vertebrates have been cloned and sequenced, the amelogenin gene, especially its splicing forms in non-mammalian species, remains largely unknown. To better understand the mechanism underlying amelogenin evolution, we previously cloned and characterized an amelogenin gene sequence from a squamate, the green iguana. In this study, we employed RT-PCR to amplify the amelogenin gene from the black spiny-tailed iguana Ctenosaura similis teeth, and discovered a novel splicing form of the amelogenin gene. The transcript of the newly identified iguana amelogenin gene (named C. Similis-T2L) is 873 nucleotides long encoding an expected polypeptide of 206 amino acids. The C. Similis-T2L contains a unique exon denominated exon X, which is located between exon 5 and exon 6. The C. Similis-T2L contains 7 exons including exon 1, 2, 3, 5, X, 6, and 7. Analysis of the secondary and tertiary structures of T2L amelogenin protein demonstrated that exon X has a dramatic effect on the amelogenin structures. This is the first report to provide definitive evidence for the amelogenin alternative splicing in non-mammalian vertebrates, revealing a unique exon X and the splicing form of the amelogenin gene transcript in Ctenosaura similis.

SUBMITTER: Wang X

PROVIDER: S-EPMC3454316 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

Wang Xinping X Deng Xuliang X Zhang Xichen X

PloS one 20120924 9

Amelogenin, the major enamel matrix protein in tooth development, has been demonstrated to play a significant role in tooth enamel formation. Previous studies have identified the alternative splicing of amelogenin in many mammalian vertebrates as one mechanism for amelogenin heterogeneous expression in teeth. While amelogenin and its splicing forms in mammalian vertebrates have been cloned and sequenced, the amelogenin gene, especially its splicing forms in non-mammalian species, remains largely ...[more]

PMID: 23029286

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Project description:In the mouse tooth organ, shortly after birth, ameloblasts acquire their secretory phenotype, which is characterized by the prominent expression and subsequent secretion of two isoforms of amelogenin, M180 and M59 (LRAP, [A-4]). Amelogenin deposition into the ameloblast extracellular matrix promotes enamel biomineralization. A complex set of intercellular signaling events, reciprocal communications between the developing oral epithelium and its underlying dental mesenchyme, guide the expression of amelogenin mRNA, and limit it to a defined period of tooth development. In tooth germ organ culture, addition of the [A-4] isoform, lacking amelogenin exon 4 and exon 6 segments a, b, c, was shown to affect ameloblast development. To understand the basis for this regulatory activity, we have studied the effects of r[A-4] on ameloblast-like LS8 cells, and the role of the putative [A-4] cell surface receptor, LAMP1, as well as the related receptor LAMP3. In the LS8 cells, the expression of the spliced isoforms of amelogenin, LAMP1, and LAMP3 were identified by RT-PCR, and real-time PCR semi-quantitative analysis assessed the modulation of M180 message. M180 mRNA was up-regulated by exogenous [A-4], and this was further increased by blockade of LAMP1, suggesting additive effects between the intracellular signaling pathways activated by the discrete agonists. Immunofluorescence staining identified the patterns of [A-4] and LAMP1 localization in LS8 cells. Internalized r[A-4] was co-localized with LAMP1 in late endosomal/lysosomal compartments. Thus, the LAMP1 and [A-4] intracellular sorting pathways are interrelated. The nitric oxide (NO) signaling pathway was activated by exogenous [A-4]. [A-4] modulated inducible nitric oxide synthase (iNOS, NOS2) and endothelial nitric oxide synthase (eNOS, NOS3) expression, albeit, to different extents. NOS2 was significantly up-regulated after 4 h, while NOS3 increased slightly after 24 h. Co-treatment of LS8 cells with r[A-4] and anti-LAMP1 antibodies further enhanced NOS2 expression. Anti-LAMP1 antibodies did not abrogate NO production in LS8 cells treated for 4 h with r[A-4], but the iNOS inhibitor, l-Nil, down-regulated both NO production and the expression of M180 mRNA. These data suggest that [A-4] modulates M180 mRNA expression, partly, via the NO signaling pathway.

Dataset Information

Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

Publications

Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

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