Dataset Information

Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta.

ABSTRACT: Amelogenin with a proline 41 to threonine mutation (P41T) is hydrolyzed at a lower rate by matrix metalloproteinase 20 (MMP20), resulting in an inherited tooth enamel defect, amelogenesis imperfecta (AI). The aim of this study was to elucidate the effect of P41T on the interactions between amelogenin and MMP20, which may contribute to the formation of this type of AI. The interactions of a recombinant wild-type human amelogenin and its P41T mutant with recombinant human MMP20 were compared by substrate competition assay, pull-down assay, and surface plasmon resonance (SPR). The results showed that the binding of the P41T mutant amelogenin for MMP20 was significantly lower than that of wild-type amelogenin. Our study supports a model in which the P41T mutation reduces the interactions between amelogenin and MMP20, leading to decreased degradation of amelogenin by MMP20, and resulting in AI.

SUBMITTER: Tanimoto K

PROVIDER: S-EPMC3677966 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta.

Tanimoto K K Le T T Zhu L L Witkowska H E HE Robinson S S Hall S S Hwang P P Denbesten P P Li W W

Journal of dental research 20080501 5

Amelogenin with a proline 41 to threonine mutation (P41T) is hydrolyzed at a lower rate by matrix metalloproteinase 20 (MMP20), resulting in an inherited tooth enamel defect, amelogenesis imperfecta (AI). The aim of this study was to elucidate the effect of P41T on the interactions between amelogenin and MMP20, which may contribute to the formation of this type of AI. The interactions of a recombinant wild-type human amelogenin and its P41T mutant with recombinant human MMP20 were compared by su ...[more]

PMID: 18434575

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Project description:Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin pre-messenger RNA (mRNA) is highly alternatively spliced, and during alternative splicing, exon4 is mostly skipped, leading to the formation of a microRNA (miR-exon4) that has been suggested to function in enamel and bone formation. While delivering the functional variation of amelogenin proteins, alternative splicing of exon4 is the decisive first step to producing miR-exon4. However, the factors that regulate the splicing of exon4 are not well understood. This study aimed to investigate the association between known mutations in exon4 and exon5 of X chromosome amelogenin that causes X-linked AI, the splicing of exon4, and miR-exon4 formation. Our results showed mutations in exon4 and exon5 of the amelogenin gene, including c.120T>C, c.152C>T, c.155C>G, and c.155delC, significantly affected the splicing of exon4 and subsequent miR-exon4 production. Using an amelogenin minigene transfected in HEK-293 cells, we observed increased inclusion of exon4 in amelogenin mRNA and reduced miR-exon4 production with these mutations. In silico analysis predicted that Ser/Arg-rich RNA splicing factor (SRSF) 2 and SRSF5 were the regulatory factors for exon4 and exon5 splicing, respectively. Electrophoretic mobility shift assay confirmed that SRSF2 binds to exon4 and SRSF5 binds to exon5, and mutations in each exon can alter SRSF binding. Transfection of the amelogenin minigene to LS8 ameloblastic cells suppressed expression of the known miR-exon4 direct targets, Nfia and Prkch, related to multiple pathways. Given the mutations on the minigene, the expression of Prkch has been significantly upregulated with c.155C>G and c.155delC mutations. Together, we confirmed that exon4 splicing is critical for miR-exon4 production, and mutations causing X-linked AI in exon4 and exon5 significantly affect exon4 splicing and following miR-exon4 production. The change in miR-exon4 would be an additional etiology of enamel defects seen in some X-linked AI.

Dataset Information

Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta.

Publications

Reduced amelogenin-MMP20 interactions in amelogenesis imperfecta.

Similar Datasets

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