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Gene
2007 May 01;3921-2:89-97. doi: 10.1016/j.gene.2006.11.014.
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Identification and characterization of matrix metalloproteinase-20 (MMP20; enamelysin) genes in reptile and amphibian.
Shintani S
,
Kobata M
,
Kamakura N
,
Toyosawa S
,
Ooshima T
.
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Matrix metalloproteinase-20 (MMP20; enamelysin) is important for proteolytic processing of extracellular matrix (ECM) proteins during the formation of enamel and plays a critical role in proteolytic processing of amelogenin (AMEL), the most abundant enamelECM protein. MMP20 might have played a role in the emergence of teeth, because jawless vertebrates with primordial teeth on their external skeletons may have possessed the MMP20 gene, and MMP20 and enamelECM proteins are thought to have evolved together in a special relationship over time. Thus, an understanding of the molecular evolution of the MMP20 gene is important for elucidating the evolution of enamel and it is necessary to identify the orthologs of the MMP20 gene in non-mammals, as it has been identified in mammals. In the present study, orthologs of the MMP20 genes from a reptile (caiman) and an amphibian (African clawed toad) were cloned and characterized. Comparisons of the orthologs revealed that the MMP20 proteins were highly conserved throughout the evolution of tetrapods. Further, the caiman, toad, and mammalian MMP20 shared several unique features specific for MMP20, but not for other matrix metalloproteinases. In addition, the toad MMP20 gene was transcribed only in the upper jaw, presumably in teeth. These results suggest that MMP20 in a common ancestor of tetrapods might have been recruited for the processing of AMEL and conserved over 350 million years of evolution.
Fig. 1.
(A) Nucleotide and translated amino acid sequences of caiman MMP20 cDNA clone. Amino acid residues are shown with the IUPAC-IUB single letter code. The stop codon is indicated by an asterisk (⁎) and the polyadenylation signals are underlined with a solid line. Primer positions and orientations are indicated by double lines with arrowheads above their boxed locations in the sequence. Vertical lines show the positions of the exon boundaries. The sequences can be accessed in the GenBank database under accession no. DQ885891. (B) Nucleotide and translated amino acid sequences of toad MMP20 cDNA clone. The signal peptide is underlined with a dotted line. Other indicators are the same as in A. The sequences can be accessed in the GenBank database under accession no. DQ885892.
Fig. 2.
Dot-plots of caiman and toad amino acid sequences (abscissa), as compared to the human MMP20 sequence (ordinate). A single dot indicates 5 matching residues in a size 7 window.
Fig. 3.
Amino acid sequence alignment of MMP20 genes from human, bovine, porcine, mouse, caiman, and toad specimens. Amino acid residues are shown with the IUPAC-IUB single letter code. Numbering shows residue positions. Identity with the simple majority consensus sequence at the top is indicated by a dash (-) and deletions introduced for optimal alignment by asterisks (⁎). Vertical lines indicate exon borders of the human, mouse, caiman, and toad MMP20 genes, and numbers on the both sides of the line denote exon numbers. The cysteine switch motif is indicated by grizzled boxes. The conserved signature for the catalytic zinc ion binding site is indicated by the open box with solid lines and the following methionine turn motif is surrounded by a box with dotted lines. The white letters on shaded backgrounds represent the unique combination of amino acid residues for the MMP20 molecules.
Fig. 4.
Neighbor-joining tree of amino acid sequences of MMP20 proteins. All MMP sequences except for MMP20 are used as representatives of human sequences. The numbers enclosed in parentheses represent an available chromosomal origin for human MMPs. The scale bar represents the distance based on the proportion of amino acid differences. Gapped sites were removed from all sequences before distance estimates were made. Numbers on the nodes indicate the percent recovery of that node in 1000 bootstrap replications.
Fig. 5.
RT-PCR analysis of toad tissues. (A) cDNA was amplified from upper jaw, lower jaw, tibia, heart, and liver specimens, using the MMP20 cDNA-specific primer sets MMPX10 and MMPX15. (B) cDNA was amplified with the primers GAPDH1 and GAPDH2, with the results used as a positive control.
