XB-ART-16485J Biol Chem 1997 May 23;27221:13527-33.
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A novel matrix metalloproteinase gene (XMMP) encoding vitronectin-like motifs is transiently expressed in Xenopus laevis early embryo development.
To study the role of matrix metalloproteinases (MMPs) in early vertebrate development, we cloned cDNAs for six different MMPs from the frog Xenopus laevis embryos at different stages of development and describe here a novel MMP called XMMP. Xenopus XMMP has 604 amino acids including a putative signal peptide of 22 residues. At the carboxyl-terminal end of the propeptide, XMMP has a 37-amino acid-long insertion domain containing a segment that is 38% identical with a rat vitronectin sequence between residues 108-135. Following this domain is an RRKR motif, a putative cleavage site for intracellular activation by furin proteinases. XMMP lacks a proline-rich linker peptide, or hinge region, typically found in other MMPs between the catalytic domain and carboxyl-terminal "hemopexin/vitronectin-like" domain. In XMMP, the carboxyl-terminal domain is composed of four tandem repeats that are 21-33% identical to a sequence (residues 213-264) encoded by vitronectin exon-5. Interestingly, XMMP gene is transiently expressed during Xenopus embryo development. XMMP mRNA of 3.0 kilobase pairs was undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo. In comparison, other Xenopus MMP genes that we have cloned show a different developmental regulation. In blastula embryo, the only MMP gene expressed was found to be 92-kDa type IV collagenase, which was also expressed in the gastrula, neurula, and pretailbud embryos. Expression of stromelysin-1, stromelysin-3, and two different membrane type-MMPs was first detected in the neurula and pretailbud embryos. These results suggest that MMPs and the novel XMMP reported here play a role in Xenopus early development.
PubMed ID: 9153198
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: furin mmp11 mmp13l mmp21 mmp3 mmp7 mmp9.1 mt4 mtnr1a mtnr1b pcyox1 rpl8 tcim
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|Figure 1 Cloning and developmental expression ofXenopus MMPs. A, top, RT-PCR with universal MMP primers using total RNA (0.5 μg) of blastula (stage 8), gastrula (stage 13), neurula (stage 15), and pretailbud (stage 24)Xenopus embryos. PCR of 40 cycles. Middle, RT-PCR with specific primers for the Xenopus ribosomal protein rpL8. PCR of 27 cycles. Bottom, RT-PCR with specific primers for the Xenopus 92-kDa type IV collagenase. PCR of 36 cycles. Lane M shows DNA size markers (100-bp ladder from Life Technologies, Inc). B, in Northern blotting, total RNA (10 μg) of blastula (8), gastrula (13), neurula (15), and pretailbud (24) Xenopus embryos was analyzed for the novelXenopus XMMP gene expression (a 3.0-kilobase pair mRNA).Numbers on the left show RNA size markers (Life Technologies, Inc.). As a control for RNA loading and transfer, the bottom panel shows 18 S ribosomal RNA on the same Northern filter visualized by EtBr staining and UV light.|
|Figure 2 Xenopus XMMP. In the cDNA-derived amino acid sequence of 604 residues of Xenopus XMMP, the first bar indicates a putative signal peptide cleavage site, and the second bar shows propeptide cleavage site.Boxed sequences mark two conserved MMP sequences that were used in primer design to clone Xenopus MMPs (see Fig. 1), and an RRKR motif, a putative cleavage site for intracellular furin-proteinases. The underlined sequence is a 37-residue-long ID. The circled GS residues refer to a missing hinge region in XMMP (see Fig. 5). The large box is a domain of four tandem repeats of a motif similar to that encoded by vitronectin exon 5 (see Fig. 4). The nine Cys residues are shown inbold, and a “signature” Cys273 iscircled. Two polyadenylation signals areunderlined.|
|Figure 3 Comparison of the frog MMPs. A sequence alignment of Tc1, Rana bullfrog tailbud collagenase (34), CL3, Xenopus collagenase-3 (GenBank™ number L49412), ST3,Xenopus stromelysin-3 (28), and XMMP. SP andPP show putative signal peptide and propeptide cleavage sites, respectively. The underlined sequence is a 37-residue ID of XMMP (residues 143–180). Boxed sequences are two conserved MMP domains and a proline-rich hinge region or linker peptide (LP). A signature Cys273 of XMMP iscircled. Consensus shows amino acids that are identical between three or all four frog MMPs.|
|Figure 4 MMP hinge. All MMPs, with the exception of XMMP, have a proline-rich hinge region (linker peptide) between the catalytic domain and COOH-terminal hemopexin/vitronectin-like domain. Representative MMP hinge sequences shown are flanked by consensus residues in bold. The sequences are from Tc1, bullfrog Rana tadpole collagenase (34); XCL3,Xenopus collagenase-3 (GenBank™ number L49412);PCL1, porcine interstitial collagenase (GenBank™ numberX54724); XST3, Xenopus stromelysin-3 (28); andSue, sea urching hatching enzyme (33). The other sequences are from human MAT, matrilysin (42), which lacks the hinge region and COOH-terminal domain; 72k, 72-kDa type IV collagenase (43); ST3, stromelysin-3 (44); MT1,MT2, MT3, and MT4, four different MT-MMPs (17-21); and 92k, 92-kDa type IV collagenase (45).|
|Figure 5 Evolutionary conservation of XMMP and vitronectin. A, alignment of ID sequence with a rat vitronectin sequence between residues 108–135. Identical residues arebold. B, four tandem XMMP repeats (see Fig. 2) are aligned with sequences encoded by rat vitronectin exons 4 and 5.Bold residues are identical between XMMP and vitronectin.C, rat vitronectin exons 4 and 5 encode sequences with 30% (16 of 53) identical amino acids shown in bold.|