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.

not in adult life, suggesting that MMPs and the degradation and remodeling of ECM play an important role in growth and development. Thus far, 14 genetically distinct MMPs have been cloned and characterized from humans. They include three different collagenases, which are the only enzymes that can cleave fibril-forming collagens such as collagen types I, II, and III, the most abundant proteins in the body. Gelatinases A and B, also known as 72-and 92-kDa type IV collagenases, digest denatured collagen (gelatin) and basement membranes, a special type of ECM of epithelial, endothelial, fat, muscle, and peripheral nerve cells (8). Stromelysins are three different MMPs with wide substrate specificity, and stromelysin-1 degrades almost any ECM component including cartilage proteoglycans (9).
Recently, five groups have described four new members of the MMP family (17)(18)(19)(20)(21)(22). These new MMPs are not secreted but remain anchored to the cell surface through their COOH-terminal transmembrane domain, and accordingly, they were named membrane type-MMPs (MT-MMPs). Studies on the human MT1-MMP indicate that it activates gelatinase A (MMP2), suggesting that MT-MMPs may facilitate pericellular matrix degradation at the cell surface and promote tumor cell invasion and metastasis (12,17). Our in situ mRNA hybridization studies on the developmental expression of the chicken MT3-MMP gene show that it is expressed at high levels in several embryonic tissues such as neural tube, dorsal root ganglia, respiratory epithelium, and developing cartilage and muscle (22).
To gain insight on the role of MMPs in early vertebrate development, we used two "universal" MMP primers (22) and RT-PCR to clone cDNAs for six different MMPs from Xenopus laevis embryos of different developmental stages. Here we describe the cloning and developmental expression of a novel MMP called XMMP. Xenopus XMMP contains 604 amino acids including a putative 22-residue signal peptide, suggesting that XMMP is a secretory protein. XMMP contains an RRKR motif at the carboxyl-terminal end of the propeptide, suggesting that it is proteolytically activated intracellularly by furin proteinases. In the propeptide, XMMP also has a 37-amino acid-long insertion domain (ID) not found in other MMPs. XMMP lacks a proline-rich hinge region typically found in other MMPs between the catalytic domain and carboxyl-terminal hemopexin/ vitronectin-like domain. Interestingly, the XMMP gene is transiently expressed in gastrula and neurula stage embryos. In comparison, other Xenopus MMPs that we have cloned show a different pattern of gene expression. The 92-kDa type collagenase (MMP9) mRNA is inherited as a maternal mRNA in the blastula stage embryo and expressed throughout the later developmental stages. Stromelysin-1, stromelysin-3, and two different MT-MMP genes (MT2-MMP and MT3-MMP) are first abundantly expressed in pretailbud embryo.

MATERIALS AND METHODS
RT-PCR-Xenopus blastula (stage 8), gastrula (stage 13), neurula (stage 15), and pretailbud (stage 24) embryos were staged according to Nieuwkoop and Faber (23). Total RNA was isolated by a modified acid guanidinium thiocyanate-phenol-chloroform extraction method (24,25). Briefly, the embryos were frozen in liquid nitrogen and pulverized to fine powder. Subsequently, the embryo material was solubilized by adding the lysis solution and total RNA was isolated as described (25). Because Xenopus embryos are very enriched in collagen and other insoluble proteins (26), one more phenol-chloroform extraction was carried out before isopropanol precipitation of RNA. RT-PCR was performed as described before (22,25). Two inosine-containing degenerate oligodeoxynucleotides, called universal MMP primers (22) (22) and then heat denatured, and 1 l of the reaction was used for PCR with primers Y35 and Y36. PCR was initiated by a first cycle: 1 min at 95°C, 30 s of annealing at 60°C, and 30 s of elongation at 70°C, using Taq DNA polymerase (Perkin-Elmer). Subsequently, 40 cycles of amplification were carried out by the following program: 4 s of denaturation at 95°C, 30 s of annealing 60°C and 30 s of elongation at 70°C. 5 l of the PCR products was used for analysis using 0.5 ϫ TBE 6% polyacrylamide gel electrophoresis. In control PCR experiments, to monitor RNA input and the efficiency of reverse transcription, we used two primers designed from the Xenopus ribosomal protein rpL8 sequence (27), forward: 5Ј-CTGCTAAGCTTCGGGCTATCGACT-3Ј, and reverse: 5Ј-CAACAGGATTCATAGCCACACCAC-3Ј. The PCR product is 466-bp. We have consistently observed that rpL8 gene expression is up-regulated ( Fig. 1) during the neurula and pretailbud stages of Xenopus embryo development (27,28). Specific primers for the Xenopus 92 kDa type IV collagenase are, forward: 5Ј-GGAATTCCTTGACCCT-GAAGTGGCTGAT-3Ј, and reverse: 5Ј-GGTCTAGAGGTCTTGGTCAT-AGCTGGAG-3Ј. The PCR product is 760 bp.
cDNA Cloning and Sequence Analysis-The PCR products were cloned into the ScrfI site of pCR-Script Direct SK(ϩ) vector (Stratagene) and initially sequenced using the dideoxynucleotide method (Sequenase kit version 2.0 DNA sequencing kit, U. S. Biochemical Corp.). Later, cycle sequencing was carried out using Taq DyeDeoxy (Applied Biosystems), and the samples were run on a model 373A DNA Sequencer (Applied Biosystems). To determine a full-length cDNA sequence for the novel Xenopus XMMP, the following rapid amplification of 5Ј and 3Ј cDNA ends strategy was used. For the 5Ј end of the mRNA, we used the degenerate primer Y36 and Superscript II RNase H-Reverse transcriptase (Life Technologies, Inc.) and constructed a cDNA library by the Anchor Ligation method as described by Apte and Siebert (29). Briefly, 2 g of total RNA from neurula stage Xenopus embryos was reverse transcribed with Y36 and Superscript, and the single-stranded cDNA was purified with a clean-up column (Promega) and ligated overnight to the Anchor. 1 l of the ligation reaction was used for amplification with the Anchor primer and Y36 primer to construct a 5Ј end cDNA sublibrary for MMPs. The 5Ј end cDNA sublibrary was then amplified using the Anchor primer and a nested specific primer designed from the Xenopus XMMP sequence: 5Ј-GCTGCTGTCGGTT-TCTCCGTCTCT-3Ј. The specific PCR products were cloned and sequenced to obtain the 5Ј end sequence of XMMP. For the 3Ј end of the mRNA, the "lock-docking" NN-1 oligo(dT) primer was used as described in the CLONTECH 3ЈAmpliFinder rapid amplification of cDNA ends kit. The first PCR was carried out with the primers from the kit and the degenerate Y35 primer to construct a 3Ј end cDNA sublibrary. The specific PCR products were amplified using the 3Ј Anchor primer and a nested specific primer derived from the Xenopus XMMP sequence: 5Ј-CCAGTAGTGAGCATGGGATTAGTC-3Ј. We confirmed the XMMP sequence ( Fig. 2) by independent cloning and sequencing of full-length clones generated by PCR and specific primers for XMMP. Sequences were analyzed using the MacVector program (IBI/Kodak). In Figs. 2 and 3, putative signal peptides (30) were predicted using on-line server. 2 We note that the 5Ј-untranslated nucleotide sequence of XMMP (Fig. 2) contains three methionine codons upstream of the putative initiator methione. However, the indicated methione is likely to be the initiator because it translates to a typical signal peptide; the residues upstream of this methionine would form a hydrophilic peptide, suggesting that it is not synthesized (30). Multiple sequence alignments were performed using the Wisconsin GCG progam PILUP. Data shown in Fig. 3 were obtained using a gap weight of 4.0 and gap length weight of 0.010, which were found to be the most stringent setting that introduced the least number of gaps yet aligned the two conserved MMP sequence domains in all four frog MMPs. The data were further edited to remove a number of "artificial" gaps flanking 1-3 amino acids, to align cysteines and to highlight the hinge region and other conserved features of MMPs. To find similar sequences in the public data base, both blastn and blastx searches were performed against the nonredundant and dbEST data bases in the National Center for Biotechnology Information (31). Also, in a BLAST search, the Xenopus XMMP amino acid sequence was compared with 215,495 protein sequences.
Northern Hybridization-Total RNA (10 g) isolated from different stage Xenopus embryos by the modified acid-phenol procedure (24,25) was run on a 1% agarose-formaldehyde gel in 1 ϫ MOPS buffer. The RNA from the agarose gel was blotted in 10 ϫ SSC (1.5 M NaCl, 0.15 M sodium citrate) to Nytran membrane. The membrane was photographed under UV light to visualize the 28 and 18 S ribosomal RNA bands that served as a control for the amount of RNA loaded and transferred. A 0.55-kilobase pair Xenopus cDNA for XMMP was 32 Plabeled by PCR (32) and hybridized overnight at 65°C in 5 ϫ SSC, 5 ϫ Denhardt, 0.1 mg/ml denatured salmon sperm DNA, 0.1% SDS. The membrane was washed at 65°C in 0.1 ϫ SSC, 0.1% SDS and exposed to Kodak x-ray film. thesized two universal MMP primers for these domains (22) and used RT-PCR to clone cDNAs for six different MMPs from Xenopus embryos at different stages of development. From the blastula embryos (stage 8), only one major PCR product of about 900-bp was detected (Fig. 1A, top). This was cloned, sequenced, and identified as the Xenopus homologue of 92-kDa type IV collagenase (data not shown). Using the universal MMP primers, the 900-bp product was barely detectable (yet visible in the original gel) in later developmental stages of gastrula, neurula, and pretailbud embryos. However, using RT-PCR and specific primers for the Xenopus 92-kDa type IV collagenase, it was detected at similar levels in all developmental stages (Fig. 1A, bottom). Apparently, in later stages other more abundant MMP mRNA templates compete for the same FIG. 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 in bold, and a "signature" Cys 273 is circled. Two polyadenylation signals are underlined. universal MMP primers in PCR. In a control experiment, we also found throughout the development comparable levels of expression of rpL8 mRNA encoding a Xenopus ribosomal protein (Fig. 1A, middle).

RESULTS
From the Xenopus embryos of gastrula (stage 13), neurula (stage 15), and pre-tailbud (stage 24) stages, a prominent PCR product of about 550 bp was detected (Fig. 1A, top). Cloning and sequencing of this product showed that it contained several different MMP-related sequences. From gastrula and neurula embryos, this product contained a sequence derived from a novel member of the MMP family (called XMMP) that was further characterized as described below. From pretailbud embryos, this product contained two different sequences derived from two different Xenopus MT-MMPs that are, respectively, similar to MT2-MMP and MT3-MMP from human and chicken (Refs. 19, 20, and 22; data not shown).
A second prominent PCR product of 350 bp from pretailbud embryos, visible also from neurula embryos (Fig. 1, top), was found to contain two additional MMP-related sequences. One sequence represents a Xenopus homologue for stromelysin-1 (data not shown), and the other is identical to the Xenopus stromelysin-3 sequence (data not shown) as described previously by Patterton et al. (28).
In Northern blotting, the novel Xenopus XMMP gene was found to encode a single 3.0-kilobase pair mRNA. It is particularly striking that this transcript, which was undetected in blastula stage embryo, is first induced at gastrula and continues to be expressed at high level at neurula stage. In comparison, this transcript almost completely disappears in pretailbud embryo, suggesting that XMMP gene is down-regulated in later Xenopus development (Fig. 1B).
Using rapid amplification of cDNA ends, a 5Ј and 3Ј end RT-PCR strategy of cDNA cloning (see "Materials and Methods"), allowed us to determine 3217 nucleotides and the complete 604-amino acid sequence for the novel Xenopus XMMP (Fig. 2, deposited as GenBank TM accession number U82541). XMMP contains a putative signal peptide of 22 amino acids with a characteristic hydropathy profile (30), and no other significant hydrophobic seqments, suggesting that it is a secretory protein. The sequence contains five potential sites for N-linked sugars (Asn 173 , Asn 267 , Asn 419 , Asn 422 , and Asn 588 ) and nine Cys residues, four of them in the putative signal peptide. Of note is that Cys 273 (circled in Fig. 2), which is an insertion, is not found in any other MMPs except NMMP, another novel MMP that we recently cloned from cultured primary chicken embryo fibroblasts. 3 Excluding the signal peptide and any post-translational modifications, Xenopus XMMP has a calculated molecular weight of 70,115.
In comparison with the bullfrog Rana catesbeiana tadpole collagenase (34), Xenopus collagenase-3 (MMP13, GenBank accession number L49412), and Xenopus stromelysin-3 (28), XMMP is most related to stromelysin-3 (20% identical amino acids). As shown in Fig. 3, a sequence alignment reveals 21% (131/635) identical residues between all four frog MMPs. One conspicuous feature of the Xenopus XMMP that makes it different from all other MMPs is a 37-amino acid-long ID, related to vitronectin (see below), that is situated at the end of the propeptide after the conserved PRCGVPD domain. In stromelysin-3 and MT-MMPs, a similarly located insertion is 8 -13 residues long (19 -22, 28). After the insertion domain there is an RRKR motif (Fig. 3), which is a putative cleavage site for intracellular furin, a Golgi-associated proteinase family. This and a related motif are also found in stromelysin-3 (RQKR) and MT-MMPs (RRKR or RRRR). Pei and Weiss (11,12) recently showed that this motif leads to an unusual intracellular activation of the human stromelysin-3 and MT1-MMP by a furindependent mechanism. Accordingly, Xenopus XMMP may be proteolytically activated by a similar intracellular mechanism.
Another conspicuous feature of XMMP is that it lacks a proline-rich linker peptide or hinge region typically found in all other MMPs between the catalytic domain and COOH-terminal hemopexin/vitronectin-like domain (6,7). Matrilysin (MMP7) lacks the linker peptide and the carboxyl-terminal domain altogether. A recent crystal structure study of porcine interstitial collagenase (MMP1) revealed that the hinge region is a highly exposed 17-residue peptide IYGPSENPVQPSGPQT-PQVCD (residues 261-277 underlined) with no secondary structure (35). As shown in Fig. 4, we suggest that in other MMPs, a homologously placed hinge region is 17-72 residues long and most pronounced in the 92-kDa type IV collagenase. We also note that a common feature of the hinge region is that it begins with glycine and ends with proline three residues upstream of the conserved cysteine residue. The hinge region, which appears to be the most variable region of MMPs, plays a role in MMP activity and substrate selection (for references, see Ref. 35).
Similar to other MMPs, the COOH-terminal domain of XMMP contains four tandem repeats of a motif similar to that in vitronectin, an abundant cell adhesion protein in plasma and tissues (36). The COOH-terminal domain plays a role in the substrate specificity and possibly other protein-protein interactions of MMPs (6,7). In the crystal structure of porcine collagenase this domain is folded as a four-bladed ␤-propeller (35). As shown in Fig. 5, we have discovered that XMMP repeats 2, 3, and 4 are 21-33% identical to a sequence encoded by vitronectin exon-5 (residues 213-264), whereas repeat 1 is more related to a sequence encoded by vitronectin exon-4 (residues 166 -212). The similarity of the four tandem repeats is also obvious in the consensus MMP sequence (49); they are 24 -39% identical with the sequence encoded by vitronectin exon-5. We note that vitronectin exons 4 and 5 code for related sequences (30% identical), suggesting that they evolved from a common ancestral exon (50). Furthermore, we find that the ID sequence of XMMP (residues 146 -173) is 38% identical and 62% chemically similar with a rat vitronectin sequence between residues 108 -135. The biological significance, if any, of the ID of XMMP is not known at present. In comparison with the sequences encoded by vitronectin exons 4 and 5, which are highly conserved in evolution, the vitronectin sequence of residues 108 -135 is not conserved between rat, rabbit, mouse, and human (36). It is also of interest that the COOH-terminal hemopexin/vitronectin-like domain of MMPs is highly conserved in the COOH-terminal end of nectinepsin from quail. Nectinepsin is a novel ECM protein with an RGD cell binding motif and an aspartyl proteinase domain (37).
When we searched the 3217-nucleotide sequence of XMMP for human and mouse ESTs in data bases, we could not find any significant matches by these analyses. This is interesting, especially because there are already 465,858 ESTs from human 3  and 63,404 ESTs from mouse (August, 1996). Because these ESTs are derived from a variety of tissues, mainly from adult tissues, it is reasonable to propose that the XMMP gene is expressed specifically during early vertebrate development. This is in line with our recent results by Northern blotting and RT-PCR indicating only low levels of XMMP transcript in select adult Xenopus tissues such as heart, liver, and ovaries. 4 Thirty-five years ago Jerry Gross described the first evidence for MMPs as "a diffusible collagenolytic factor" from bullfrog tadpole tissues in culture (38), and then asked how tadpoles lose their tails. (39). Part of the answer may involve collagenase-1 (MMP1) that Oofusa et al. (34) recently cloned from metamorphosing bullfrog tadpoles. During frog metamorphosis, also the small intestine of tadpoles goes through a rapid tissue remodeling, epithelial cell death and renewal by adultlike epithelial cells. When Shi and colleagues (28) studied this in more detail by in situ hybridization, they found that stromelysin-3 mRNA was abundantly expressed in several stromal cells surrounding the dying intestinal epithelial cells. These data, as well as studies by Werb and Bissell and their colleagues on the role of stromelysin-1 expression during the involution of mouse mammary glands (40,41), suggest that proteolytic degradation of the ECM by MMPs plays an important morphogenetic role in vertebrate development and tissue remodeling.
In conclusion, we have studied here only a few early stages of Xenopus embryo development for the expression of MMP genes (blastula, gastrula, neurula, and pretailbud) and cloned cDNAs for six different MMPs, including a novel MMP called XMMP. Using the universal MMP primers (22) and RT-PCR we expect to clone other members of the MMP family from other stages of Xenopus development, morphogenesis, and tissue remodeling. Our results make it clear that MMPs are an evolutionary conserved family of ECM degrading enzymes in Xenopus. Future experiments will explore in more detail the role of MMPs in early vertebrate development.