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Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases
Gene 211 (1998) 57–62
Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases Kazuhiro Wada a, Hiroshi Sato a,*, Hiroaki Kinoh b, Masahiro Kajita b, Hiroshi Yamamoto c, Motoharu Seiki b a Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920, Japan b Department of Cancer Cell, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Tokyo 108, Japan c Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa 920, Japan Received 16 October 1997; received in revised form 13 January 1998; accepted 13 January 1998; Received by T. Sekiya
Abstract Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Extracellular matrix; Remodeling; Morphogenesis; Genome database; TIMP
1. Introduction Matrix metalloproteinases (MMPs) are zinc-binding endopeptidases that collectively degrade the constituent macromolecules of the extracellular matrix (ECM ) (Matrisian, 1990; Woessner, 1991). Thus, the MMPs are thought to participate in ECM remodeling and degradation, and have been implicated in playing important roles during organ morphogenesis, embryonic development and pathological processes (Stetler* Corresponding author. Tel: +81 76 265 2750; Fax: +81 76 234 4505; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); C., Caenorhabditis; cDNA, complementary DNA; ECM, extracellular matrix; kb, kilobase pairs; MMP, matrix metalloproteinase; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reversetranscription PCR; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TIMP, tissue inhibitor of MMPs. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 07 6 - 6
Stevenson et al., 1993; Tryggvason et al., 1993; Sato and Seiki, 1996). An overall view of the MMP family is important for a better understanding of the biological role, and a growing number of human MMPs have been identified corresponding to the complexity of ECM components in recent years (Sato et al., 1994; Takino et al., 1995a,b; Will and Hinzmann, 1995; Cossins et al., 1996; Pendas et al., 1997). Caenorhabditis elegans is a simple multicellular organism with approximately 1000 cells per body and has ECM components, including collagens, similar to human counterparts (Johnstone, 1994; Sibley et al., 1994). Thus, C. elegans is expected to have a similar ECM metabolizing system to human and will be a good system for analyzing the biological roles of MMP. In this study, we identified three putative MMP genes of C. elegans by sequence similarity searching of genome database. These C. elegans MMP cDNAs were cloned, and gene products expressed in E. coli were biochemically studied.
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2. Materials and methods
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plied by K. Iwata of Fuji Chemical Industries, Ltd, Takaoka, Japan.
2.1. Database search A Caenorhabditis elegans Database (ACeDB) was searched for similarity to the amino acid (aa) sequence of MT1-MMP zinc-binding domain (GNDIFLVAVHELGHALGLEHSSDPSAIMAPF ) using the program tblastn (Altshul et al., 1990). Approximately 7-kb regions surrounding the sequences encoding homologous aa sequences were further analyzed by the program GeneFinder for C. elegans genome to predict the gene transcripts. The criteria used to identify putative MMP family members were the existence of the PRCGVPD motif and the zinc binding site (HEGHXXGXXHS) at an appropriate distance (Birkedal-Hansen et al., 1993). Predicted MMP genes were amplified by polymerase chain reaction (PCR) using C. elegans mixed stage complementary DNA (cDNA) library as a template, which was supplied by Dr Kohara of National Institute of Genetics, Mishima, Japan. 2.2. Expression of MMPS in E. coli C. elegans MMPs were expressed in E. coli as fusion proteins with FLAG peptide at the carboxyl terminus. DNA fragments encoding the region spanning the catalytic to carboxyl-terminal domains were PCR-amplified using primers containing extra HindIII site and were inserted at HindIII site of pFLAG.CTC vector ( Eastman Chemical Company). Recombinant proteins with FLAG peptide at carboxyl terminus were extracted from E. coli strain XL1-Blue with 20 mM Tris–HCl buffer (pH 7.5) containing 150 mM NaCl and 0.5% Triton X-100 and absorbed to anti-FLAG M2 affinity gel ( Eastman Kodak). Recombinant proteins were eluted with 6 M urea and dialyzed overnight against 20 mM Tris–HCl buffer containing 150 mM NaCl. The enzyme activities of recombinant MMPs were assayed using human type I gelatin and fluorescence-quenching substrate for MMP, Mca–Pro–Leu–Gly–Leu–Gly–Leu–Dpa–Ala– Arg–NH (Peptide Inst., Osaka, Japan). Synthetic MMP 2 inhibitors BB-94 and CT543 were chemically synthesized as described previously ( Kinoshita et al., 1996). Recombinant human TIMP-1 and TIMP-2 were sup-
3. Results and discussion 3.1. Cloning of C. elegans MMP genes; domain structure of the deduced products C. elegans genome cloned in cosmid clones C31B8, H19M22 and Y19A7 were predicted to express mRNA transcripts containing long open reading frames (ORFs) which encode the MMP family, including a pro-peptide domain with the activation locus and a catalytic domain with the zinc-binding site. The MMP cDNAs were amplified by PCR with C. elegans cDNA library as a template, which generated a single band for each amplification. The nucleotide sequences of these cDNAs have been submitted to the DDBJ/EMBL/GenBank DNA Databases under the Accession Nos AB007815, AB007816 and AB007817. The nt sequences of cloned cDNAs matched quite well with the sequences predicted by the program GeneFinder, except for a few splicing junctions. The deduced aa sequences were aligned to human MMP-1/interstitial collagenase and MT1-MMP ( Fig. 1) (Goldberg et al., 1986; Sato et al., 1994; Takino et al., 1995a). These C. elegans MMPs have the characteristic domain organization of known MMPs. The PRCGVPD motif in the pro-peptide domain, which is the cysteine switch region responsible for maintenance of enzyme latency, is well conserved in human MMPs ( Van Wart and Birkedal-Hansen, 1990). Fifteen human MMPs have the PRCGVPD sequence, whereas MMP-2/gelatinase A and MMP-18 have PRCGNPD and PRCGLED, respectively (Collier et al., 1988; Cossins et al., 1996). C. elegans MMPs have rather diverse sequences for this motif; PRCTQTD in MMPH19, PRCGHPD in MMP-Y19 and SRCGVTD in MMP-C31. Two of three C. elegans MMPs, MMP-H19 and MMP-Y19 have a short aa insert between the propeptide and the catalytic enzyme domain that contains the RXKR motif, a furin-like cleavage site. Human stromelysin-3 and membrane-type MMP-1,-2,-3 (MT1-, MT2- and MT3-MMP) have a similar insert containing the RXKR motif (Basset et al., 1990; Sato et al., 1994; Will and Hinzmann, 1995; Takino et al., 1995b) and
Fig. 1. C. elegans MMPs aligned with MMP-1 and MT1-MMP. The ORFs that initiate from ATG and terminate at TAA codon were translated. Each domain indicated in the figure was assigned according to Birkedal-Hansen et al. (1993). PRCGVPD and RXKR motifs are indicated by closed squares and closed circles, respectively. The zinc binding site is double-underlined. Methods: Amplification of the MMP gene identified in C. elegans genome C31B8 cosmid clone was performed with a forward primer starting from base position 15716 and a reverse primer from base position 11975 of cosmid C31B8. MMP gene identified in cosmid H19M22 was amplified with a forward primer from base position 1434 and a reverse primer from 5653 of cosmid H19M22. Amplified fragments were cloned into the pCRII plasmid and sequenced by an ABI autosequencer. A part of the MMP gene identified in cosmid clone Y19A7 was amplified with a forward primer from base position 4297 and a reverse primer from 4133, which generated a 156-bp fragment. The C. elegans cDNA library was screened with this PCR product.
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were shown to be processed by furin (Pei and Weiss, 1995, 1996; Sato et al., 1996). The aa sequence of the catalytic domain, particularly the binding site for zinc (HEXGHXXGXXHS), is well conserved between C. elegans and human MMPs. A serine residue on the right side of third histidine residue distinguishes MMP family members from members of the astacin, snake venom metalloproteinase and disintegrin families (Jiang and Bond, 1992). Members of the astacin family contain a glutamic acid, and members of snake venom metalloproteinase and disintegrin families have an aspartic acid residue at this position. The aa sequence homology with MMP-1 in the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively, and the aa sequence homology with other human MMPs is at a similar level. Thus, they cannot be readily assigned to a particular subclass of human MMP from aa sequence homology. The carboxyl-terminal domain of known MMP consists of four repeats that share some limited sequence homology with modules also found in hemopexin and in vitronectin, and the four tandem modules are held together by a single disulfide bond between two cysteine residues that flank the hemopexin-like domain. None of C. elegans MMPs shows an apparent sequence similarity with hemopexin in this region, but MMP-C31 and -Y19 contain two cysteine residues that can flank this domain and may form a similar structure. Unlike other MMPs, MMP-H19 has two cysteine residues in the hinge domain and two cysteine residues in the carboxyl-terminal domain, and the last cysteine residue resides at the middle of carboxyl-terminal domain. Thus, it may be possible that the structure of MMP-H19 carboxyl-terminal domain is quite different from that of hemopexin-like domain. Up to the present time, about 50% of C. elegans genome sequence remains to be determined, and thus C. elegans should have more genes for MMPs. 3.2. Characterization of recombinant C. elegans MMPS C. elegans MMPs were expressed in E. coli as fusion proteins with FLAG peptide at carboxyl terminus and purified using anti-FLAG antibody columns. Human membrane-type MMP-3 (MT3-MMP) was also synthesized in E. coli for comparison. Purified proteins showed expected molecular weights as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) ( Fig. 2A). Among the three C. elegans MMPs, only MMP-C31 digested gelatin (Fig. 2B). None of the C. elegans MMPs hydrolyzed either native type I collagen or carboxymethylated transferrin, and none of them has proteolytic activity on gelatin and casein zymogrohies (data not shown). The enzyme activity of purified MMPs was also assayed using a fluorescence-quenching substrate for MMP ( Table 1). MMP-C31 cleaved the peptide at an efficiency comparable with MT3-MMP.
A
B
Fig. 2. SDS–PAGE of recombinant MMPs (A) and of their gelatin digestion products (B). (A) Purified proteins were separated on a 10% polyacrylamide gel and stained with Coomassie Brilliant Blue. (B) Human type I gelatin (10 mg) was incubated in the absence ( lane −) or presence of 50 ng recombinant MMP proteins as indicated ( lanes C31, H19 and Y19, respectively) at 37°C for 12 h. The digestion products were analyzed by SDS–PAGE under reducing conditions. Methods: MMP-C31 cDNA fragment encoding from Phe 138 to carboxyl terminal Cys 501, MMP-H19 from Phe 66 to Lys 424, MMPY19 from Tyr 100 to Phe 520 and MT3-MMP from Tyr112 to Ser 557 were PCR-amplified using primers containing extra HindIII site and were inserted at HindIII site of pFLAG.CTC vector. Recombinant proteins were purified as described in Materials and methods.
MMP-H19 cleaved less effectively than MMP-C31, and MMP-Y19 showed only a trace activity against this substrate. It is noteworthy that C. elegans MMPs showed enzyme activity in proportion to aa homology with human MMPs. Namely, MMP-C31, which has 45% homology with MMP-1 at the catalytic domain, has the highest enzyme activity against gelatin and peptide substrate, and MMP-H19 and -Y19, which have 34% and 23% homology at the catalytic domain with human MMP-1, respectively, showed 55% and 4.9%
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K. Wada et al. / Gene 211 (1998) 57–62 Table 1 Enzyme activity of recombinant MMPs MMP Inhibitor: MT3-MMP MMP-C31 MMP-H19 MMP-Y19
− 100 124.1 68.3 6.1
C. elegans MMPs morphogenesis.
TIMP-1
TIMP-2
BB94
CT543
105.5 16.8 11.5 ND
15.2 14.3 8.0 ND
3.3 2.8 3.7 ND
4.5 3.4 2.8 ND
Recombinant MMP proteins (0.1 mg) were incubated in a total volume of 0.5 ml with fluorescence-quenching substrate (1 mM ) for MMP at 37°C for an hour in the absence ( lane, −) or presence of inhibitors, TIMP-1 (1 mg), TIMP-2 (1 mg), BB94 (1×10-8M ) and CT543 (1×10-8M ). Activity of MT3-MMP without inhibitor was set to 100%. ND, not determined.
activity of MMP-C31 against only peptide substrate, respectively. Interactions of MT3-MMP and C. elegans MMPs, MMP-C31 and -H19 with human tissue inhibitors of MMP ( TIMP)-1 and -2 were tested. Both TIMP-1 and -2 effectively inhibited the enzyme activity of these C. elegans MMPs. BB94 and CT543, specific inhibitors of human MMPs, also efficiently interfered with C. elegans MMPs, MMP-C31 and -H19. These results suggest that the structure of the C. elegans MMPs (MMP-C31 and -H19) catalytic center is closely related to that of human MMPs. MT3-MMP was inhibited only by TIMP-2 but not by TIMP-1 as was previously observed with MT1-MMP (Sato et al., 1996). An attempt is being made to obtain nematodes that have an inactivated MMP gene by insertion of transposon Tc1. So far, some clones were isolated that had an inactivated MMP-C31 gene, but none of them showed any apparent phenotypical change. In contrast, inactivation of the MMP-H19 gene seems to be lethal, because only nematodes with MMP-H19 gene deficient heterozygote were obtained from the progeny of deficient heterozygote (unpublished data). 3.3. Conclusion In summary, we have identified three C. elegans MMPs. MMP-C31, which has the highest aa homology with human MMPs, digested gelatin and peptide substrate. MMP-H19, which shares a lower aa homology with human MMPs, only digested peptide substrate, and less efficiently than MMP-C31. The enzyme activity of these two C. elegans MMPs was inhibited by specific inhibitors of human MMPs, suggesting that the catalytic center structure of these MMP resembles that of human MMPs as expected from their aa homology with human MMPs. The enzyme activity of MMP-Y19, which shares the lowest aa homology with human MMPs, was not detected with common MMP substrates. Studies are now under way to determine the expression profile of
and
their
roles
in
organ
Acknowledgement We thank Dr R. Hosono (School of Medicine, Kanazawa University) for helpful discussion, Dr Y. Kohara (National Institute of Genetics, Mishima, Japan) for supplying the C. elegans cDNA library, and Dr K. Iwata (Fuji Chemical Industries, Ltd, Takaoka, Japan) for supplying recombinant human TIMP-1 and TIMP-2. This work was supported by a grant-in-aid for Cancer Research, from the Ministry of Education, Science Technology of Japan.
References Altshul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tools. J. Mol. Biol. 215, 403–410. Basset, P., Bellocq, J.P., Wolf, C., Stoll, I., Hutin, P., Limacher, J.M., Podhajcer, O.L., Chenard, M.P., Rio, M.C., Chambon, P., 1990. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348, 699–704. Birkedal-Hansen, H., Moore, W.G., Bodden, M.K., Windsor, L.J., Birkedal-Hansen, B., DeCarlo, A., Engler, J.A., 1993. Matrix metalloproteinases: a review. Crit. Rev. Oral Biol. Med. 4, 197–250. Collier, I.E., Wilhelm, S.M., Eisen, A.Z., Marmer, B.L., Grant, G.A., Seltzer, J.L., Kronberger, A., He, C., Bauer, E.A., Goldberg, G.I., 1988. H-ras oncogene-transformed human bronchial epithelial cells ( TBE-1) secrete a single metalloproteinase capable of degrading basement membrane collagen. J. Biol. Chem. 263, 6579–6587. Cossins, J., Dudgeon, T.J., Catlin, G., Gearing, A.J.H., Clements, J.M., 1996. Identification of MMP-18, a putative novel human matrix metalloproteinase. Biochem. Biophys. Res. Commun. 228, 494–498. Goldberg, G.I., Wilhelm, S.M., Kronberger, A., Bauer, E.A., Grant, G.A., Eisen, A.Z., 1986. Human fibroblast collagenase: complete primary structure and homology to an oncogene transformationinduced rat protein. J. Biol. Chem. 261, 6600–6605. Jiang, W., Bond, J.S., 1992. Families of metalloendopeptidases and their relationships. FEBS 312, 110–114. Johnstone, I.L., 1994. The cuticle of the nematode Caenorhabditis elegans: a complex collagen structure. Bioessays 16, 171–178. Kinoshita, T., Sato, H., Takino, T., Itoh, M., Akizawa, T., Seiki, M., 1996. Processing of a precursor of 72-kilodalton type IV collagenase/ gelatinase A by a recombinant membrane-type 1 matrix metalloproteinase. Cancer Res. 56, 2535–2538. Matrisian, L.M., 1990. Metalloproteinases and their inhibitors in matrix remodeling. Trends Genet. 6, 121–125. Pei, D., Weiss, S.J., 1995. Furin-dependent intracellular activation of the human stromelysin-3 zymogen. Nature 375, 244–247. Pei, D., Weiss, S.J., 1996. Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J. Biol. Chem. 271, 9135–9140. Pendas, A.M., Knauper, V., Puente, X.S., Llano, E., Mattei, M.G., Apte, S., Murphy, G., Lopez, O.C., 1997. Identification and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location, and tissue distribution. J. Biol. Chem. 272, 4281–4286. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto,
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E., Seiki, M., 1994. A matrix metalloproteinase expressed on the surface of invasive tumour cells [see comments]. Nature 370, 61–65. Sato, H., Kinoshita, T., Takino, T., Nakayama, K., Seiki, M., 1996. Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinases (TIMP)-2. FEBS Lett. 393, 101–104. Sato, H., Seiki, M., 1996. Membrane-type matrix metalloproteinases (MT-MMPs) in tumor metastasis. J. Biochem. 119, 209–215. Sibley, M.H., Graham, P.L., von Mende, N., Kramer, J.M., 1994. Mutations in the alpha 2(IV ) basement membrane collagen gene of Caenorhabditis elegans produce phenotypes of differing severities. EMBO J. 13, 3278–3285. Stetler-Stevenson, W., Aznavoorian, S., Liotta, L.A., 1993. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu. Rev. Cell Biol. 9, 541–573. Takino, T., Sato, H., Yamamoto, E., Seiki, M., 1995a. Cloning of a
gene potentially encoding a novel matrix metalloproteinase having a trans-membrane domain at the C-terminus. Gene 115, 293–298. Takino, T., Sato, H., Yamamoto, E., Seiki, M., 1995b. Cloning of a human gene potentially encoding a novel matrix metalloproteinase having a C-terminal transmembrane domain. Gene 155, 293–298. Tryggvason, K., Hoyhtya, M., Pyke, C., 1993. Type IV collagenases in invasive tumors. Breast Cancer Res. Treat. 24, 209–218. Van Wart, H., Birkedal-Hansen, H., 1990. The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. USA 87, 5578–5582. Will, H., Hinzmann, B., 1995. cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment. Eur. J. Biochem. 231, 602–608. Woessner, J.F.J., 1991. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 5, 2145–2154.
Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases Kazuhiro Wada a, Hiroshi Sato a,*, Hiroaki Kinoh b, Masahiro Kajita b, Hiroshi Yamamoto c, Motoharu Seiki b a Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920, Japan b Department of Cancer Cell, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Tokyo 108, Japan c Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa 920, Japan Received 16 October 1997; received in revised form 13 January 1998; accepted 13 January 1998; Received by T. Sekiya
Abstract Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Extracellular matrix; Remodeling; Morphogenesis; Genome database; TIMP
1. Introduction Matrix metalloproteinases (MMPs) are zinc-binding endopeptidases that collectively degrade the constituent macromolecules of the extracellular matrix (ECM ) (Matrisian, 1990; Woessner, 1991). Thus, the MMPs are thought to participate in ECM remodeling and degradation, and have been implicated in playing important roles during organ morphogenesis, embryonic development and pathological processes (Stetler* Corresponding author. Tel: +81 76 265 2750; Fax: +81 76 234 4505; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); C., Caenorhabditis; cDNA, complementary DNA; ECM, extracellular matrix; kb, kilobase pairs; MMP, matrix metalloproteinase; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reversetranscription PCR; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TIMP, tissue inhibitor of MMPs. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 07 6 - 6
Stevenson et al., 1993; Tryggvason et al., 1993; Sato and Seiki, 1996). An overall view of the MMP family is important for a better understanding of the biological role, and a growing number of human MMPs have been identified corresponding to the complexity of ECM components in recent years (Sato et al., 1994; Takino et al., 1995a,b; Will and Hinzmann, 1995; Cossins et al., 1996; Pendas et al., 1997). Caenorhabditis elegans is a simple multicellular organism with approximately 1000 cells per body and has ECM components, including collagens, similar to human counterparts (Johnstone, 1994; Sibley et al., 1994). Thus, C. elegans is expected to have a similar ECM metabolizing system to human and will be a good system for analyzing the biological roles of MMP. In this study, we identified three putative MMP genes of C. elegans by sequence similarity searching of genome database. These C. elegans MMP cDNAs were cloned, and gene products expressed in E. coli were biochemically studied.
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2. Materials and methods
59
plied by K. Iwata of Fuji Chemical Industries, Ltd, Takaoka, Japan.
2.1. Database search A Caenorhabditis elegans Database (ACeDB) was searched for similarity to the amino acid (aa) sequence of MT1-MMP zinc-binding domain (GNDIFLVAVHELGHALGLEHSSDPSAIMAPF ) using the program tblastn (Altshul et al., 1990). Approximately 7-kb regions surrounding the sequences encoding homologous aa sequences were further analyzed by the program GeneFinder for C. elegans genome to predict the gene transcripts. The criteria used to identify putative MMP family members were the existence of the PRCGVPD motif and the zinc binding site (HEGHXXGXXHS) at an appropriate distance (Birkedal-Hansen et al., 1993). Predicted MMP genes were amplified by polymerase chain reaction (PCR) using C. elegans mixed stage complementary DNA (cDNA) library as a template, which was supplied by Dr Kohara of National Institute of Genetics, Mishima, Japan. 2.2. Expression of MMPS in E. coli C. elegans MMPs were expressed in E. coli as fusion proteins with FLAG peptide at the carboxyl terminus. DNA fragments encoding the region spanning the catalytic to carboxyl-terminal domains were PCR-amplified using primers containing extra HindIII site and were inserted at HindIII site of pFLAG.CTC vector ( Eastman Chemical Company). Recombinant proteins with FLAG peptide at carboxyl terminus were extracted from E. coli strain XL1-Blue with 20 mM Tris–HCl buffer (pH 7.5) containing 150 mM NaCl and 0.5% Triton X-100 and absorbed to anti-FLAG M2 affinity gel ( Eastman Kodak). Recombinant proteins were eluted with 6 M urea and dialyzed overnight against 20 mM Tris–HCl buffer containing 150 mM NaCl. The enzyme activities of recombinant MMPs were assayed using human type I gelatin and fluorescence-quenching substrate for MMP, Mca–Pro–Leu–Gly–Leu–Gly–Leu–Dpa–Ala– Arg–NH (Peptide Inst., Osaka, Japan). Synthetic MMP 2 inhibitors BB-94 and CT543 were chemically synthesized as described previously ( Kinoshita et al., 1996). Recombinant human TIMP-1 and TIMP-2 were sup-
3. Results and discussion 3.1. Cloning of C. elegans MMP genes; domain structure of the deduced products C. elegans genome cloned in cosmid clones C31B8, H19M22 and Y19A7 were predicted to express mRNA transcripts containing long open reading frames (ORFs) which encode the MMP family, including a pro-peptide domain with the activation locus and a catalytic domain with the zinc-binding site. The MMP cDNAs were amplified by PCR with C. elegans cDNA library as a template, which generated a single band for each amplification. The nucleotide sequences of these cDNAs have been submitted to the DDBJ/EMBL/GenBank DNA Databases under the Accession Nos AB007815, AB007816 and AB007817. The nt sequences of cloned cDNAs matched quite well with the sequences predicted by the program GeneFinder, except for a few splicing junctions. The deduced aa sequences were aligned to human MMP-1/interstitial collagenase and MT1-MMP ( Fig. 1) (Goldberg et al., 1986; Sato et al., 1994; Takino et al., 1995a). These C. elegans MMPs have the characteristic domain organization of known MMPs. The PRCGVPD motif in the pro-peptide domain, which is the cysteine switch region responsible for maintenance of enzyme latency, is well conserved in human MMPs ( Van Wart and Birkedal-Hansen, 1990). Fifteen human MMPs have the PRCGVPD sequence, whereas MMP-2/gelatinase A and MMP-18 have PRCGNPD and PRCGLED, respectively (Collier et al., 1988; Cossins et al., 1996). C. elegans MMPs have rather diverse sequences for this motif; PRCTQTD in MMPH19, PRCGHPD in MMP-Y19 and SRCGVTD in MMP-C31. Two of three C. elegans MMPs, MMP-H19 and MMP-Y19 have a short aa insert between the propeptide and the catalytic enzyme domain that contains the RXKR motif, a furin-like cleavage site. Human stromelysin-3 and membrane-type MMP-1,-2,-3 (MT1-, MT2- and MT3-MMP) have a similar insert containing the RXKR motif (Basset et al., 1990; Sato et al., 1994; Will and Hinzmann, 1995; Takino et al., 1995b) and
Fig. 1. C. elegans MMPs aligned with MMP-1 and MT1-MMP. The ORFs that initiate from ATG and terminate at TAA codon were translated. Each domain indicated in the figure was assigned according to Birkedal-Hansen et al. (1993). PRCGVPD and RXKR motifs are indicated by closed squares and closed circles, respectively. The zinc binding site is double-underlined. Methods: Amplification of the MMP gene identified in C. elegans genome C31B8 cosmid clone was performed with a forward primer starting from base position 15716 and a reverse primer from base position 11975 of cosmid C31B8. MMP gene identified in cosmid H19M22 was amplified with a forward primer from base position 1434 and a reverse primer from 5653 of cosmid H19M22. Amplified fragments were cloned into the pCRII plasmid and sequenced by an ABI autosequencer. A part of the MMP gene identified in cosmid clone Y19A7 was amplified with a forward primer from base position 4297 and a reverse primer from 4133, which generated a 156-bp fragment. The C. elegans cDNA library was screened with this PCR product.
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were shown to be processed by furin (Pei and Weiss, 1995, 1996; Sato et al., 1996). The aa sequence of the catalytic domain, particularly the binding site for zinc (HEXGHXXGXXHS), is well conserved between C. elegans and human MMPs. A serine residue on the right side of third histidine residue distinguishes MMP family members from members of the astacin, snake venom metalloproteinase and disintegrin families (Jiang and Bond, 1992). Members of the astacin family contain a glutamic acid, and members of snake venom metalloproteinase and disintegrin families have an aspartic acid residue at this position. The aa sequence homology with MMP-1 in the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively, and the aa sequence homology with other human MMPs is at a similar level. Thus, they cannot be readily assigned to a particular subclass of human MMP from aa sequence homology. The carboxyl-terminal domain of known MMP consists of four repeats that share some limited sequence homology with modules also found in hemopexin and in vitronectin, and the four tandem modules are held together by a single disulfide bond between two cysteine residues that flank the hemopexin-like domain. None of C. elegans MMPs shows an apparent sequence similarity with hemopexin in this region, but MMP-C31 and -Y19 contain two cysteine residues that can flank this domain and may form a similar structure. Unlike other MMPs, MMP-H19 has two cysteine residues in the hinge domain and two cysteine residues in the carboxyl-terminal domain, and the last cysteine residue resides at the middle of carboxyl-terminal domain. Thus, it may be possible that the structure of MMP-H19 carboxyl-terminal domain is quite different from that of hemopexin-like domain. Up to the present time, about 50% of C. elegans genome sequence remains to be determined, and thus C. elegans should have more genes for MMPs. 3.2. Characterization of recombinant C. elegans MMPS C. elegans MMPs were expressed in E. coli as fusion proteins with FLAG peptide at carboxyl terminus and purified using anti-FLAG antibody columns. Human membrane-type MMP-3 (MT3-MMP) was also synthesized in E. coli for comparison. Purified proteins showed expected molecular weights as judged by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) ( Fig. 2A). Among the three C. elegans MMPs, only MMP-C31 digested gelatin (Fig. 2B). None of the C. elegans MMPs hydrolyzed either native type I collagen or carboxymethylated transferrin, and none of them has proteolytic activity on gelatin and casein zymogrohies (data not shown). The enzyme activity of purified MMPs was also assayed using a fluorescence-quenching substrate for MMP ( Table 1). MMP-C31 cleaved the peptide at an efficiency comparable with MT3-MMP.
A
B
Fig. 2. SDS–PAGE of recombinant MMPs (A) and of their gelatin digestion products (B). (A) Purified proteins were separated on a 10% polyacrylamide gel and stained with Coomassie Brilliant Blue. (B) Human type I gelatin (10 mg) was incubated in the absence ( lane −) or presence of 50 ng recombinant MMP proteins as indicated ( lanes C31, H19 and Y19, respectively) at 37°C for 12 h. The digestion products were analyzed by SDS–PAGE under reducing conditions. Methods: MMP-C31 cDNA fragment encoding from Phe 138 to carboxyl terminal Cys 501, MMP-H19 from Phe 66 to Lys 424, MMPY19 from Tyr 100 to Phe 520 and MT3-MMP from Tyr112 to Ser 557 were PCR-amplified using primers containing extra HindIII site and were inserted at HindIII site of pFLAG.CTC vector. Recombinant proteins were purified as described in Materials and methods.
MMP-H19 cleaved less effectively than MMP-C31, and MMP-Y19 showed only a trace activity against this substrate. It is noteworthy that C. elegans MMPs showed enzyme activity in proportion to aa homology with human MMPs. Namely, MMP-C31, which has 45% homology with MMP-1 at the catalytic domain, has the highest enzyme activity against gelatin and peptide substrate, and MMP-H19 and -Y19, which have 34% and 23% homology at the catalytic domain with human MMP-1, respectively, showed 55% and 4.9%
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K. Wada et al. / Gene 211 (1998) 57–62 Table 1 Enzyme activity of recombinant MMPs MMP Inhibitor: MT3-MMP MMP-C31 MMP-H19 MMP-Y19
− 100 124.1 68.3 6.1
C. elegans MMPs morphogenesis.
TIMP-1
TIMP-2
BB94
CT543
105.5 16.8 11.5 ND
15.2 14.3 8.0 ND
3.3 2.8 3.7 ND
4.5 3.4 2.8 ND
Recombinant MMP proteins (0.1 mg) were incubated in a total volume of 0.5 ml with fluorescence-quenching substrate (1 mM ) for MMP at 37°C for an hour in the absence ( lane, −) or presence of inhibitors, TIMP-1 (1 mg), TIMP-2 (1 mg), BB94 (1×10-8M ) and CT543 (1×10-8M ). Activity of MT3-MMP without inhibitor was set to 100%. ND, not determined.
activity of MMP-C31 against only peptide substrate, respectively. Interactions of MT3-MMP and C. elegans MMPs, MMP-C31 and -H19 with human tissue inhibitors of MMP ( TIMP)-1 and -2 were tested. Both TIMP-1 and -2 effectively inhibited the enzyme activity of these C. elegans MMPs. BB94 and CT543, specific inhibitors of human MMPs, also efficiently interfered with C. elegans MMPs, MMP-C31 and -H19. These results suggest that the structure of the C. elegans MMPs (MMP-C31 and -H19) catalytic center is closely related to that of human MMPs. MT3-MMP was inhibited only by TIMP-2 but not by TIMP-1 as was previously observed with MT1-MMP (Sato et al., 1996). An attempt is being made to obtain nematodes that have an inactivated MMP gene by insertion of transposon Tc1. So far, some clones were isolated that had an inactivated MMP-C31 gene, but none of them showed any apparent phenotypical change. In contrast, inactivation of the MMP-H19 gene seems to be lethal, because only nematodes with MMP-H19 gene deficient heterozygote were obtained from the progeny of deficient heterozygote (unpublished data). 3.3. Conclusion In summary, we have identified three C. elegans MMPs. MMP-C31, which has the highest aa homology with human MMPs, digested gelatin and peptide substrate. MMP-H19, which shares a lower aa homology with human MMPs, only digested peptide substrate, and less efficiently than MMP-C31. The enzyme activity of these two C. elegans MMPs was inhibited by specific inhibitors of human MMPs, suggesting that the catalytic center structure of these MMP resembles that of human MMPs as expected from their aa homology with human MMPs. The enzyme activity of MMP-Y19, which shares the lowest aa homology with human MMPs, was not detected with common MMP substrates. Studies are now under way to determine the expression profile of
and
their
roles
in
organ
Acknowledgement We thank Dr R. Hosono (School of Medicine, Kanazawa University) for helpful discussion, Dr Y. Kohara (National Institute of Genetics, Mishima, Japan) for supplying the C. elegans cDNA library, and Dr K. Iwata (Fuji Chemical Industries, Ltd, Takaoka, Japan) for supplying recombinant human TIMP-1 and TIMP-2. This work was supported by a grant-in-aid for Cancer Research, from the Ministry of Education, Science Technology of Japan.
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