Identification and Enzymatic Characterization of Two Diverging Murine Counterparts of Human Interstitial Collagenase (MMP-1) Expressed at Sites of Embryo Implantation
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m007674200
ISSN1083-351X
AutoresMilagros Balbı́n, Antonio Fueyo, Vera Knäuper, José M. López, Jesús Álvarez, Luis Sánchez‐Pulido, Vı́ctor Quesada, Javier Bordallo, Gillian Murphy, Carlos López‐Otín,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoRemodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murinecollagenase-like A andB). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-Bgenes are located in the A1–A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.AJ278461AJ278462 Remodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murinecollagenase-like A andB). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-Bgenes are located in the A1–A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.AJ278461AJ278462 matrix metalloproteinase membrane-type polyacrylamide gel electrophoresis reverse transcriptase-polymerase chain reaction tissue inhibitor of metalloproteinases P1 artificial chromosome days postcoitum base pair(s) kilobase pair(s) (7-methoxycoumarin-4-yl)-acetic acid norvaline l-dinitrophenyl-diamino propionic acid cyclohexyl alanine Controlled degradation of the extracellular matrix is an essential event in a variety of physiological conditions involving connective tissue remodeling such as embryonic growth and development, uterine involution, ovulation, bone growth and resorption, and wound healing (1Nagase H. Woessner Jr., F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3788) Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2602) Google Scholar). In addition, excessive breakdown of connective tissue plays an important role in a number of pathological processes such as rheumatoid arthritis, atherosclerosis, pulmonary emphysema, and tumor invasion and metastasis (1Nagase H. Woessner Jr., F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3788) Google Scholar, 2Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2602) Google Scholar). Among the diverse proteolytic enzymes potentially involved in these physiological and pathological processes, many studies have focused on matrix metalloproteinases (MMPs),1 a family of structurally related endopeptidases collectively capable of degrading the major protein components of the extracellular matrix and basement membranes. At present, 20 different human MMPs have been characterized at the amino acid sequence level (3Urı́a J.A. López-Otı́n C. Cancer Res. 2000; 60: 4745-4751PubMed Google Scholar). According to structural and functional characteristics, these human MMPs can be classified into at least six different subfamilies of closely related members: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and other MMPs. The collagenase subfamily of human MMPs consists of three distinct members: fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13). An additional collagenase called collagenase-4 has been identified in Xenopus laevis (4Stolow M.A. Bauzon D.D. Li J. Sedgwick T. Liang V.C.T. Sang Q.A. Shi Y.B. Mol. Biol. Cell. 1996; 7: 1471-1483Crossref PubMed Scopus (125) Google Scholar), but to date the putative orthologues of this enzyme in other vertebrate species have not been described. Biochemical characterization of all these collagenases has revealed that they share the ability to cleave fibrillar collagens at a specific peptide bond, resulting in the generation of fragments of about three-fourths and one-fourth the size of the intact molecule. Then, the resulting fragments denature spontaneously to gelatin in physiological temperature and become susceptible to degradation by other MMPs (5Freije J.P. Dı́ez-Itza I. Balbı́n M. Sánchez L.M. Blasco R. Tolivia J. López-Otı́n C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar, 6Welgus H.G. Jeffrey J.J. Eisen A.Z. J. Biol. Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Google Scholar, 7Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar, 8Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). Interestingly, kinetic studies have revealed that each human collagenase shows distinct substrate preferences toward the diverse fibrillar collagens. Thus, MMP-1 degrades preferentially type III collagen (6Welgus H.G. Jeffrey J.J. Eisen A.Z. J. Biol. Chem. 1981; 256: 9511-9515Abstract Full Text PDF PubMed Google Scholar), MMP-8 prefers type I collagen (7Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar), and MMP-13 degrades type II collagen 6-fold more effectively than type I and type III collagens (8Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). It is also remarkable that MMP-13 displays about 40-fold stronger gelatinolytic activity than MMP-1 and MMP-8 (8Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). On the basis of these data, we have previously proposed that the different human collagenases have evolved as specialized enzymes to participate in the remodeling of tissues with different collagen composition (8Knäuper V. López-Otı́n C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar). The observation that the three human collagenases exhibit distinct tissue distribution and are subjected to different regulatory mechanisms (9Urı́a J.A. Jiménez M.G. Balbı́n M. Freije J.M.P. López-Otı́n C. J. Biol. Chem. 1998; 273: 9769-9777Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 10Pendás A.M. Urı́a J.A. Jiménez M.G. Balbı́n M. Freije J.P. López-Otı́n C. Clin. Chim. Acta. 2000; 291: 137-155Crossref PubMed Scopus (73) Google Scholar) is also consistent with the idea that they may play different functional roles in both physiological and pathological processes. To provide further experimental support to this proposal, it is essential that animal models be available in which the activity of the different enzymes can be selectively manipulated. However, these studies have been seriously hampered by the inability to detect the murine orthologue of MMP-1. In fact, to date only murine MMP-8 and MMP-13 have been identified and characterized at the amino acid sequence level (11Balbı́n M. Fueyo A. Knäuper V. Pendás A.M. López J.M. Jiménez M.G. Murphy G. López-Otı́n C. J. Biol. Chem. 1998; 273: 23959-23968Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 12Quinn C.O. Scott D.K. Brinckerhoff C.E. Matrisian L.M. Jeffrey J.J. Partridge N.C. J. Biol. Chem. 1990; 265: 22342-22347Abstract Full Text PDF PubMed Google Scholar, 13Henriet P. Rousseau G.G. Eeckout Y. FEBS Lett. 1992; 310: 175-178Crossref PubMed Scopus (116) Google Scholar), whereas all attempts from many different groups to isolate murine MMP-1 have been repeatedly unsuccessful. These data have suggested that MMP-1 may be functionally substituted in murine tissues by other enzymes with collagenolytic activity such as MMP-8 and MMP-13. Nevertheless, the possibility that additional as yet unidentified murine enzymes could be structurally or functionally related to human MMP-1 cannot be definitively ruled out. To evaluate this possibility, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. As a direct result of this work, we report herein the identification of two novel members of the MMP gene family originally selected by their positive hybridization with the human MMP-1 probe, and contained within the cluster of MMP genes located at murine chromosome 9. We also describe the expression of the genes in Escherichia coli and perform an analysis of the enzymatic activity of the recombinant proteins. Finally, we analyze the expression of these novel MMPs in murine tissues with the finding of their predominant presence at sites of embryo implantation. A high density gridded mouse P1 artificial chromosome (PAC) genomic library was supplied by the Human Genome Mapping Resource Center (Cambridgeshire, UK). Restriction endonucleases and other reagents used for molecular cloning were from Roche Molecular Biochemicals (Mannheim, Germany). Oligonucleotides were synthesized in an Applied Biosystems (Foster City, CA) model 392A DNA synthesizer. Double-stranded DNA probes were radiolabeled with [α-32P]dCTP (3000 Ci/mmol) from Amersham Pharmacia Biotech (Buckinghamshire, UK) using a commercial random-priming kit purchased from the same company. The mouse PAC genomic library was hybridized with a [α-32P]dCTP-labeled cDNA probe corresponding to full-length human MMP-1 probe (ATCC number 57684). Hybridization and washes were performed at 60 °C. After autoradiographic exposure of the filters, 24 positive clones were detected, and 7 of them were further analyzed by extensive Southern blotting and DNA sequencing of isolated fragments. Oligonucleotides derived from the coding yexons of the previously isolated genomic DNA sequences were used as primers for RT-PCR amplification of RNA from mouse embryos using the RNA-PCR kit from PerkinElmer Life Sciences. All PCR assays were carried out in a GeneAmp 2400 or 9700 PCR system from PerkinElmer Life Sciences. Full-length cDNA of Mcol-A and Mcol-B was obtained by RT-PCR amplification and further assembly of two overlapping fragments of each gene, covering from the ATG sequence to the stop codon of the previously identified genomic fragments. DNA fragments of interest were sequenced by the dideoxy chain termination method, using the Sequenase Version 2.0 kit (U.S. Biochemicals, Cleveland, OH), and the ABI-Prism DNA sequencer (Applied Biosystems). Computer analysis of DNA and protein sequences was performed with the GCG software package of the University of Wisconsin Genetics Computer Group. A phylogenetic tree directed to examine the evolutionary relationships between human and mouse MMPs clustered in human chromosome 11 and mouse chromosome 9 was constructed on-line at the United Kingdom Human Genome Mapping Project Resource Center, using PIE, which provides awww interface to programs included in the PHYLIP software package. Labeling of the probes was performed by using 2 μg of PAC or BAC DNA in a nick translation reaction with biotin-16-dUTP. Biotinylated probes were hybridized to mouse male metaphase chromosomes and detected using two avidin-fluorescein layers. Chromosomes were diamine-2-phenylindole dihydrochloride-banded, and images were captured in a Zeiss axiophot fluorescence microscope equipped with a charge-coupled device camera (Photometrics). The specific probe for mouse chromosome 9 was the telomeric probe BAC 55J6 corresponding to the marker D9Mit152 (14Korenberg J.R. Chen X.N. Devon K.L. Noya D. Oster-Granite M.L. Birren B.W. Genome Res. 1999; 9: 514-523Crossref PubMed Scopus (48) Google Scholar). Nylon filters containing 20 μg of RNA of murine tissues were prehybridized at 42 °C for 3 h in 50% formamide, 5× SSPE (1× = 150 mm NaCl, 10 mm NaH2PO4, 1 mm EDTA, pH 7.4), 10× Denhardt's solution, 2% SDS, and 100 μg/ml denatured herring sperm DNA, and then hybridized for 20 h under the same conditions. Filters were washed with 0.1× SSC, 0.1% SDS for 2 h at 50 °C and exposed to autoradiography. RNA integrity and equal loading was assessed by hybridization with an actin probe. Digoxigenin-11-UTP-labeled single-stranded RNA probes were prepared with digoxigenin RNA-labeling mix and the corresponding T3 or T7 RNA polymerase (Roche Molecular Biochemicals) according to the manufacturer's instructions. Mcol-A probe was a 770-bp BamHI fragment, Mcol-B probe was a 700-bpBamHI/HindIII fragment, and MMP-9 probe was a 1353-bp BamHI fragment, and all of them were subcloned in pBluescript (Stratagene) vector. In situ hybridization was performed on paraffin-embedded tissue sections from 9.5-day postcoitum (dpc) mouse embryos or 10.5-dpc rat embryos, essentially as described (15Jiménez M.J. Balbı́n M. López J.M. Álvarez J. Komori T. López-Otı́n C. Mol. Cell. Biol. 1999; 19: 4431-4442Crossref PubMed Google Scholar). 1.3-kbp fragments of the cDNAs encoding the prodomain, catalytic domain, and hemopexin domains of these proteins were generated by PCR amplification with primers 5′-ggctcgagaTTCCCTGTGATTCAGGAT-3′ and 5′-ggaattcTTAGCAGTTGAACCAAGTATTAAT-3′ for Mcol-A, and 5′-ggctcgagaTTCCCTGTGTTTCACAACG-3′ and 5′-ggaattcTTTCCATTAACTTGATAAGG-3′, for Mcol-B. The PCR-amplified product was cloned in the pRSETB expression vector and transformed into BL21(DE3)pLysS-competent E. coli cells. After induction with isopropyl-1-thio-β-d-galactopyranoside (0.5 mm final concentration), inclusion bodies were prepared and the enzymes were refolded as described previously (16Butler G.S. Apte S.S. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 10846-10851Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). For comparative purposes we refolded human MMP-13 and -14 using this protocol and established that these enzymes displayed collagenolytic activity, as an indication of correct folding of the C-terminal hemopexin-like domain, which determines collagenolytic ability in all known human collagenases. Enzymatic activity of purified recombinant Mcol-A and Mcol-B against fibrillar collagens was followed by SDS-PAGE. All assays were performed in 50 mm Tris/HCl, 5 mm CaCl2, 150 mm NaCl, and 0.05% (v/v) Brij-35, pH 7.6, for 16 h at 37 °C (17Willembrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M.I. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (213) Google Scholar). The enzyme/substrate ratio (w/w) used in these experiments was 1/10. Enzymatic activity was also analyzed using the synthetic fluorescent substrates Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (QF-24), Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 (QF-35), and Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2 (QF-41) (provided by C. G. Knight, University of Cambridge, UK). Routine assays were performed at 37 °C at substrate concentrations of 1 μm in an assay buffer of 50 mm Tris/HCl, 5 mm CaCl2, 150 mm NaCl, 0.05% (v/v) Brij-35, pH 7.6, with a final concentration of Me2SO of 1% (17Willembrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M.I. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (213) Google Scholar). The fluorometric measurements were performed using an LS50B PerkinElmer Life Sciences spectrofluorometer. Enzyme concentrations were determined by active site titration using a standard TIMP-1 solution following 4-h preincubation to allow complex formation (17Willembrock F. Crabbe T. Slocombe P.M. Sutton C.W. Docherty A.J.P. Cockett M.I. O'Shea M.I. Brocklehurst K. Phillips I.R. Murphy G. Biochemistry. 1993; 32: 4330-4337Crossref PubMed Scopus (213) Google Scholar). Collagenolytic activity was determined by incubating soluble rat type I collagen (18Cawston T.E. Murphy G. Methods Enzymol. 1981; 80: 711-722Crossref PubMed Scopus (80) Google Scholar) or acid-soluble bovine type II collagen (Biogenesis Inc., Poole, UK) with the recombinant enzymes at 25 °C, and the degradation products were demonstrated by SDS-PAGE. Additionally, we determined the specific collagenolytic activity of Mcol-A using14C-labeled rat type I collagen in a fibrillar assay at 35 °C essentially as described (18Cawston T.E. Murphy G. Methods Enzymol. 1981; 80: 711-722Crossref PubMed Scopus (80) Google Scholar). The activity of both murine orthologues against 14C-labeled gelatin and casein was determined by overnight incubation at 37 °C. Three-dimensional models of catalytic domains of Mcol-A and Mcol-B were calculated using a semiautomated modeling server (19Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9259) Google Scholar) and analyzed with the Swiss-PdbViewer. Briefly, the amino acid sequences of the respective catalytic domains were compared with the sequences of the macromolecules deposited in the Protein Data Bank to identify suitable templates. We chose nonredundant proteins that had the highest structural quality, and high similarity with Mcol-A and Mcol-B. The pdb files corresponding to these proteins are 2TCL (human MMP-1), 1JAN (human MMP-8), the B chain of file 830C (human MMP-13), and 1SLM (human MMP-3). The templates were superimposed and aligned structurally. Then, the target sequences were automatically threaded over the structure, built with ProMod II, and energy-minimized with Gromos96. The models were analyzed with Swiss-Pdb Viewer, whereas the electrostatic calculations were performed with MolMol (20Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6425) Google Scholar). Charges of conserved ions were also included in the calculations: two Zn2+ and two Ca2+ for the catalytic domain, as present in 2TCL. The figures were modeled with MolMol and rendered with Megapov and POV-Ray (from the POV-Ray site on the Web). To identify putative murine MMPs structurally related to human interstitial collagenase (MMP-1), we screened a mouse PAC genomic library using as a probe a full-length cDNA coding for this human protease. After hybridization under low stringency conditions, several PAC clones were selected on the basis of positive hybridization to the probe. The inserts contained in these clones were characterized by endonuclease restriction analysis and selected fragments showing hybridization with the MMP-1 cDNA probe were cloned and subjected to nucleotide sequencing. This analysis allowed the identification of two DNA fragments, derived from PAC 528 C11, whose nucleotide sequences were similar to those previously determined for other murine MMPs. Further sequence analysis of these fragments and comparison with the exon-intron distribution of other MMP genes led us to identify several putative exons of a presumably novel MMP gene. To try to determine the complete structure of this MMP, studies were undertaken to isolate a full-length cDNA encoding this enzyme. To do that, two primers covering the start and stop codons identified in the putative first and last exons of the cloned MMP gene were synthesized and used for RT-PCR amplification of total RNA obtained from mouse embryos. The PCR-amplified product was cloned, and its identity was confirmed by nucleotide sequencing. Computer analysis of the obtained sequence (Fig.1A) revealed an open reading frame coding for a protein of 464 amino acids with a predicted molecular mass of 53.5 kDa, which was tentatively called Mcol-A (Murine collagenase-likeA).Figure 1Nucleotide sequence and deduced amino acid sequences of mouse Mcol-A and Mcol-B. The deduced amino acid sequences for Mcol-A (A) and Mcol-B (B) are shown below the nucleotide sequences. Potential sites forN-glycosylation are underlined. C, comparison of the amino acid sequences of mouse Mcol-A and Mcol-B with mouse collagenases (MMP-8 and MMP-13), human MMP-1, and stromelysins. The multiple alignment was performed with the PILEUP program of the GCG package. Identical residues in all sequences are shadowed ingray. RGD residues exclusive of MMP-1 areunderlined. Residues specific of collagenases are inbold and marked with an asterisk. Numbering refers to Mcol-A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further analysis, of additional clones obtained by RT-PCR amplification of murine embryos RNA with oligonucleotides derived from the sequence determined for Mcol-A, revealed the presence of sequences highly related to but distinct from that determined for this novel MMP. A full-length cDNA for this apparently distinct MMP was isolated following the same strategy as above and then characterized by nucleotide sequencing. Analysis of the resulting sequence (Fig.1B) allowed the finding of an open reading frame encoding a protein of 463 residues, with a calculated molecular mass of 53.5 kDa, and tentatively called Mcol-B. Genomic clones for this second MMP gene were also identified from DNA fragments obtained from PAC 519 F1 and allowed to confirm the sequence determined by analysis of the cDNA amplified by RT-PCR of murine embryos RNA. A comparison of the deduced amino acid sequences determined for Mcol-A and Mcol-B showed that they were closely related, exhibiting about 82% identities between them. Pairwise comparisons for sequence similarities between the identified amino acid sequences (Fig. 1C) and those determined for other murine MMPs showed that the maximum percentage of identities was found with mouse neutrophil collagenase (MMP-8) (48% and 45% with Mcol-A and Mcol-B, respectively). Interestingly, a higher percentage of identities (58% in amino acids and 74% in nucleotides) was found with human interstitial collagenase (MMP-1). This comparative sequence analysis also revealed that both Mcol-A and Mcol-B display all structural features characteristic of archetypal MMPs, including signal sequences, prodomain regions with the conserved Cys residues in the conserved PRCGVPD motif (at positions 87–93), catalytic, and hemopexin domains (Fig. 1C). The percentage of identities of each domain of the murine proteins with human MMP-1 is 53% (prodomain), 63% (catalytic), and 59% (hemopexin) in the case of Mcol-A, and 53% (prodomain), 58% (catalytic), and 61% (hemopexin) in the case of Mcol-B. The amino acid sequence deduced for Mcol-A and McolB contains three and two potential sites of N-glycosylation, respectively, including the one at position 117 absolutely conserved in the catalytic domain of collagenases, macrophage metalloelastases, stromelysin-1 and -2, gelatinase B, and MT-MMPs. To further explore the structural relationship between human MMP-1 and murine Mcol-A and Mcol-B, we next performed a more detailed sequence analysis with special emphasis aimed at comparing a series of residues conserved in all collagenases described to date and proposed as essential determinants of collagenase specificity. These residues include Tyr-210, Asp-231, and Gly-233 according to human MMP-1 numbering (5Freije J.P. Dı́ez-Itza I. Balbı́n M. Sánchez L.M. Blasco R. Tolivia J. López-Otı́n C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar, 21Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1986; 261: 6600-6605Abstract Full Text PDF PubMed Google Scholar). The equivalent residues at these three positions in Mcol-A are Phe-208, Asp-229, and Gly-231, whereas in Mcol-B these residues are Phe-208, Asp-229, and Glu-231, respectively (Fig.1C). Therefore, it seems that Mcol-A is more related to collagenases than Mcol-B at least in terms of occurrence of residues important for this activity. This structural analysis also revealed that both Mcol-A and Mcol-B contain an RGD (Arg-Gly-Asp) motif in the catalytic domain. This motif is present at equivalent position in the MMP-1 sequence from all species in which this protein has been characterized, but not in other MMPs, providing additional evidence on the structural relationship between MMP-1 and the newly identified family members Mcol-A and Mcol-B. By contrast, both enzymes lack the nine-residue insertion present in the hinge region of all stromelysins. They also lack the fibronectin-like domain present in gelatinases, the C-terminal extension rich in hydrophobic residues characteristic of MT-MMPs, and the furin activation motif (RX(R/K)R) mediating the intracellular activation of MT-MMPs and stromelysin-3 (22Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (523) Google Scholar, 23Sato H. Kinoshita T. Takino T. Nakayama K. Seiki M. FEBS Lett. 1996; 393: 101-104Crossref PubMed Scopus (292) Google Scholar). In summary, and taking collectively all these structural comparisons, most data point to the inclusion of Mcol-A and Mcol-B as members of the collagenase subfamily, although they cannot be unequivocally classified within this group on the exclusive basis of their amino acid sequence characteristics. To determine the chromosomal location of murine genes encoding Mcol-A and Mcol-B, metaphase spreads from a male mouse were hybridized with the biotinylated PACs 528 C11 and 519 F1 enclosing these genes and with the telomeric marker of chromosome 9, BAC 55J6. After single- and double-fluorescent in situ hybridization experiments with both probes, fluorescent signal corresponding to Mcol-A andMcol-B genes was located to the A1–A2 region of chromosome 9 (Fig. 2). Other murine MMP genes (MMP-7, -12, -13, and -20) have been already mapped to this region (24Shapiro S.D. Kobayashi D.K. Ley T.J. J. Biol. Chem. 1993; 268: 23824-23829Abstract Full Text PDF PubMed Google Scholar, 25Schorpp M. 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To establish the relative order of Mcol-A and Mcol-B loci within the cluster of MMP genes in mouse chromosome 9, DNA was isolated from the YAC clone I139A1, which contains murine MMP-8 andMMP-13 (11Balbı́n M. Fueyo A. Knäuper V. Pendás A.M. López J.M. Jiménez M.G. Murphy G. López-Otı́n C. J. Biol. Chem. 1998; 273: 23959-23968Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) as well as the new Mcol-A andMcol-B,
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