Gene, 120 (1992) 321-322 0 1992 Elsevier Science Publishers
B.V. All rights reserved.
321
0378-l 119/92/$05.00
GENE 06739
Cloning and sequencing of a cDNA encoding mouse stromelysin (Dideoxy sequencing; matrix metalloproteinase;
Khalil Hammani,
Patrick Henriet
l*
collagenase; homology)
and Yves Eeckhout
Laboratoire de Chimie Physiologique (Connective Tissue Group), Universitkde Louvain, and International Institute of Cellular and Molecular Pathology, B-1200 Bruxelles, Belgium Received
by D.T. Denhardt:
23 June 1992; Accepted:
30 June 1992; Received
at publishers:
16 July 1992
SUMMARY
A cDNA encoding mouse stromelysin 1 was cloned and the 1740-bp sequence was determined. The deduced amino acid (aa) sequence was compared with stromelysin 1 sequences of other mammals. Comparison with a previously published incomplete aa sequence of mouse stromelysin 1 revealed three single aa differences.
Stromelysin 1 (MMP3; EC 3.4.24.17) is a member of a family of MMPs implicated in normal and pathological degradation of connective tissues and in the activation of procollagenase (Woessner, 1991). An incomplete aa sequence of mouse MMP3 has been derived previously (Ostrowski et al., 1988) from an unpublished nt sequence of a cDNA cloned from squamous sarcoma cells. Poly(A)+mRNA was purified from NMRI mouse calvaria cultured in the presence of heparin (Vaes, 1972) and reverse transcribed. Primers derived from the two most conserved regions of the MMPs allowed the selective PCR amplification of a 0.4-kb cDNA fragment encoding mouse MMP3. Northern blotting analysis of total RNAs purified from calvaria revealed a single 1.8-kb RNA under the most stringent conditions when using this fragment as probe
Correspondence to: Dr. Y. Eeckhout, (Connective
Tissue Group),
stitute of Cellular and Molecular crate, 75, B-1200 Bruxelles,
Laboratoire
Universite
de Chimie Physiologlque
de Louvain
Pathology,
and International
In-
UCL 7539, Avenue Hippo-
Belgium.
Tel. (32-2)7647555; Fax (32-2)7647573. *Upon request, the authors will supply experimental
details for this Brief
Note. Abbreviations:
aa, amino
acid(s);
bp, base pair(s);
cDNA,
DNA
com-
plementary; kb, kilobase or 1000 bp; MMP, matrix metalloproteinase; NMRI, Naval Medical Research Institute; PCR, polymerase chain reaction.
(data not shown). With the same probe, a 1.7-kb cDNA was isolated from a cDNA library constructed from poly(A)+mRNA of the cultured mouse calvaria. The sequence shows a 24-bp 5’-untranslated region and two initial ATGs separated by one codon as in rabbit MMP3 cDNA (Fini et al., 1987). The second ATG is probably the start codon because it is located within a more favorable context for initiation (Kozak, 1991). The coding sequence (1431 nt) shares 92% identity with the rat stromelysin 1 sequence (Matrisian et al., 1985) but only 77% with the rat stromelysin 2 (MMPlO) sequence (Breathnach et al., 1987), indicating that it codes for mouse stromelysin 1 (MMP3). The deduced aa sequence (477 aa) differs by three aa from the incomplete (440 aa) sequence deduced by Ostrowski et al. (1988) and exhibits the domain structure common to MMPs: a signal peptide (aa l-17), a propeptide (aa 18-99) containing the Cys switch, a catalytic domain (aa 100-264) containing the zinc-binding region and an hemopexin-like domain (aa 265-477). The deduced aa sequence of mouse MMP3 was also compared to those reported previously for (I) human MMP3 (Whitham et al., 1986), (2) rabbit MMP3 (Fini et al., 1987), (3) rat collagenase (Quinn et al., 1990), and (4) human collagenase (Goldberg et al., 1986). The y0 identical aa was 77, 78, 48 and 55, respectively, indicating that the two rodent MMP3s are not distinct from the other mammalian MMP3s.
322 This work was supported by the Fund for Medical Scientific Research (Belgium) and by the Belgian State Prime Minister’s Office, Science Policy Pro~~m~g (Interuniversity Attraction Poles, Grant 7bis, and concerted actions, Grant 88/93-122). P.H. is a Research Fellow of the Institut pour 1’Encouragement de la Recherche Scientifique dans 1’Industrie et 1’Agriculture. The authors are grateful to Drs. G.G. Rousseau and J.N. Octave for their methodological advice, and to M.C. Baelden, Y. Marchand and E. Stevens for their expert assistance.
REFERENCES
Fig. 1. The nt MMP3 cDNA and aligned aa sequence of mouse preprostromelysin 1. The single site for potential N-glycosylation and the consensus sequence for poly(A) are underlined. The two aa residues differing from the Ostrowski et al. (1988) sequence are doubly underlined; the third difference is that our sequence does not contain a Leu between Va1274 and Val”s. EMBL accession No. is X66402.
Breathnach, R., Matrisian, L.M., Gesnel, M.C., Staub, A. and Leroy, P.: Sequences coding for part of oncogene-induced transin are highly conserved in a related rat gene. Nucleic Acids Res. 15 (1987) 11391151. Fini, M.E., Karmilowicz, M.J., Ruby, P.L., Beeman, A.M., Borges, K.A. and Brinckerhoff, C.E.: Cloning of a complementary DNA for rabbit proactivator. A metalloproteinase that activates synovial cell collagenase, shares homology with stromelysin and trausin, and is coordinately regulated with collagenase. Arthr. Rheum. 30 (1987) 12541264. Goldberg, G.I., Wilhelm, SM., Kronberger, A., Bauer, E.A., Grant, G.A. and Eisen, AZ.: Human fibroblast collagenase. Complete primary strncture and homology to an oncogene ~ansfo~ation-induced rat protein. J. Biol. Chem. 261 (1986) 6600-6605. Kozak, M.: Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem. 266 (1191) 19867-19870. Matrisian, L.M., Glaichenhaus, N., Gesnel, M.C. and Breathnach, R.: Epidermal growth factor and oncogenes induce transcription of the same cellular mRNA in rat fibroblasts. EMBO J. 4 (1985) 1435-1440. Ostrowski, L.E., Finch, J., Krieg, P., Matrisian, L., Patskan, G., O’Connell, J.F., Phillips, J., Slaga, T.J., Breathnach, R. and Bowden, G.T.: Expression pattern of a gene for a secreted metahoproteinase during late stages of tumor progression. Mol. Carcinogen. 1 (1988) 13-19. Quinn, CO., Scott, D.K., Brinckerhoff, C.E., Matrisian, L.M., Jeffrey, J.J. and Partridge, NC.: Rat collagenase. Cloning, amino acid sequence comparison and parathyroid hormone regulation in osteoblastic cells. J. Bid. Chem. 265 (1990) 22342-22347. Vaes, G.: The release of collagenase as an inactive proenzyme by bone explants in culture. B&hem. J. 126 (1972) 275-289. Whitham, S.E., Murphy, G., Angel, P., Rahmsdorf, H.J., Smith, B.S., Lyons, A., Harris, T.J.R., Reynolds, J.J., Herrlich, P. and Docherty, A.J.P.: Comparison of human stromelysin and collagenase by cloning and sequence analysis. Biochem. J. 240 (1986) 913-916. Woessner Jr., J.F.: Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 5 (1991) 2145-2154.
B.V. All rights reserved.
321
0378-l 119/92/$05.00
GENE 06739
Cloning and sequencing of a cDNA encoding mouse stromelysin (Dideoxy sequencing; matrix metalloproteinase;
Khalil Hammani,
Patrick Henriet
l*
collagenase; homology)
and Yves Eeckhout
Laboratoire de Chimie Physiologique (Connective Tissue Group), Universitkde Louvain, and International Institute of Cellular and Molecular Pathology, B-1200 Bruxelles, Belgium Received
by D.T. Denhardt:
23 June 1992; Accepted:
30 June 1992; Received
at publishers:
16 July 1992
SUMMARY
A cDNA encoding mouse stromelysin 1 was cloned and the 1740-bp sequence was determined. The deduced amino acid (aa) sequence was compared with stromelysin 1 sequences of other mammals. Comparison with a previously published incomplete aa sequence of mouse stromelysin 1 revealed three single aa differences.
Stromelysin 1 (MMP3; EC 3.4.24.17) is a member of a family of MMPs implicated in normal and pathological degradation of connective tissues and in the activation of procollagenase (Woessner, 1991). An incomplete aa sequence of mouse MMP3 has been derived previously (Ostrowski et al., 1988) from an unpublished nt sequence of a cDNA cloned from squamous sarcoma cells. Poly(A)+mRNA was purified from NMRI mouse calvaria cultured in the presence of heparin (Vaes, 1972) and reverse transcribed. Primers derived from the two most conserved regions of the MMPs allowed the selective PCR amplification of a 0.4-kb cDNA fragment encoding mouse MMP3. Northern blotting analysis of total RNAs purified from calvaria revealed a single 1.8-kb RNA under the most stringent conditions when using this fragment as probe
Correspondence to: Dr. Y. Eeckhout, (Connective
Tissue Group),
stitute of Cellular and Molecular crate, 75, B-1200 Bruxelles,
Laboratoire
Universite
de Chimie Physiologlque
de Louvain
Pathology,
and International
In-
UCL 7539, Avenue Hippo-
Belgium.
Tel. (32-2)7647555; Fax (32-2)7647573. *Upon request, the authors will supply experimental
details for this Brief
Note. Abbreviations:
aa, amino
acid(s);
bp, base pair(s);
cDNA,
DNA
com-
plementary; kb, kilobase or 1000 bp; MMP, matrix metalloproteinase; NMRI, Naval Medical Research Institute; PCR, polymerase chain reaction.
(data not shown). With the same probe, a 1.7-kb cDNA was isolated from a cDNA library constructed from poly(A)+mRNA of the cultured mouse calvaria. The sequence shows a 24-bp 5’-untranslated region and two initial ATGs separated by one codon as in rabbit MMP3 cDNA (Fini et al., 1987). The second ATG is probably the start codon because it is located within a more favorable context for initiation (Kozak, 1991). The coding sequence (1431 nt) shares 92% identity with the rat stromelysin 1 sequence (Matrisian et al., 1985) but only 77% with the rat stromelysin 2 (MMPlO) sequence (Breathnach et al., 1987), indicating that it codes for mouse stromelysin 1 (MMP3). The deduced aa sequence (477 aa) differs by three aa from the incomplete (440 aa) sequence deduced by Ostrowski et al. (1988) and exhibits the domain structure common to MMPs: a signal peptide (aa l-17), a propeptide (aa 18-99) containing the Cys switch, a catalytic domain (aa 100-264) containing the zinc-binding region and an hemopexin-like domain (aa 265-477). The deduced aa sequence of mouse MMP3 was also compared to those reported previously for (I) human MMP3 (Whitham et al., 1986), (2) rabbit MMP3 (Fini et al., 1987), (3) rat collagenase (Quinn et al., 1990), and (4) human collagenase (Goldberg et al., 1986). The y0 identical aa was 77, 78, 48 and 55, respectively, indicating that the two rodent MMP3s are not distinct from the other mammalian MMP3s.
322 This work was supported by the Fund for Medical Scientific Research (Belgium) and by the Belgian State Prime Minister’s Office, Science Policy Pro~~m~g (Interuniversity Attraction Poles, Grant 7bis, and concerted actions, Grant 88/93-122). P.H. is a Research Fellow of the Institut pour 1’Encouragement de la Recherche Scientifique dans 1’Industrie et 1’Agriculture. The authors are grateful to Drs. G.G. Rousseau and J.N. Octave for their methodological advice, and to M.C. Baelden, Y. Marchand and E. Stevens for their expert assistance.
REFERENCES
Fig. 1. The nt MMP3 cDNA and aligned aa sequence of mouse preprostromelysin 1. The single site for potential N-glycosylation and the consensus sequence for poly(A) are underlined. The two aa residues differing from the Ostrowski et al. (1988) sequence are doubly underlined; the third difference is that our sequence does not contain a Leu between Va1274 and Val”s. EMBL accession No. is X66402.
Breathnach, R., Matrisian, L.M., Gesnel, M.C., Staub, A. and Leroy, P.: Sequences coding for part of oncogene-induced transin are highly conserved in a related rat gene. Nucleic Acids Res. 15 (1987) 11391151. Fini, M.E., Karmilowicz, M.J., Ruby, P.L., Beeman, A.M., Borges, K.A. and Brinckerhoff, C.E.: Cloning of a complementary DNA for rabbit proactivator. A metalloproteinase that activates synovial cell collagenase, shares homology with stromelysin and trausin, and is coordinately regulated with collagenase. Arthr. Rheum. 30 (1987) 12541264. Goldberg, G.I., Wilhelm, SM., Kronberger, A., Bauer, E.A., Grant, G.A. and Eisen, AZ.: Human fibroblast collagenase. Complete primary strncture and homology to an oncogene ~ansfo~ation-induced rat protein. J. Biol. Chem. 261 (1986) 6600-6605. Kozak, M.: Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem. 266 (1191) 19867-19870. Matrisian, L.M., Glaichenhaus, N., Gesnel, M.C. and Breathnach, R.: Epidermal growth factor and oncogenes induce transcription of the same cellular mRNA in rat fibroblasts. EMBO J. 4 (1985) 1435-1440. Ostrowski, L.E., Finch, J., Krieg, P., Matrisian, L., Patskan, G., O’Connell, J.F., Phillips, J., Slaga, T.J., Breathnach, R. and Bowden, G.T.: Expression pattern of a gene for a secreted metahoproteinase during late stages of tumor progression. Mol. Carcinogen. 1 (1988) 13-19. Quinn, CO., Scott, D.K., Brinckerhoff, C.E., Matrisian, L.M., Jeffrey, J.J. and Partridge, NC.: Rat collagenase. Cloning, amino acid sequence comparison and parathyroid hormone regulation in osteoblastic cells. J. Bid. Chem. 265 (1990) 22342-22347. Vaes, G.: The release of collagenase as an inactive proenzyme by bone explants in culture. B&hem. J. 126 (1972) 275-289. Whitham, S.E., Murphy, G., Angel, P., Rahmsdorf, H.J., Smith, B.S., Lyons, A., Harris, T.J.R., Reynolds, J.J., Herrlich, P. and Docherty, A.J.P.: Comparison of human stromelysin and collagenase by cloning and sequence analysis. Biochem. J. 240 (1986) 913-916. Woessner Jr., J.F.: Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 5 (1991) 2145-2154.
