- Email: [email protected]
Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) matrix metalloproteinase-9
Fish & Shellfish Immunology 17 (2004) 499e503 www.elsevier.com/locate/fsi
Short sequence report
Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) matrix metalloproteinase-9 Mark C. Johnson, Amaia Sangrador-Vegas1, Terry J. Smith2, Michael T. Cairns) National Diagnostics Centre, National University of Ireland, Galway, Ireland Received 20 March 2003; revised 28 April 2004; accepted 19 May 2004
Keywords: Rainbow trout; MMP-9; mRNA expression; Immune stimulation; cDNA sequence
Matrix metalloproteinase 9 (MMP-9) belongs to a family of zinc-dependent endopeptidases [1]. Due to its ability to cleave structural extracellular matrix (ECM) molecules, mammalian MMP-9 has been associated with tissue remodelling and regeneration [1]. MMP-9 is among a subset of MMPs expressed in regenerating newt limb tissue [2] and is expressed in tandem with MMP-2 during ovulation in medaka fish (Oryzias latipes) [3]. However, in comparison with the levels of expression in mammalian leucocytes, the connective tissue cells are only moderate producers of MMP-9 [4]. Recently, MMP-9 has been implicated in the functioning of a number of mechanisms within the mammalian immune system. Human MMP-9 has been shown to activate or potentiate a number of cytokines and chemokines by cleavage, such as IL-1 and IL-8 [5,6]. Potentiation of IL-8 by MMP-9 can lead to increased chemoattraction of neutrophils [7]. This has direct implications in the regulation of leucocytosis, haematopoietic stem cell mobilisation and migration of leucocytes to sites of infection and inflammation [8,9]. MMP-9 is also important in these processes due to its ability to cleave all types of denatured collagens ( gelatins), native type IV and V collagens as well as elastin and other matrix proteins, allowing movement of leucocytes [10]. MMP-9 expression has been detected in the migration of human B- and T-lymphocytes, neutrophils, eosinophils and natural killer cells [4,11]. Transcriptional regulation of mammalian MMP-9 in leucocytes is mediated by cytokines, particularly TNFa, as well as bacterial LPS, dsRNA and lectins [5,12,13]. In this report we describe the cloning of a MMP-9 cDNA in rainbow trout, demonstrate that the MMP-9 gene has a differential tissue distribution and that trout MMP-9 expression is upregulated by the combined stimulus of the pro-inflammatory cytokine TNFa and bacterial LPS. To identify genes associated with leucocyte development and maturation, a cDNA library derived from 4 h PHA-stimulated rainbow trout head kidney leucocytes [14] was screened by differential plaque
) Corresponding author. Tel.: C353-91-512405; fax: C353-91-586570. E-mail address: [email protected] (M.T. Cairns). 1 Present address: Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland. 2 Present address: National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.05.005
1
CAAGAGCCGGCCACAACAAAGAGATTGAACAGCAACAAGAGCCAAGGGAGAAACTGTGCTTTTTTTGTGTGTTTGTGTGTGTGTAGGCTGTCATTGACTG
101
TCTATCTGGCACATTCTTGCATCTTCTCTGTCACCTTTGGTCTCACCATGAGAAGAGTTCTGGCTTTGTTTGTGTTGGGGATGTGTATGCTGAGTGGATG
201
GTGCGTTCCCCTGAAGAAGTCTGTGTCCGTCACGTTCCCTGGAGATGTCCTCAAGAACATGACGGATACGGAGCTGGCAAATAGCTACTTGCAGAGGTTT
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GGCTACATGGACGTTCAGCACCGCAGCGGTTTCCAGTCCATGGCGTCCACCTCCAAGGCTCTGATGAGAATGCAGAGGCAGATGGGGTTGGAGGAGACAG
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GGACGCTGGACAAGTCTACGGTAGCAGCCATGAAGGCACCTCGCTGCGGGGTCCCTGATGTACGCTCCTACCAGACCTTCCAGGGGGACCTGAAATGGGA
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CCATCACGACATTACGTACAGGATCCTGAACTACTCTCCAGACATGGGAGCCTCTCTGATAGACGACGCCTTCGCCAGAGCCTTCAAGGTGTGGAGTGAT
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GTCACCCCTCTCACCTTCACGCGACTCTTCGACGGCATCGCTGACATCATGGTGTCATTTGGAAAAGCAGACCATGGAGACGGCTACCCCTTCGATGGTA
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AGGACGGCCTTCTGGCCCACGCCTTCCCCCCTGGTGAGGGCATACAGGGAGACGCCCACTTTGACGATGACGAAAACTGGACCCTCGGCAAAGGAGCAGC
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TGTGAAGACTAGCTTTGGCAACGCAGAGGGTGCTCTGTGCCACTTCCCTTTTTCCTTCGGGGGCAAGCAGTACTCCACCTGCACCACAGAGGGGCGCTCT
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GACAACCTGCCCTGGTGTGCCACCACAGCCGACTACGGCAGAGACAAGAAATTCGGCTTCTGTCCAAGTGAACTTCTGTACACATTCGACGGAAACAGCA
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1001 ACGGCAAAGCATGTGTGTTCCCCTTTGTGTTTCTCGGGGAGACATATGAAGGTTGCACGACGGAAGGCCGCAGCGATGGATACCGCTGGTGTTCCACCAC G
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1101 AGAGAACTTTGACAAGGATAAGAAATTTGGCTTCTGTCCCAACAGAGATACGGCTGTGATTGGTGGAAACTCTGAGGGAGAGCCGTGTCACTTCCCCTTC E
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1201 GTCTTCCTGGGAAATAAGTATGACTCATGCACCAGCGAGGGACGGGGAGACGGCAGGCTGTGGTGCGCTACCACCAGCAACTTTGACACAGACACGAAAT V
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1301 GGGGCTTCTGCCAAGATAGAGGCTACAGTCTGTTCCTGGTTGCGGCCCATGAGTTTGGTCATGCCCTGGGTCTGGACCACTCCAACATCAGAAACGCCCT G
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1401 CATGTATCCCATGTACAGCTACGTGGAAGACTTCTCCCTGCATAAAGATGATGTTGAAGGCATTCACTATCTCTATGGGTCCAAGACAGGCCCTGACCCC M
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1501 ATCCCCACCATTCCTTCACCTGGCCCTGATCCCAAGCCTGACACCACTACCACTAAGTCCACTACCACTACCACCACACACCCTGTGGACCCATCCCAGA I
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1601 GCCCCTGCCAGATTAACAAATTCGACACCATCACAGAAATTGATGGAGACCTGCACTTCTTCAAAGATGGGCAATATTGGAGGATGTCGAGCAAGACTGA P
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1701 TGGTGGCCTGGAGGGTCCGTTCTCCATGTCAAAGAGGTGGCCGGCTGTGCCAGTCGTCGTCGACACGGCCTTCGAGGACCTTGCGACCAAGAAAATCTAC G
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1801 TTCTTCTCAGGCACCAGGTTCTGGGTGTATACAGGACAGAGTGTCCTGGGACCCCGCAGCATTGAGAAACTGGGCCTGTCCTCCACTGTTGAGAAGGTGG F
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1901 AGGGAGCACTGCAGAGAGGGAAAGGCAAGGTGCTCCTCTTCAATGGAGAGAACTACTGGAGGCTGGATGTGGAGGGCCAGAAAATCGACACGGGATATCC E
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2001 TCGCTTCACAGACCTGGTCTTTGGAGGAGTTCCCCTCGATTCCCACGATGTGTTCCAATTCAAGGGCAACTCCTACTTCTGCCGGGATAGTTTCTACTGG R
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2101 CGAATGAACTCCAACAGACAGGTGGACCGTGTGGGATACGTGAAGTACGATCTCCTGAACTGCAGTGGCTCTTAACTGTGGGGGCTGAAGATTGAAGAAC R
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2201 CCTTTCAATAGGGCCTCTTAGAGTAGGGTCCCATATCTGTTTGTTGACGTCTTGCCAACTTATATATTTAAATTGAAAGTAATTTAGCAGACAACTCTTA 2301 TCCAGAGCGACTTGAGAATGACGGCGAATGTCTTACGGTACGGTTTGTGTTTATGCAGTACCTTTTGGAAAGTTGAAACGTCACCCCAAAGATTGTGTTT 2401 TGTAAAATGATTGGCCACCTTCCATGTACCAACAGTAGAATGGGTACTTTTCTTCAAACGGCGTGTGAGTTAACAGGTTCCTGTCATTTAACCTAGACTA 2501 GGGTCCAGAGAGATATAACCACAGCCCTGAAATAACCACAGCTCCAGCAGAATCGATTCAAAATCTGTTGTGGCTTTCACCTGTCTCCCTGGCACAGTCT 2601 ATTTTGGATGAGACATGAATTTATATTTATTTATATTGCTCTTCAGTTTTATTTTTTGTGTTCAACAATACAAGTTATTTTAGTATGTAAATCAATGTTT 2701 TGTATTAAAACAGAACTGAAAAAGCAATAAACAAATAGCACTTCAAAAAAAAAAAAAAAAA
Fig. 1. Compiled full-length rainbow trout MMP-9 sequence. The deduced amino acid sequence is reported in one-letter code. The start and stop codons and the potential polyadenylation signal sequence are in bold. The putative N-glycosylation sites are highlighted by background shading. The cell attachment sequence RGD and the mRNA instability motifs ATTTA are designated with underlined, bold type. The sequence is available from the EMBL/GenBank databases under accession number AJ320533.
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hybridisation using first strand cDNA probes derived from head kidney and a mixed tissue probe. Twentytwo positives were identified after screening 8000 clones, and from these two positives were identified as MMP-9. Plasmids were excised from pure bacteriophage using ExAssist helper phage according to the manufacturer’s protocol (Stratagene, UK), and inserts sequenced using T3 and T7 vector primers (MWG Biotech, Germany). The full-length cDNA of 2761 bp contains an ORF coding for a putative protein of 675 residues, with a predicted molecular weight of 75 kDa for the full-length latent protein and 64 kDa for the mature enzyme (Fig. 1). Matches for all MMP-9 domains and motifs were identified by ProfileScan [15], including a signal peptide, propeptide domain, a catalytic domain (interspersed by three fibronectin type II repeats), a hinge region and a hemopexin-like repeat binding domain (data not shown). Using the ScanProsite [16] program three potential N-glycosylation sites and a RGD cell attachment motif [17] were detected. In contrast, mammalian MMP-9s tend to be heavily O-glycosylated. The RGD tripeptide sequence is central for the ability of mammalian MMP-9s to interact with cell surface receptors and adhesion to substrates. The mRNA instability motif (ATTTA) was found repeated six times downstream of the stop codon, suggesting that this transcript has only short-term stability. To examine whether trout MMP-9 would respond to the stimulus of TNFa and LPS in a similar fashion to its mammalian equivalentsdsuggesting a possible role for trout MMP-9 in leucocyte activationdhead kidney leucocytes were prepared by density-gradient centrifugation over a 51% percoll gradient [18], plated at a density of 1!107 cells ml ÿ1 in 90 mm tissue culture plates and incubated at 15 (C in modified L-15 growth media (2% foetal calf serum, 0.1% heparin, supplemented with penicillin and streptomycin) and treated with recombinant human TNFa (12.5 units ml ÿ1) and LPS (25 mg ml ÿ1). Control (unstimulated) cultures were incubated concurrently under identical conditions. The cultured leucocytes were harvested at 4, 24 and 48 h timepoints. Northern blots with 15 mg of total RNA per sample timepoint were hybridised with a [a32P]UTP labelled antisense RNA probe derived from rainbow trout MMP-9 (Riboprobe System, Promega) (Fig. 2). Densitometric analysis was performed, normalisation of the results carried out with a b-actin control, and the ratios of stimulated sample compared to control were calculated. Normalised expression of trout MMP-9 was marginally higher 4 h after stimulation, four-fold higher 24 h poststimulation and 11-fold higher in the stimulated sample compared to the control sample at 48 h. This result is in agreement with the MMP-9 response in mammals. Further evidence agreeing with this MMP-9 response was obtained when an SSH library enriched in sequences upregulated after immune stimulation with LPS and TNFa was generated [19]. Random sequencing of 50 clones led to the identification of
Fig. 2. Northern blot analysis of RNA isolated from primary cultures of rainbow trout leucocytes. The blot was hybridised with rainbow trout MMP-9 riboprobe (a). Loading and integrity of the RNA was checked by rehybridisation with a rainbow trout b-actin riboprobe using a 1 kb fragment corresponding to the 3# region of the b-actin cDNA sequence (accession number: AJ438158) (b). Lane 1, 0 h control; lane 2, 4 h control; lane 3, 4 h stimulated; lane 4, 24 h control; lane 5, 24 h stimulated; lane 6, 48 h control; lane 7, 48 h stimulated.
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M.C. Johnson et al. / Fish & Shellfish Immunology 17 (2004) 499e503
Fig. 3. Tissue-specific expression of MMP-9 in rainbow trout examined by RT-PCR. cDNA synthesised from total RNA was subjected to 29 PCR cycles with specific primers for MMP-9 (a). A pair of intron-spanning b-actin primers (5#-ATGGAAGATGAAATCGCCGC-3# and 5#-CGACATGGAGAAGATCTGGCA-3#) were also used as an internal control (21 cycles of amplification) (b).
a partial MMP-9 clone. The conservation of this response between mammals and fish, together with the previous finding that MMP-9 expression in medaka is associated with tissue remodelling during ovulation [3], suggests that teleost MMP-9 may operate within the same contexts described in mammals. RNA from nine tissues from trout (head kidney, blood, brain, gill, gonad, heart, liver, muscle and spleen) were isolated. RT-PCR was used to study the tissue expression profile using two primers (5#CGACGGAAACAGCAACGGC-3# and 5#-TCCCAGAGCCCCTGCCAA-3#) designed to amplify a 624 bp fragment of trout MMP-9. The result illustrated that the MMP-9 gene is expressed in head kidney, whole blood and spleen, but appears weak in the remaining tissues (Fig. 3). This analysis indicates that constitutive trout MMP-9 expression is largely restricted to tissues of the immune system. This finding is in agreement with other reports suggesting that teleost MMP-9 expression, like mammalian expression, is observed in tissues abundant in leucocytes and blood cellsdkidney and spleen in carp (Cyprinus carpio) and kidney, spleen, gill and heart in Japanese flounder (Paralicthys olivaceus) [20,21]. Interestingly, the findings of this study taken together with the reports for carp and flounder are in contrast to a study suggesting expression of MMP-9 in medaka is absent in these tissues and is restricted to ovarian tissue [3]. Acknowledgements This work was supported by grants from the European Community (BIO2 CT 97-0554), BioResearch Ireland and Cork Co. Co. References [1] Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behaviour. Annu Rev Cell Dev Biol 2001;17:463e516. [2] Miyazaki K, Uchiyama K, Imokawa Y, Yoshizato K. Cloning and characterisation of cDNAs for matrix metalloproteinases of regenerating newt limbs. Proc Nat Acad Sci U S A 1996;93:6819e24.
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[3] Matsui H, Ogiwara K, Ohkura R, Yamashita M, Takahashi T. Expression of Gelatinases A and B in the ovary of the medaka fish Oryzias latipes. Euro J Biochem 2000;267:4658e67. [4] Opdenakker G, Van den Steen PE, Van Damme J. Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol 2001;22:571e9. [5] Opdenakker G, Van den Steen PE, Dubois B, Nelissen I, Van Collie E, Masure S, et al. Gelatinase B functions as regulator and effector in leukocyte biology. J Leukocyte Biol 2001;69:851e9. [6] Scho¨nbeck U, Mach F, Libby P. Generation of biologically active IL-1b by matrix metalloproteinases: a novel caspase-1-independent pathway of IL-1b processing. J Immunol 1998;161:3340e6. [7] Starckx S, Van den Steen PE, Wuyts A, Van Damme J, Opdenakker G. Neutrophil gelatinase B and chemokines in leukocytosis and stem cell mobilisation. Leukemia Lymphoma 2002;43:233e41. [8] Laterveer L, Lindley IJ, Heemskerk DP, Camps JA, Pauwels EK, Willemze R, et al. Rapid mobilisation of haematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8. Blood 1996;87:781e8. [9] Fibbe WE, Pruijt JF, Van Kooyk Y, Figdor CG, Opdenakker G, Willemze R. The role of metalloproteinases and adhesion molecules in interleukin-8-induced stem cell mobilisation. Semin Hematol 2000;37:19e24. [10] Trocme´ C, Gaudin P, Berthier S, Barro C, Zaoui P, Morel F. Human B lymphocytes synthesise the 92-kDa gelatinase, matrix metalloproteinase-9. J Biol Chem 1998;273:20677e84. [11] Okada S, Kita H, George TJ, Gleich GJ, Leiferman KM. Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9. Am J Res Cell Mol Biol 1997;17:519e28. [12] Robinson SC, Scott KA, Balkwill FR. Chemokine stimulation of monocyte matrix metalloproteinase-9 requires endogenous TNF-a. Euro J Immunol 2002;32:404e12. [13] Mun-Bryce S, Rosenberg GA. Gelatinase B modulates selective opening of the blood-brain barrier during inflammation. Am J Physiol 1998;274:1203e11. [14] Davidson J, Smith TJ, Martin SAM. Cloning and sequence analysis of rainbow trout LMP2 cDNA and differential expression of the mRNA. Fish Shellfish Immunol 1999;9:621e32. [15] Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 1997;10:1e6. [16] Hofmann K, Bucher P, Falquet L, Bairoch A. The PROSITE database, its status in 1999. Nucl Acids Res 1999;27:215e9. [17] D’Souza SE, Ginsberg MH, Plow EF. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci 1991;16: 246e50. [18] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in fish immunology. Fair Haven, NJ: SOS; 1990. p. 137e54. [19] Sangrador-Vegas A, Lennington J, Smith T. Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridisation. Cytokine 2002;17:66e70. [20] Takeuchi K, Kubota S, Kinoshita M, Toyohara H, Sakaguchi M. Cloning and characterization of cDNA for carp matrix metalloproteinase 9. Fisheries Sci 2002;68:610e7. [21] Kinoshita M, Yabe T, Kubota M, Takeuchi K, Kubota S, Toyohara H, et al. cDNA cloning and characterization of two gelatinases from Japanese flounder. Fisheries Sci 2002;68:618e26.
Short sequence report
Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) matrix metalloproteinase-9 Mark C. Johnson, Amaia Sangrador-Vegas1, Terry J. Smith2, Michael T. Cairns) National Diagnostics Centre, National University of Ireland, Galway, Ireland Received 20 March 2003; revised 28 April 2004; accepted 19 May 2004
Keywords: Rainbow trout; MMP-9; mRNA expression; Immune stimulation; cDNA sequence
Matrix metalloproteinase 9 (MMP-9) belongs to a family of zinc-dependent endopeptidases [1]. Due to its ability to cleave structural extracellular matrix (ECM) molecules, mammalian MMP-9 has been associated with tissue remodelling and regeneration [1]. MMP-9 is among a subset of MMPs expressed in regenerating newt limb tissue [2] and is expressed in tandem with MMP-2 during ovulation in medaka fish (Oryzias latipes) [3]. However, in comparison with the levels of expression in mammalian leucocytes, the connective tissue cells are only moderate producers of MMP-9 [4]. Recently, MMP-9 has been implicated in the functioning of a number of mechanisms within the mammalian immune system. Human MMP-9 has been shown to activate or potentiate a number of cytokines and chemokines by cleavage, such as IL-1 and IL-8 [5,6]. Potentiation of IL-8 by MMP-9 can lead to increased chemoattraction of neutrophils [7]. This has direct implications in the regulation of leucocytosis, haematopoietic stem cell mobilisation and migration of leucocytes to sites of infection and inflammation [8,9]. MMP-9 is also important in these processes due to its ability to cleave all types of denatured collagens ( gelatins), native type IV and V collagens as well as elastin and other matrix proteins, allowing movement of leucocytes [10]. MMP-9 expression has been detected in the migration of human B- and T-lymphocytes, neutrophils, eosinophils and natural killer cells [4,11]. Transcriptional regulation of mammalian MMP-9 in leucocytes is mediated by cytokines, particularly TNFa, as well as bacterial LPS, dsRNA and lectins [5,12,13]. In this report we describe the cloning of a MMP-9 cDNA in rainbow trout, demonstrate that the MMP-9 gene has a differential tissue distribution and that trout MMP-9 expression is upregulated by the combined stimulus of the pro-inflammatory cytokine TNFa and bacterial LPS. To identify genes associated with leucocyte development and maturation, a cDNA library derived from 4 h PHA-stimulated rainbow trout head kidney leucocytes [14] was screened by differential plaque
) Corresponding author. Tel.: C353-91-512405; fax: C353-91-586570. E-mail address: [email protected] (M.T. Cairns). 1 Present address: Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland. 2 Present address: National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.05.005
1
CAAGAGCCGGCCACAACAAAGAGATTGAACAGCAACAAGAGCCAAGGGAGAAACTGTGCTTTTTTTGTGTGTTTGTGTGTGTGTAGGCTGTCATTGACTG
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TCTATCTGGCACATTCTTGCATCTTCTCTGTCACCTTTGGTCTCACCATGAGAAGAGTTCTGGCTTTGTTTGTGTTGGGGATGTGTATGCTGAGTGGATG
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GTGCGTTCCCCTGAAGAAGTCTGTGTCCGTCACGTTCCCTGGAGATGTCCTCAAGAACATGACGGATACGGAGCTGGCAAATAGCTACTTGCAGAGGTTT
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GGCTACATGGACGTTCAGCACCGCAGCGGTTTCCAGTCCATGGCGTCCACCTCCAAGGCTCTGATGAGAATGCAGAGGCAGATGGGGTTGGAGGAGACAG
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GGACGCTGGACAAGTCTACGGTAGCAGCCATGAAGGCACCTCGCTGCGGGGTCCCTGATGTACGCTCCTACCAGACCTTCCAGGGGGACCTGAAATGGGA
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CCATCACGACATTACGTACAGGATCCTGAACTACTCTCCAGACATGGGAGCCTCTCTGATAGACGACGCCTTCGCCAGAGCCTTCAAGGTGTGGAGTGAT
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GTCACCCCTCTCACCTTCACGCGACTCTTCGACGGCATCGCTGACATCATGGTGTCATTTGGAAAAGCAGACCATGGAGACGGCTACCCCTTCGATGGTA
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L
V
R
I
S
S
D
F
Y
D
G
Q
A
K
T
F
A
F
A
D
Q
R
H
G
A
G
D
F
D
L
K
G
K
V
Y
W
W
P
D
S
F
D
D
G
K
701
AGGACGGCCTTCTGGCCCACGCCTTCCCCCCTGGTGAGGGCATACAGGGAGACGCCCACTTTGACGATGACGAAAACTGGACCCTCGGCAAAGGAGCAGC
801
TGTGAAGACTAGCTTTGGCAACGCAGAGGGTGCTCTGTGCCACTTCCCTTTTTCCTTCGGGGGCAAGCAGTACTCCACCTGCACCACAGAGGGGCGCTCT
901
GACAACCTGCCCTGGTGTGCCACCACAGCCGACTACGGCAGAGACAAGAAATTCGGCTTCTGTCCAAGTGAACTTCTGTACACATTCGACGGAAACAGCA
D
G
V
L
K
D
L
T
N
A
S
L
H
F
P
A
G
W
F
N
C
P
A
A
P
E
T
G
G
T
A
E
A D
G
L Y
I
C G
Q
H R
G
F D
D
P K
A
F K
H
S F
F
F G
D
G F
D
G C
D
K P
E
Q S
N
Y E
W
S L
T
T L
L
C Y
G
T T
K
T F
G
E D
A
G G
A
R N
S S
N
1001 ACGGCAAAGCATGTGTGTTCCCCTTTGTGTTTCTCGGGGAGACATATGAAGGTTGCACGACGGAAGGCCGCAGCGATGGATACCGCTGGTGTTCCACCAC G
K
A
C
V
F
P
F
V
F
L
G
E
T
Y
E
G
C
T
T
E
G
R
S
D
G
Y
R
W
C
S
T
T
1101 AGAGAACTTTGACAAGGATAAGAAATTTGGCTTCTGTCCCAACAGAGATACGGCTGTGATTGGTGGAAACTCTGAGGGAGAGCCGTGTCACTTCCCCTTC E
N
F
D
K
D
K
K
F
G
F
C
P
N
R
D
T
A
V
I
G
G
N
S
E
G
E
P
C
H
F
P
F
1201 GTCTTCCTGGGAAATAAGTATGACTCATGCACCAGCGAGGGACGGGGAGACGGCAGGCTGTGGTGCGCTACCACCAGCAACTTTGACACAGACACGAAAT V
F
L
G
N
K
Y
D
S
C
T
S
E
G
R
G
D
G
R
L
W
C
A
T
T
S
N
F
D
T
D
T
K
W
1301 GGGGCTTCTGCCAAGATAGAGGCTACAGTCTGTTCCTGGTTGCGGCCCATGAGTTTGGTCATGCCCTGGGTCTGGACCACTCCAACATCAGAAACGCCCT G
F
C
Q
D
R
G
Y
S
L
F
L
V
A
A
H
E
F
G
H
A
L
G
L
D
H
S
N
I
R
N
A
L
1401 CATGTATCCCATGTACAGCTACGTGGAAGACTTCTCCCTGCATAAAGATGATGTTGAAGGCATTCACTATCTCTATGGGTCCAAGACAGGCCCTGACCCC M
Y
P
M
Y
S
Y
V
E
D
F
S
L
H
K
D
D
V
E
G
I
H
Y
L
Y
G
S
K
T
G
P
D
P
1501 ATCCCCACCATTCCTTCACCTGGCCCTGATCCCAAGCCTGACACCACTACCACTAAGTCCACTACCACTACCACCACACACCCTGTGGACCCATCCCAGA I
P
T
I
P
S
P
G
P
D
P
K
P
D
T
T
T
T
K
S
T
T
T
T
T
T
H
P
V
D
P
S
Q
S
1601 GCCCCTGCCAGATTAACAAATTCGACACCATCACAGAAATTGATGGAGACCTGCACTTCTTCAAAGATGGGCAATATTGGAGGATGTCGAGCAAGACTGA P
C
Q
I
N
K
F
D
T
I
T
E
I
D
G
D
L
H
F
F
K
D
G
Q
Y
W
R
M
S
S
K
T
D
1701 TGGTGGCCTGGAGGGTCCGTTCTCCATGTCAAAGAGGTGGCCGGCTGTGCCAGTCGTCGTCGACACGGCCTTCGAGGACCTTGCGACCAAGAAAATCTAC G
G
L
E
G
P
F
S
M
S
K
R
W
P
A
V
P
V
V
V
D
T
A
F
E
D
L
A
T
K
K
I
Y
1801 TTCTTCTCAGGCACCAGGTTCTGGGTGTATACAGGACAGAGTGTCCTGGGACCCCGCAGCATTGAGAAACTGGGCCTGTCCTCCACTGTTGAGAAGGTGG F
F
S
G
T
R
F
W
V
Y
T
G
Q
S
V
L
G
P
R
S
I
E
K
L
G
L
S
S
T
V
E
K
R
V
1901 AGGGAGCACTGCAGAGAGGGAAAGGCAAGGTGCTCCTCTTCAATGGAGAGAACTACTGGAGGCTGGATGTGGAGGGCCAGAAAATCGACACGGGATATCC E
G
A
L
Q
G
K
G
K
V
L
L
F
N
G
E
N
Y
W
R
L
D
V
E
G
Q
K
I
D
T
G
Y
P
2001 TCGCTTCACAGACCTGGTCTTTGGAGGAGTTCCCCTCGATTCCCACGATGTGTTCCAATTCAAGGGCAACTCCTACTTCTGCCGGGATAGTTTCTACTGG R
F
T
D
L
V
F
G
G
V
P
L
D
S
H
D
V
F
Q
F
K
G
N
S
Y
F
C
R
D
S
F
Y
W
2101 CGAATGAACTCCAACAGACAGGTGGACCGTGTGGGATACGTGAAGTACGATCTCCTGAACTGCAGTGGCTCTTAACTGTGGGGGCTGAAGATTGAAGAAC R
M
N
S
N
R
Q
V
D
R
V
G
Y
V
K
Y
D
L
L
N
C
S
G
S
*
2201 CCTTTCAATAGGGCCTCTTAGAGTAGGGTCCCATATCTGTTTGTTGACGTCTTGCCAACTTATATATTTAAATTGAAAGTAATTTAGCAGACAACTCTTA 2301 TCCAGAGCGACTTGAGAATGACGGCGAATGTCTTACGGTACGGTTTGTGTTTATGCAGTACCTTTTGGAAAGTTGAAACGTCACCCCAAAGATTGTGTTT 2401 TGTAAAATGATTGGCCACCTTCCATGTACCAACAGTAGAATGGGTACTTTTCTTCAAACGGCGTGTGAGTTAACAGGTTCCTGTCATTTAACCTAGACTA 2501 GGGTCCAGAGAGATATAACCACAGCCCTGAAATAACCACAGCTCCAGCAGAATCGATTCAAAATCTGTTGTGGCTTTCACCTGTCTCCCTGGCACAGTCT 2601 ATTTTGGATGAGACATGAATTTATATTTATTTATATTGCTCTTCAGTTTTATTTTTTGTGTTCAACAATACAAGTTATTTTAGTATGTAAATCAATGTTT 2701 TGTATTAAAACAGAACTGAAAAAGCAATAAACAAATAGCACTTCAAAAAAAAAAAAAAAAA
Fig. 1. Compiled full-length rainbow trout MMP-9 sequence. The deduced amino acid sequence is reported in one-letter code. The start and stop codons and the potential polyadenylation signal sequence are in bold. The putative N-glycosylation sites are highlighted by background shading. The cell attachment sequence RGD and the mRNA instability motifs ATTTA are designated with underlined, bold type. The sequence is available from the EMBL/GenBank databases under accession number AJ320533.
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hybridisation using first strand cDNA probes derived from head kidney and a mixed tissue probe. Twentytwo positives were identified after screening 8000 clones, and from these two positives were identified as MMP-9. Plasmids were excised from pure bacteriophage using ExAssist helper phage according to the manufacturer’s protocol (Stratagene, UK), and inserts sequenced using T3 and T7 vector primers (MWG Biotech, Germany). The full-length cDNA of 2761 bp contains an ORF coding for a putative protein of 675 residues, with a predicted molecular weight of 75 kDa for the full-length latent protein and 64 kDa for the mature enzyme (Fig. 1). Matches for all MMP-9 domains and motifs were identified by ProfileScan [15], including a signal peptide, propeptide domain, a catalytic domain (interspersed by three fibronectin type II repeats), a hinge region and a hemopexin-like repeat binding domain (data not shown). Using the ScanProsite [16] program three potential N-glycosylation sites and a RGD cell attachment motif [17] were detected. In contrast, mammalian MMP-9s tend to be heavily O-glycosylated. The RGD tripeptide sequence is central for the ability of mammalian MMP-9s to interact with cell surface receptors and adhesion to substrates. The mRNA instability motif (ATTTA) was found repeated six times downstream of the stop codon, suggesting that this transcript has only short-term stability. To examine whether trout MMP-9 would respond to the stimulus of TNFa and LPS in a similar fashion to its mammalian equivalentsdsuggesting a possible role for trout MMP-9 in leucocyte activationdhead kidney leucocytes were prepared by density-gradient centrifugation over a 51% percoll gradient [18], plated at a density of 1!107 cells ml ÿ1 in 90 mm tissue culture plates and incubated at 15 (C in modified L-15 growth media (2% foetal calf serum, 0.1% heparin, supplemented with penicillin and streptomycin) and treated with recombinant human TNFa (12.5 units ml ÿ1) and LPS (25 mg ml ÿ1). Control (unstimulated) cultures were incubated concurrently under identical conditions. The cultured leucocytes were harvested at 4, 24 and 48 h timepoints. Northern blots with 15 mg of total RNA per sample timepoint were hybridised with a [a32P]UTP labelled antisense RNA probe derived from rainbow trout MMP-9 (Riboprobe System, Promega) (Fig. 2). Densitometric analysis was performed, normalisation of the results carried out with a b-actin control, and the ratios of stimulated sample compared to control were calculated. Normalised expression of trout MMP-9 was marginally higher 4 h after stimulation, four-fold higher 24 h poststimulation and 11-fold higher in the stimulated sample compared to the control sample at 48 h. This result is in agreement with the MMP-9 response in mammals. Further evidence agreeing with this MMP-9 response was obtained when an SSH library enriched in sequences upregulated after immune stimulation with LPS and TNFa was generated [19]. Random sequencing of 50 clones led to the identification of
Fig. 2. Northern blot analysis of RNA isolated from primary cultures of rainbow trout leucocytes. The blot was hybridised with rainbow trout MMP-9 riboprobe (a). Loading and integrity of the RNA was checked by rehybridisation with a rainbow trout b-actin riboprobe using a 1 kb fragment corresponding to the 3# region of the b-actin cDNA sequence (accession number: AJ438158) (b). Lane 1, 0 h control; lane 2, 4 h control; lane 3, 4 h stimulated; lane 4, 24 h control; lane 5, 24 h stimulated; lane 6, 48 h control; lane 7, 48 h stimulated.
502
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Fig. 3. Tissue-specific expression of MMP-9 in rainbow trout examined by RT-PCR. cDNA synthesised from total RNA was subjected to 29 PCR cycles with specific primers for MMP-9 (a). A pair of intron-spanning b-actin primers (5#-ATGGAAGATGAAATCGCCGC-3# and 5#-CGACATGGAGAAGATCTGGCA-3#) were also used as an internal control (21 cycles of amplification) (b).
a partial MMP-9 clone. The conservation of this response between mammals and fish, together with the previous finding that MMP-9 expression in medaka is associated with tissue remodelling during ovulation [3], suggests that teleost MMP-9 may operate within the same contexts described in mammals. RNA from nine tissues from trout (head kidney, blood, brain, gill, gonad, heart, liver, muscle and spleen) were isolated. RT-PCR was used to study the tissue expression profile using two primers (5#CGACGGAAACAGCAACGGC-3# and 5#-TCCCAGAGCCCCTGCCAA-3#) designed to amplify a 624 bp fragment of trout MMP-9. The result illustrated that the MMP-9 gene is expressed in head kidney, whole blood and spleen, but appears weak in the remaining tissues (Fig. 3). This analysis indicates that constitutive trout MMP-9 expression is largely restricted to tissues of the immune system. This finding is in agreement with other reports suggesting that teleost MMP-9 expression, like mammalian expression, is observed in tissues abundant in leucocytes and blood cellsdkidney and spleen in carp (Cyprinus carpio) and kidney, spleen, gill and heart in Japanese flounder (Paralicthys olivaceus) [20,21]. Interestingly, the findings of this study taken together with the reports for carp and flounder are in contrast to a study suggesting expression of MMP-9 in medaka is absent in these tissues and is restricted to ovarian tissue [3]. Acknowledgements This work was supported by grants from the European Community (BIO2 CT 97-0554), BioResearch Ireland and Cork Co. Co. References [1] Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behaviour. Annu Rev Cell Dev Biol 2001;17:463e516. [2] Miyazaki K, Uchiyama K, Imokawa Y, Yoshizato K. Cloning and characterisation of cDNAs for matrix metalloproteinases of regenerating newt limbs. Proc Nat Acad Sci U S A 1996;93:6819e24.
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[3] Matsui H, Ogiwara K, Ohkura R, Yamashita M, Takahashi T. Expression of Gelatinases A and B in the ovary of the medaka fish Oryzias latipes. Euro J Biochem 2000;267:4658e67. [4] Opdenakker G, Van den Steen PE, Van Damme J. Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol 2001;22:571e9. [5] Opdenakker G, Van den Steen PE, Dubois B, Nelissen I, Van Collie E, Masure S, et al. Gelatinase B functions as regulator and effector in leukocyte biology. J Leukocyte Biol 2001;69:851e9. [6] Scho¨nbeck U, Mach F, Libby P. Generation of biologically active IL-1b by matrix metalloproteinases: a novel caspase-1-independent pathway of IL-1b processing. J Immunol 1998;161:3340e6. [7] Starckx S, Van den Steen PE, Wuyts A, Van Damme J, Opdenakker G. Neutrophil gelatinase B and chemokines in leukocytosis and stem cell mobilisation. Leukemia Lymphoma 2002;43:233e41. [8] Laterveer L, Lindley IJ, Heemskerk DP, Camps JA, Pauwels EK, Willemze R, et al. Rapid mobilisation of haematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8. Blood 1996;87:781e8. [9] Fibbe WE, Pruijt JF, Van Kooyk Y, Figdor CG, Opdenakker G, Willemze R. The role of metalloproteinases and adhesion molecules in interleukin-8-induced stem cell mobilisation. Semin Hematol 2000;37:19e24. [10] Trocme´ C, Gaudin P, Berthier S, Barro C, Zaoui P, Morel F. Human B lymphocytes synthesise the 92-kDa gelatinase, matrix metalloproteinase-9. J Biol Chem 1998;273:20677e84. [11] Okada S, Kita H, George TJ, Gleich GJ, Leiferman KM. Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9. Am J Res Cell Mol Biol 1997;17:519e28. [12] Robinson SC, Scott KA, Balkwill FR. Chemokine stimulation of monocyte matrix metalloproteinase-9 requires endogenous TNF-a. Euro J Immunol 2002;32:404e12. [13] Mun-Bryce S, Rosenberg GA. Gelatinase B modulates selective opening of the blood-brain barrier during inflammation. Am J Physiol 1998;274:1203e11. [14] Davidson J, Smith TJ, Martin SAM. Cloning and sequence analysis of rainbow trout LMP2 cDNA and differential expression of the mRNA. Fish Shellfish Immunol 1999;9:621e32. [15] Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 1997;10:1e6. [16] Hofmann K, Bucher P, Falquet L, Bairoch A. The PROSITE database, its status in 1999. Nucl Acids Res 1999;27:215e9. [17] D’Souza SE, Ginsberg MH, Plow EF. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci 1991;16: 246e50. [18] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in fish immunology. Fair Haven, NJ: SOS; 1990. p. 137e54. [19] Sangrador-Vegas A, Lennington J, Smith T. Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridisation. Cytokine 2002;17:66e70. [20] Takeuchi K, Kubota S, Kinoshita M, Toyohara H, Sakaguchi M. Cloning and characterization of cDNA for carp matrix metalloproteinase 9. Fisheries Sci 2002;68:610e7. [21] Kinoshita M, Yabe T, Kubota M, Takeuchi K, Kubota S, Toyohara H, et al. cDNA cloning and characterization of two gelatinases from Japanese flounder. Fisheries Sci 2002;68:618e26.