Submitted to: Annual Eastern Fish Health Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 5/15/2007
Publication Date: 6/20/2007
Citation: Yeh, H., Klesius, P.H. 2007. Molecular Cloning, Expression and Genome Organization of Channel Catfish (Ictalurus punctatus) Matrix Metalloproteinase-9. Annual Eastern Fish Health Workshop. June 2007. page 79.
Technical Abstract: In the course of studying pathogenesis of enteric septicemia of catfish, we noted that channel catfish matrix metalloproteinase-9 (MMP-9) gene was up-regulated after Edwardsiella ictaluri infection. In this study, we cloned, sequenced using the RACE (rapid amplification of cDNA ends) method and characterized MMP-9. The sequence of the CC MMP-9 cDNA gene consisted of 2562 nucleotides that potentially encode a 686 amino acid peptide with a calculated molecular mass (without glycosylation) of approximate 77.4 kDa. In addition, analyzed by the SiganlIP 3.0 program, the CC MMP-9 included a signal peptide with a cleavage site at positions 20 and 21 (AWS-HP). CC MMP-9 analyzed by the NetOGlyc 3.1 program had potentially heavy O-glycosylation sites between positions 447 and 490. CC MMP-9 consisted of many structural domains, including a signal sequence, matrix metalloprotease domain, matrixin, fibronectin type II domain 1, fibronectin type II domain 2, fibronectin type II domain 3 and hemopexin-like repeats. In addition, the pair-wise comparison of amino acid sequences showed that the MMP-9 was highly conserved among fish species (70% - 74%), but not in mammalian MMP-9 (e.g. 55% with human MMP-9). To further determine evolutionary relatedness of MMP-9 among animals, phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.1 program based on the ClustalW alignment data. MMP-9 from various fish formed a single well supported clade, distinguishable from mammalian MMP-9. In addition, MMP-9 of common carp and zebrafish branched with CC MMP-9, but had a distant evolutionary relationship. The complete sequence of the MMP-9 genomic DNA consisted of 5663 nucleotides. Analysis of the sequence revealed that channel catfish MMP-9 contains 13 exons (#1 - #13). The numbers of base pairs of each exon are as follows (exon 1 – exon 13): 160, 236, 151, 130, 177, 174, 177, 159, 236, 142, 155, 107 and 108, respectively.