Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and atlantic salmon (Salmo salar)

Abstract

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon IV. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

FIG. 1.

Nucleotide and deduced amino acid sequences of the precursors of asCATH_1 (A), asCATH_2 (B), and rtCATH_2 (C). Numbering is on the left for nucleotides and on the right for amino acids. Exons are shown in uppercase letter, and introns are shown in lowercase letters. The predicted translation of the exon-coding regions is given. The putative mature antimicrobial sequences are underlined. The arrows show the respective putative cleavage site for signal peptidase and elastase. Asterisks mark the stop codons. Polyadenylation signals are underlined.

FIG. 1.

Nucleotide and deduced amino acid sequences of the precursors of asCATH_1 (A), asCATH_2 (B), and rtCATH_2 (C). Numbering is on the left for nucleotides and on the right for amino acids. Exons are shown in uppercase letter, and introns are shown in lowercase letters. The predicted translation of the exon-coding regions is given. The putative mature antimicrobial sequences are underlined. The arrows show the respective putative cleavage site for signal peptidase and elastase. Asterisks mark the stop codons. Polyadenylation signals are underlined.

FIG. 1.

Nucleotide and deduced amino acid sequences of the precursors of asCATH_1 (A), asCATH_2 (B), and rtCATH_2 (C). Numbering is on the left for nucleotides and on the right for amino acids. Exons are shown in uppercase letter, and introns are shown in lowercase letters. The predicted translation of the exon-coding regions is given. The putative mature antimicrobial sequences are underlined. The arrows show the respective putative cleavage site for signal peptidase and elastase. Asterisks mark the stop codons. Polyadenylation signals are underlined.

FIG. 2.

Multiple-sequence alignment of the predicted translation of rainbow trout cathelicidins rtCATH_1 and rtCATH_2 and Atlantic salmon cathelicidins asCATH_1 and asCATH_2. Identical residues (indicated by asterisks) and similar residues (indicated by periods or colons) identified by the CLUSTAL program are indicated. The conserved motifs encoded by exon IV in each sequence are boxed. The cysteines involved in disulfide bond formation are in boldface text and are indicated with a percent sign. The predicted mature peptides are in italics, and the signal peptide is indicated by a bar above the alignment.

FIG. 3.

Unrooted phylogram showing the phylogenetic relationships between known cathelicidin genes that have been sequenced within the vertebrates. Phylogeny was estimated by using the amino acid sequences of precursors of cathelicidins with the neighbor-joining method. The resulting tree was bootstrapped 1,000 times, and the bootstrap values are shown as percentages.

FIG. 4.

Northern blot analysis of rtCATH_1 and rtCATH_2 mRNA expression: exposed film (A) and densitometric reading of the film (B). The mRNA levels were normalized by the 18S RNA level and expressed as a percentage of the mRNA level in the gill of a fish challenged for 24 h (100%). Con, control fish; 8 h, fish injected intraperitoneally intraperitoneally with 2 × 10⁶ CFU of Aeromonas salmonicida MT423 for 8 h; 24 h, fish injected intraperitoneally with 2 × 10⁶ CFU of A. salmonicida for 24 h; G, gill; HK, head kidney; I, intestine; L, liver; SK, skin; SP, spleen.

FIG. 5.

Time-dependent cleavage of prtCATH_1 by elastase. Shown is a Western blot of a 4 to 12% polyacrylamide gel after incubation with an anti-six-His tag antibody. Five micrograms of prtCATH_1-6His was incubated with 5 μl Tris buffer (lane 1) or elastase (50 ng in 5 μl Tris buffer) for 10 min (lane 2), 60 min (lane 3), and 120 min (lane 4). The reactions were stopped by acidification with an equal volume of 5% acetic acid, and the samples were run on the gel.

FIG. 6.

Mass spectroscopic analysis of the mature peptide of rtCATH_1. The mature peptide was generated from elastase digestion of the recombinant rtCATH_1 proprotein with a six-His tag at the C terminus and was recovered from the 4 to 12% polyacrylamide gel after separation. The mass of the peptide was determined by MALDI mass spectrometry to be 8,214.42 Da.