Discovery of Teleost Plasma Kallikrein/Coagulation Factor XI-Like Gene from Channel Catfish (Ictalurus punctatus) and the Evidence that the Protein Encoded by it Acts as a Lectin.

Mammalian plasma kallikrein (PK) and coagulation factor XI (fXI) are serine proteases that play in the kinin-kallikrein cascade and in the blood clotting pathway. These proteases share sequence homology and have four apple domains (APDs) and a serine protease domain (SPD) from their N-terminus to C-terminus. No homologs of these proteases are believed to be present in fish species, except for lobe-finned fish. Fish, however, have a unique lectin, named kalliklectin (KL), which is composed of APDs only. In the present study, we found genomic sequences encoding a protein with both APDs and SPD in a few cartilaginous and bony fishes, including the channel catfish Ictalurus punctatus, using bioinformatic analysis. Furthermore, we purified two ~ 70 kDa proteins from the blood plasma of the catfish using mannose-affinity and gel filtration chromatography sequentially. Using de novo sequencing with quadrupole time-of-flight tandem mass spectrometry, several internal amino acid sequences in these proteins were mapped onto possible PK/fXI-like sequences that are thought to be splicing variants. Exploration of APD-containing proteins in the hagfish genome database and phylogenetic analysis suggested that the PK/fXI-like gene originated from hepatocyte growth factor, and that the gene was acquired in a common ancestor of jawed fish. Synteny analysis provided evidence for chromosomal translocation around the PK/fXI-like locus that occurred in the common ancestor of holosteans and teleosts after separation from the lobe-finned fish lineage, or gene duplication into two chromosomes, followed by independent gene losses. This is the first identification of PK/fXI-like proteins in teleosts.

Molecular evolution of kalliklectin in teleost and identification of the novel type with eight apple domains in channel catfish, Ictalurus punctatus.

Kalliklectin is a unique fish-specific lectin that demonstrates sequence similarity to mammalian plasma kallikrein and coagulation factor XI, which are not lectins but proteases. Reported fish kalliklectins and these mammalian proteases comprise four characteristic "apple domains" (APDs). Bioinformatics analysis revealed that Siluriformes species possess anomalous kalliklectins comprising 6 to 16 APDs. Complementary DNA cloning showed that the full-length nucleotide sequence of Ictalurus punctatus consists of 2240 bp that encode 720 amino acid residues to produce a mature protein with a putative 18 amino acid N-terminus peptide sequence. This protein has a predicted molecular mass of 83,417.23 Da. Reverse transcription-polymerase chain reaction (RT-PCR) showed that this lectin gene expresses in the liver but not in any other tissues, including the mucosal tissues. This differential expression pattern makes this lectin unique compared to other lectins described in previous studies. We successfully detected an 85-kDa protein in the serum using western blotting analysis, suggesting that this lectin protein is produced by the liver and secreted into the bloodstream. We characterized a novel cDNA sequence encoding a new type of kalliklectin with eight APDs isolated from channel catfish, I. punctatus. Based on phylogenetic analysis, we speculated that there was a duplication of the third and fourth APD set in a common Siluriformes ancestor at some point after its separation from the common teleost ancestor and that these duplications then underwent independent repeats in different lineages resulting in the generation of the [APD1]-[APD2]-{[APD3]-[APD-4]} × n structure in modern catfishes.