Isolation and identification of the major plasma fibronectin cDNA from Japanese catfish Silurus asotus

Abstract

Major plasma fibronectin from Japanese catfish was isolated using affinity chromatography, and the fibronectin was digested with thermolysin. Peptide sequences of the fragments were obtained by peptide sequencer. Complete fibronectin cDNA was obtained from Japanese catfish liver cells using 5'-rapid amplification of cDNA end (RACE) and 3'-RACE based on the peptide sequences. It consists of a 6885 bp open reading frame, which is putatively translated to a protein of 2295 amino acids resides. The catfish fibronectin has 12 type-I modules, 2 type-II modules and 15 type-III modules, and variable sites V and lacks both EIIIA and EIIIB sites. Homology of the entire amino acids residues of catfish fibronectin with those of mammals (Homo sapiens, Rattus norvegicus, Bos taurus) is only 47-48% and 57% with that of Danio rerio. However, amino acid sequence of type-I module 3 and type-I module12 are highly conserved and homology exceeds 80% with corresponding regions of the mammals, Xenopus laevis and fish species (Silurus asotus and D. rerio). Phylogenetic analysis indicates that only type-I module 4 shows a different pattern of phylogenetic tree. One major fibronectin mRNA was detected in whole liver and hepatocytes by northern hybridization, however, five to six other bands were also detected in both samples. © 2006 Elsevier Inc. All rights reserved.