A C1 inhibitor ortholog from rock bream (Oplegnathus fasciatus): Molecular perspectives of a central regulator in terms of its genomic arrangement, transcriptional profiles and anti-protease activities of recombinant peptide

Abstract

C1 inhibitor (C1Inh), a member of serpin superfamily, is a crucial regulator of the activation of various plasmatic cascades associated with immunity and inflammation. This study describes the identification and characterization of a C1Inh gene from rock bream Oplegnathus fasciatus (OfC1Inh) at structural, expressional and functional levels. The cDNA-(2245 bp) and corresponding gDNA-sequences (5.2 kbp) of OfC1Inh were isolated from rock bream transcriptome- and BAG-libraries, respectively. Predicted amino acid sequence of OfC1Inh revealed a two-domain architecture composed of an N-terminal region with two Ig-like domains and a C-terminal region with a serpin domain. Tertiary model of OfC1Inh disclosed its active site topology. In the multi-exonic genomic arrangement of OfC1Inh, it consisted of eleven exons disjoined by ten introns as observed in few other fish homologs. Our comparative analysis indicated that the teleostean C1Inhs were distinct from their non-teleostean vertebrate counterparts in terms of their (1) extended N-terminal domains, (2) evolutionary divergence and (3) exon-intron distribution. The OfC1Inh had a TATA-deficient promoter with a putative initiator element, and two tandemly arranged downstream promoter elements. Several components associated with the immune and inflammatory transcriptional activation were also predicted to exist in 5' flanking region of OfC1Inh. The exclusive mRNA levels in liver and moderate levels in extra-hepatic tissues intimated the diversified importance of OfC1Inh in rock bream physiology. We also provide an evidence for the involvement of OfC1Inh in immune balance, based on its modulated transcription upon different PAMP (lipopolysaccharide and poly I:C)- or pathogen (Streptococcus iniae and rock bream irido virus)-challenges. A recombinantly expressed fusion protein [(r)OfC1Inh] was employed in demonstrating the anti-protease function of OfC1Inh. The (r)OfC1Inh exhibited detectable inhibitory activity against Cl esterase and thrombin, where the anti-C1 esterase role was shown to be potentiated by heparin. Taken together, the results of this study provide the first line of evidence for the possible involvement of a teleostean C1Inh in fish immunity, based on its expressional response(s) and inhibitory properties against two enzymes involved in biological cascades. (C) 2013 Elsevier Ltd. All rights reserved.