MOLECULAR, TRANSCRIPTIONAL AND FUNCTIONAL INSIGHTS INTO C1 INHIBITOR, MAIN REGULATOR OF THE COMPLEMENT SYSTEM, FROM Sebastes schlegelii.  

The complement system plays a crucial role in innate and adaptive immunity against microbial infection. Since uncontrolled activation of the complement pathway leads to serious pathological conditions, tight regulation of the activation of this system is vital in assuring homeostasis. This system is activated through classical, alternative and lectin pathways. Complement component 1 inhibitor inhibits the activation of classical and lectin pathways. C1 inhibitor ortholog from S. schlegelii was identified from previously constructed databases and designated as SsC1inh. Identified cDNA sequence was 2161 bp long with an ORF of 1797 bp, encoding 598 amino acids with a signal peptide of 20 amino acids. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that serpin domain of C1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. Theoretical molecular weight and isoelectric point of SsC1inh were 64.8 kDa and 5.38, respectively. SsC1inh showed the highest identity (76.3%) and similarity (87.8%) with Oplegnathus fasciatus. Transcriptional analysis showed the highest expression level in liver, with 588699-fold compared to heart, in which the lowest expression was recorded, under normal physiological conditions. In RT-PCR analysis, after immune challenge experiments with lipopolysaccharide, Streptococcus iniae, and polyinosinic: polycytidylic acid, SsC1inh mRNA expression was significantly downregulated from 6 to 24 hours, post injection for all three stimuli. After 24 hours, the levels came back to normal but in LPS challenge, the level remained downregulated until 72 hours, post injection. To check the protease inhibitory potential of C1inh invitro the complete open reading frame (ORF) was cloned into pMAL-c5X, transformed into Escherichia coli BL21, recombinant SsC1inh (rSsC1inh) protein was over expressed by IPTG induction and purified by maltose affinity chromatography. Protease inhibition potential of rSsC1inh was assessed against C1 esterase and thrombin, two serine proteases, using colorimetric assays. In protease inhibition assays, approximately 30% and 57.6% of human C1esterase and thrombin activity was inhibited by 50 µg rSsC1inh. The results in this study prove the potential of SsC1inh to act as a regulator of the complement system to maintain homeostasis.

Keywords: Rockfish, complement system, protease inhibitor, homeostasis