Molecular characterization and functional analysis of a complement C3 molecule in the orange-spotted grouper (Epinephelus coioides)

Abstract

Complement component C3 is a key molecule in the complement system whose activation is essential for all the important functions performed by this system. In this study, we examined a temperature-subtracted library from the orange-spotted grouper (Epinephelus coioides), and identified a sequence with high similarity to other complement C3 proteins. Rapid amplification of the cDNA ends (RACE) yielded the full open reading frame of this protein, and subsequent analysis indicated that the Ec-C3 (E. coioides-C3) gene encodes a protein of 1657 amino acid residues with a molecular mass of 184.56 kDa. The deduced amino acid sequence showed that Ec-C3 has conserved residues and domains known to be critical for C3 function. RT-PCR assays showed that under normal physiological conditions Ec-C3 mRNA is most strongly expressed in the liver, but relatively high levels were also found in many other tissues, including intestine, muscles, gills, hemocytes, heart, spleen, head, kidney and brain. Further analysis of Ec-C3 gene expression in liver tissue by quantitative real-time PCR demonstrated that Ec-C3 transcript levels increased when the fish were exposed to both pH and temperature stress, but the time when its expression level peaked differed under these stresses. The results show that Ec-C3 mRNA expression in the orange-spotted grouper is influenced by pH and temperature stress and that Ec-C3 may play an important role in antioxidation mechanisms.