Characterization And Functional Analysis Of The Complement Factor I (CFI) And Acute Phase Response Of CFH Family Members In Zebrafish

The complement system in vertebrates plays a crucial role in the elimination ofpathogens. To regulate complement on self-tissue and to prevent spontaneousactivation and systemic depletion, complement is controlled by both fluid-phase andmembrane-bound inhibitors. One such inhibitor, complement factor I (CFI) regulatescomplement by proteolytic cleavage of components C3b and C4b in the presence ofspecific cofactors. Complement factor H (CFH), the main cofactor for CFI, regulatesthe alternative pathway of complement activation by acting in the breakdown of C3bto iC3b. To gain further insight into the regulators of complement activation in bonyfish, zebrafish CFI was cloned and its chariacterization and function was studied. Theexpression of CFH family genes and CFI and their regulation by LPS and LTA duringthe ontogeny of zebrafish embryos/larvae were also studied.The full-length of zebrafish CFI cDNA sequence is2384bp and encodes apredicted protein with731amino acids. The molecular weight of CFI protein is81.4kDa and the pI is6.17. It shares as high as35.5-67.1%amino acid identities withother CFI of vertebrates. There are lots of conserved Cys residues in the peptide.Similar to mammals CFI, zebrafish CFI has a leader peptide, FIMAC, SRCR, LDLr1,LDLr2and SP domains, except that there is a teleost-specific sequence between theleader peptide and the FIMAC domain. The mature CFI protein is a heterodimer witha heavy chain and a light chain. The heavy chain contains FIMAC, SRCR, LDLr1andLDLr2domains and the light chain only contains the SP domain. In the SP domain,His-Asp-Ser residues comprise the catalytic triad and the conserved Asp residue in thebottom of the catalytic pocket. As a solube glycosylated protein, the zebrafish CFIpeptide has5N-glycosylated sites which scatter on the whole peptide and8O-glycosylated sites which locate on the region between the leader peptide and the FIMAC domain. The3D structure of zebrafish CFI is quite similar with that of human,indicating that it may perform the same function. The phylogenetic trees show thatCFI proteins are highly conserved during evolution and the zebrafish CFI is clusteredwith other fish CFI. The CFI gene of zebrafish expressed widely in various tissuesand mainly in ovary, testis, liver, brain, intestines and eyes. CFHs and CFI havedifferent expression patterns during the ontogeny of zebrafish embryos/larvae andthey respond differently to LPS and LTA challenge. We speculate that CFH familygenes play important roles through different ways and regulate the complementactivation pathways accurately. Whole mount in situ hybridization shows that CFI hasa dynamic expression pattern in early stage, it expresses mainly in YSL in early stage,and later on, it specifically expresses in pineal gland and YSL. Overexpression of CFImRNA shows no visible phenotype, but whether CFI is involved in early developmentstill needs further investigation. In vitro expression of the recombinant proteinCFI-28a has been performed in E. coli and the protein we obtained is64.2kDa (MW),this layes a basis for further study of zebrafish CFI function.