Cloning and structural analysis of complement component 3d in wild birds provides insight into its functional evolution

Complement component 3 d (C3d) is the final cleavage product of the complement component C3 and serves as a crucial role in link innate and adaptive immunity, and increase B-cell sensitivity to an antigen by 1000-10000 fold. The crystal structure of human C3d revealed there are two distinct surfaces, a convex surface containing the thioester-constituting residues that mediate covalent binding to the target antigen, and a concave surface with an acidic pocket responsible for interaction with CR2. In this study, we cloned and sequenced cDNA fragment encoding C3d region from 15 wild bird species. Then, the C3d sequences from wild birds, chicken and mammals were aligned to construct phylogenetic trees. Phylogenetic tree displayed two main branches, indicating mammals and birds, but the bird C3d branch was divided into two main parts, with five wild birds (Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus) clustering much closer to mammals. In addition, the C3d proteins of Ardeola bacchus, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus contained a Glu163 residue at the position at which Lys163 was found in other birds. However, Glu163 have the same charge polarity as Asp163, which is the key amino acid residue comprising the acidic pocket combined with CR2 found at this position in mammals, and Zoothera also possessed Asp163 at this position. Structure modeling analyses also verified that the C3ds of these five wild bird species exhibited the amino acid sequence and structure comprising the typical acidic pocket found in mammals that is required for combination with B cell surface receptors, which contribute electrostatic forces to interact with CR2. Our investigations indicate that some bird C3ds may already have the ability to bind with CR2 by electrostatic force, like mammals. As Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus have more typical C3d concave acid pockets and thus a stronger ability to bind CR2, we speculate that these five wild birds may have a solider immunity against pathogens. Our phylogenetic and structural analyses of bird C3ds provide insights on the evolutionary divergence in the function of immune factors of avian and mammalian.

Automated summary

C3d is an important protein in the immune system, connecting innate and adaptive immunity. Phylogenetic and structural analyses show that some wild birds have C3d proteins with characteristics closer to mammals, potentially leading to stronger immunity against pathogens.