Abstract
As the central component in the complement system, complement component 3 (C3) plays essential roles in both the innate and adaptive immune responses. Here, a C3 gene (designated as pf-C3) was obtained from the pearl oyster Pinctada fucata by RT-PCR and rapid amplification of cDNA ends (RACE). The pf-C3 cDNA consists of 5,634 bp with an open reading frame (ORF) of 5,193 bp encoding a protein of 1,730 amino acids with a 19 residue signal peptide. The deduced pf-C3 protein possessed the characteristic structural features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Phylogenetic analysis revealed that pf-C3 is closely related to the C3s from other mollusks. Pf-C3 mRNA was expressed in all examined tissues including gill, digestive gland, adductor muscle, mantle and foot, while the highest expression was found in the digestive gland. Following the challenge with Vibrio alginolyticus, pf-C3 expression was significantly induced in hemocytes. Luciferase reporter assays indicated that pf-C3a could activate the NF-κB signal pathway in HEK293T cells. Further knockdown of pf-C3 by specific siRNA could significantly reduce the phagocytosis of V. alginolyticus by hemocytes in vitro. These results would help increase understanding of the function of C3 in the invertebrate immune system and therefore provide new insights into the roles of the primitive complement system in invertebrates.
Highlights
The innate immune system in all metazoans is an evolutionarily ancient form, which offers the non-specific defense and exerts an important role in the first-line defense to resist foreign invaders or pathogens
To address the relationship between pf-component 3 (C3) expression and hemocyte phagocytosis, the in vitro phagocytosis assay was performed through flow cytometry analysis (Figure 7), and the result revealed that knockdown of pf-C3 significantly lowered the phagocytosis of V. alginolyticus from 73.55 to 52.45% compared with the control, which means fewer V. alginolyticus could be engulfed by hemocytes treated by siRNA
The innate immune responses in mollusks have been well described in several species; the exploration of genomic and transcriptomic data is providing new insights regarding several aspects of invertebrate immunology [4]
Summary
The innate immune system in all metazoans is an evolutionarily ancient form, which offers the non-specific defense and exerts an important role in the first-line defense to resist foreign invaders or pathogens. The above three pathways are combined during the complement component 3 (C3) proteolytic activation process; activating the C3 fragments induces certain functions to resist pathogens, facilitating immune responses [1]. These mechanisms comprise opsonization of C3b coated non-self particles [4, 5], and inflammation develops by means of C3a anaphylatoxin together with C5 component activation, inducing terminal complement proteins that constitute membrane attack complex (MAC) [4, 6]. The work might provide a clue for further studies on bivalve C3 and expand the horizon to better understand the functional evolution of the ancient complement system
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