Functional Identification of Complement Factor D and Analysis of Its Expression during GCRV Infection in Grass Carp (Ctenopharyngodon idella)

Abstract

Complement factor D (Df) is a serine protease well known for activating the alternative pathway (AP) in mammals by promoting the cleavage of complement component 3 (C3), thus becoming involved in innate defense. In teleost fish, however, the functional mechanisms of Df in the AP and against pathogen infection are far from clear. In the present study, we cloned and characterized the Df gene, CiDf, from grass carp (Ctenopharyngodon idella) and analyzed its function in promoting C3 cleavage and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiDf was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. CiDf harbors a conserved Tryp_SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that CiDf shares the highest identity (96%) and similarity (98%) with Df from Anabarilius grahami. Phylogenetic analysis indicated that CiDf and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the CiDf gene structure is characterized by four introns and five exons. The incubation of recombinant CiDf protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of CiDf in the AP in promoting C3 cleavage, similar to Dfs in mammals. CiDf mRNA expression was widely detected in various tissues and levels were relatively higher in the liver, spleen, and intestine of grass carp. During GCRV infection over a 168-hour period, a high level of CiDf mRNA expression in the liver, spleen, and intestine was maintained at 144 and 168 h, suggesting AP activity at the late stage of GCRV infection. Collectively, the above results reveal the conserved structure and function of CiDf and its distinct expression patterns after GCRV infection, which provide a key basis for studying the roles of Df and AP during GCRV infection in the grass carp C. idella.

Functional and Expressional Analyses Reveal the Distinct Role of Complement Factor I in Regulating Complement System Activation during GCRV Infection in Ctenopharyngodon idella

“…The results showed that the content of CiCFI proteins continued to decline during GCRV infection from the early stage at the incubation period to the late stage at the restored period, corresponding to the results from the liver. In contrast, our previous study has revealed that Df, a well-known complement enhancer in the AP by cleaving Bf to promote the cleavage of C3 and the production of C3b, is observed with a continued up-regulated expression in the grass carp during GCRV infection, even at the late stage of infection [ 35 ]. Theoretically, the down-regulation of CFI and the up-regulation of Df should bring about an increase in the content of C3b proteins in the serum of grass carp during GCRV infection.…”

“…In our previous study, transcriptome analyses show that the AP is significantly activated in grass carps after GCRV infection, with 2.18-fold up-regulation of Df mRNA expression and 9410.14-fold down-regulation of CFI mRNA expression [ 33 ]. Df, a complement enhancer, involves the initiation and amplification loops of complement system activation by cleaving Bf to promote C3 cleavage, and its regulation on complement activation has recently been verified in the grass carp [ 35 ]. CFI, a serine protease, is well-known as the key complement inhibitor, and plays a crucial role in host-pathogen interactions in mammals [ 13 ].…”

“…Previous studies have revealed that the AP is significantly activated in grass carp after GCRV infection with transcriptome analysis, with a 2.18-fold up-regulation of complement factor D (Df) mRNA expression and a 9410.14-fold down-regulation of CFI mRNA expression [ 33 ]. Df is a serine protease for activating the AP by cleaving complement factor B (Bf) to promote the cleavage of C3 [ 34 ], and its enhancement on complement activation has recently been verified in C. idella [ 35 ]. The aims of the present study are (1) to clarify the molecular characteristics of CFI (designated as CiCFI) from C. idella , (2) to verify the function of CiCFI in the complement system, and (3) to explore its role during GCRV infection, which will add fundamental data for the full picture of the complement regulatory mechanism mediated by Df and CFI during GCRV infection.…”

Factor D

“…At this stage, it seems that most of the complement components other than FD have appeared after the duplication of primordial genes encoding C3/C4/C5, FB/C2, C1r/C1s/MASP‐1/MASP‐2/MASP‐3, and C6/C7/C8/C9 molecules 69,75 . Since FD has not been identified in lampreys and cartilaginous fish, teleost fish seems to be the first species in which FD appeared 76 . The exact role of FD in the complement system of teleost fish is unclear.…”

“…69,75 Since FD has not been identified in lampreys and cartilaginous fish, teleost fish seems to be the first species in which FD appeared. 76 The exact role of FD in the complement system of teleost fish is unclear. However, the incubation of recombinant grass carp FD with grass carp serum significantly increased the C3b content, suggesting that at least FD is involved in promoting C3 cleavage in grass carp, likely via AP activation.…”