Fish complement C4 gene evolution and gene/protein regulatory network analyses and simulated stereo conformation of C4–MASP–2 protein complex

Highlights

• Codon usage of C4 gene is much closer to genomic environment in higher vertebrates.

• C4 genes from each species groups underwent negative selection pressures.

• Functional complexities of two C4 isotypes tend to distinct in species from different evolutionary positions.

• Protein structural analysis showed that C4 may have theoretical interaction with MASP–2 protein in grass carp.

Abstract

Complement C4 is a central protein by acting as pivotal molecule in the activation of the complement system. More than a decade ago, C4 gene duplication had been found in several species including fish, revealing the evolutionary origin of C4 gene. However, the evolutionary pattern and systematic function of C4 are still limited. In this study, C4 D and H types in different species groups were completely diverged. The codon usage of C4 H type in higher vertebrates were much closer to their own genome environment, in contrast to lower vertebrates, suggesting that the evolution may provide the dynamic for homogeneous codon usage between specific gene and genome. Multiple C4 sequence alignment showed that the sequences were conserved among different species. However, sequence similarity was obviously different between species C4 D and H type. Negative selection pressure was found on C4 gene evolution and it may be one of the possible reasons for the sequence broad similarity and conservation among interspecies. Proteins from C4 protein–protein interaction (PPI) network were enriched in more hematopoiesis, infections, diseases and immune–related pathways in human than zebrafish. The result suggested that the functional complexities of C4 isotypes are distinct in species from different evolutionary positions. The simulated C4 protein structures between human and grass carp shared structural similarity and the stereo structures of grass carp C4–MASP–2 protein complexes were further simulated according to a study of human. These results suggested that the interaction between C4 and MASP–2 proteins may also exist in grass carp. Our results can provide an insight for the evolutionary process of C4 and better understanding to the potential mechanism of interaction between C4 and MASP–2 in fish species.

Introduction

The complement system is a sophisticated and huge system, consists of more than 30 complement components and exert vital roles in the innate and adaptive immune responses. Three pathways, the classical, alternative and lectin pathways, are known to be activated in complement system when organism fight against exogenous microorganisms [1]. These three pathways are activated and achieved via a series of cascade and amplification reactions: from the proteolytic activation of C5 to the formation of C5β–C9 membrane attack complex (MAC) [2,3]. The formation of MAC is able to induce the pathogenic bacteria destruction by increasing their membrane permeability. Complement–mediated killing also has the possibility to trigger and connect with adaptive immunity, it occurs when complement is activated either directly by microorganisms or by antibody−antigen (Ag−Ig) complexes [4]. In general, the antibody participates and modulates complement–mediated immune response by activation of the opsonization and immune complex clearance.

Complement C4 plays an important role in the activation of the complement system. The activation and generation of C4 is the result of the activated C1s (classical pathway) and MASP−2 (Mannan−binding lectin serine protease 2) (lectin pathway). During the activation of complement, the major fragment C4b acts as an opsonin to mark the surface of pathogens and alter self−tissue for removal. It attaches to surface nucleophiles on the activator through their reaction with the thioester (TE) exposed upon C4 cleavage [5,6]. Therefore, the characterized functional importance of C4 strictly relies on its structure and regulatory mechanism. Taking human as an example, human C4 is synthesized as a prepro single chain of 1744 amino acids. It comprises 3 subunits: the α, β and γ chains. These chains have the order β−α−γ in the precursor molecule [7]. C4 can be cleaved by one activated C1s into C4a and C4b, which binds C2a and C2b to form the enzymatic complexes to activate C3 and C5 in classical pathway [8]. The generated C4b fragment is a modulatory subunit of the classical pathway C3 (C4b2a) and C5 convertases (C4bC2aC3b).

It is similar to higher vertebrate, from previous study, two isotypes of C4 were found in fish and some mammals species which are H type (active/catalytic residue is histidine H) and D type (active/catalytic histidine H residue is replaced by an aspartic acid D) [9]. Although, two types of C4 were found in most of mammalian species, it is likely to origin from same ancestor lineage according to the phylogeny. Thioester bond exists inside of the C3 and C4 molecules. Histidine (H) can cleaves thioester bond in order to allow the covalent binding of these molecules to nearby cell surfaces when C3 and C4 are converted into C3b and C4b via proteolytic activation [10]. In fish, for instance, the substrate specificity has not been analyzed for the carp C4 isotypes. However, both isotypes are functional and mediating the hemolysis of carp serum via the classical and lectin pathways [11]. In addition, the published study showed that fish complement can lyse and opsonize foreign organisms for destruction by phagocytes. There are also indications that complement fragments can participate in inflammatory reactions [4]. However, the mechanism of regulating the opsonization and its association with adaptive immunity in fish are still poorly understood. Identification of the interaction between complement protein and receptor is a promising work for understanding the body regulation of interaction between host and pathogens. It can also provide the theoretical reference for fish vaccine fight against virulent pathogens.

Many complement receptors were initially reported to have the binding function with C3 and C4b, they are C4b–binding protein (C4BP), complement receptors type 1 (CR1) and type 2 (CR2), and decay−accelerating factor (DAF) [12]. In plasma, human C4BP is an oligomeric glycoprotein which has three major isoforms: α7/β1, α7/β0, and α6/β1. C4BP, serving as cofactor, binds nascent C4b molecules in factor I−mediated proteolysis [13,14]. However, among these receptors, CR1 is not clearly recognized outside of mammals [15]. Also no direct CR2 homologue has been found in the bony fish. Decay–accelerating factor (DAF) is a glycoprotein, anchoring to the cell membrane by phosphatidylinositol [16,17], binds to activated C3b and C4b, thereby inhibiting amplification of the complement cascade on host cell membranes [[18], [19], [20]]. Mannose–binding lectin associated proteases (MASPs) are a group members of the serine protease superfamily, play a vital role in targeting recognition processes. It has been currently demonstrated to cleave C4 in a similar way to the activation of the classical pathway and trigger the following formation of the C3 convertase via binding of C4b to C2a [[21], [22], [23]]. Although the functional importance of MASP–2 in the classical pathway's activation has been noticed, the mechanism of protein interaction between MASP–2 and C4β was still unexplored.

The grass carp (Ctenopharyngodon idella) is widely distributed in Asia. It is one of the most economical and herbivorous aquaculture freshwater species for food and water weed control [24,25]. In 2015, the genomic information of grass carp had been deciphered [26]. It provides the extremely useful resource for gene structural and functional study. The significantly expanded immune–related gene family in grass carp was observed by analyzing the genome. It was consistent with the species evolutionary adaption. Furthermore, the previous study identified a total of 64 genes from grass carp complement system via blasting against draft genome and transcriptome [[26], [27], [28]].

To better understand the evolutionary process and putative function of fish complement C4, we built the C4 phylogenetic tree. Two C4 linages in teleost and birds/reptiles clusters were isolated. In the text, species C4 sequences shared high amino acid conservation. We further performed a selective pressure analysis on different species groups, similar negative selection pressures were found in all groups. The C4 putative functional exploration was performed via building regulatory network using C4 as a network hub gene/protein. We further simulated the spatial structures of C4 protein and C4–MASP–2 protein complex. Compare to other complement members, our results indicated a unique evolutionary process of C4 among species and achieved a newly putative spatial conformation of C4–MASP–2 protein complex in grass carp. These results can also provide an insight for illustrating the potential mechanism of the interaction between C4 and MASP–2.