Full length articleN-terminal domain of EcC1INH in Epinephelus coioides can antagonize the LPS-stimulated inflammatory response
Introduction
In mammal, cell-killing mechanisms are the most important components of host immune defense against invading microorganisms, infectious tissues and malignant cells via a cytotoxic manner by activating specific proteins such as complement proteins [1]. Complement cascade is one of the central constitutes in mammalian macrophage-activating immune system [2] and serves as professional effectors capable of increasing the phagocytosis [3], generating significant amounts of superoxide production [4] as well as synchronizing nature immune system with acquired immunity [5]. Additionally, classic pathway, alternative pathway and Mannose binding lectin (MBL) pathway can produce active opsonins or recruiters of other proteins and form membrane attack complexes (MAC), thus defending against the invasion of microorganism [6]. Recent studies indicate that mammalian complement system activated by bacterial lipopolysaccharide (LPS) stimulation can effectively confer protection against the subsequent bacterial infection by enhancing phagocytic activity, whereas a destroyed complement system can significantly reduce the in vivo clearance of virulent strains, implying that complement activation participates in the host killing mechanism of microorganism [7]. In addition, complement deficiency may relate to a reduced resistance to the microorganism invasion, leading to an increasing prevalence of pyogenic infections and immune complex diseases [8].
Complement 1 inhibitor (C1INH) plays an indispensable role in modulating complement pathways as a negative modulator that inhibits the classical pathway by depressing the activities of complement 1r (C1r) and complement 1s (C1s) [9], exhibits a downregulatory effect in the lectin complement pathway by inhibiting mannose binding lectin associated serum protease (MASP) [10], as well as blocks the alternative complement cascade to decrease complement 3 (C3) cleavage by directly inhibiting the binding activity [11]. Recent findings indicate that C1INH may also inactivate surface-mediated pathway [12,13], which may appear to be associated with the regulation of inflammatory response [14]. In addition, C1INH, a key inhibitor of plasma serine proteases, harboring the characteristic structure of serpin superfamily, may serve as a major downregulator of inflammatory processes in mammal [15].
Mounting evidences indicate that the innate immune defense system in invertebrates harbors pathogen recognition units, but it is only a teleostean basic defense mechanism [16]. In contrast, bony fish contain developed complement cascades [17], and the structures of teleostean complements are homologous to those of mammals [18]. Despite previous researches pay more attention to complement cascades in mammals, only a few reports study on the teleostean complement system. In recent years, teleostean C1INHs have been identified in several fish species, including Oreochromis niloticus [19,20], Pseudosciaena croceav [21], Oplegnathus fasciatus [22] and Sebastes schlegelii [23]. Most of these studies focus on the gene structure and gene analysis of teleostean C1INHs, but the possible function of teleostean C1INHs in LPS-mediated inflammatory response is still unclear.
Stressors such as various pathogenic diseases may exert a harmful effect on the immune system in grouper [24,25]. The recent emergence of global climate anomaly arising a wide public concern may be one of the abnormal phenomena, which can aggravate the expansion of vibrio infection via the long-distance geographical transportation [26]. Vibrio alginolyticus is one of vibrio strains can produce a extracellular toxin [27]. In addition, fecal population may facilitate the increasing level of vibrio population [28]. Vibrio strains also contain a TonB/ExbB/ExbD complex that can regulate the iron uptake processes, thus rendering the invading bacteria less susceptible to a microenvironmental condition of limited iron availability [29]. Recent findings indicate that in vivo injection of V. alginolyticus or its extracellular products can significantly induce toxicological effect in grouper, leading to a slight exophthalmia with corneal opaqueness in moribund/dead fish [27]. Thus, a better understanding of the immunity-related mechanism underlying the change of EcC1INH expression to vibrio stimulation is of great importance and may contribute to the sustainable aquaculture. In this research, we mainly investigated the binding activity of N-terminal domain of EcC1INH to LPS and its protective effect on LPS-induced inflammatory responses.
Section snippets
Fish preparation
Healthy groupers (approximately 12.50 g) were obtained from a fish farm (Guangdong, China). Groupers were fed twice and acclimatized in aerated seawater before vibrio challenge.
Cloning
Cloning methods and protocols of full-length EcC1INH cDNA was based on our previous studies using a rapid amplification of cDNA ends (Clontech, China) [30] and the primers for gene cloning were shown in Table 1.
Bioinformatics analysis
Based on our pervious studies, the domain structures of predicted EcC1INH amino acid sequences were analyzed by
Bioinformatics analysis of EcC1INH cDNA
In Supplementary Fig. 1A, EcC1INH cDNA of 2219 bp comprised an ORF of 1797 bp with a poly(A) tail. In addition, the molecular mass of the deduced EcC1INH polypeptide (598 amino acids) was about 66.46 KDa.
In Supplementary Fig. 1B, EcC1INH was a member of serpin superfamily and the deduced amino acid sequence of EcC1INH contained two potential functional domains: Ig domain (amino acids 28–106 and amino acids 116–208, respectively) and serpin domain (amino acids 234–598). GenBank analysis
Discussion
The activation of mammalian complement cascades is involved in the host defense mechanism against microorganism infection [49]. Generally speaking, microorganism infection can activate the human alternative pathway by targeting to C3 receptor-bearing cells [50]. Among the known complement cascades, alternative pathway is an antibody-independent complement cascade, which can be directly activated by LPS stimulation [[51], [52], [53]]. Recent findings demonstrate that mammalian C1INH plays a
Acknowledgements
This project was supported by Guangzhou science and technology plan project (grant nos. 201707010135) and National Natural Science Foundation of China (Grant No.31670423).
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These authors contributed equally to this work.