Full Length ArticleTwo fibrinogen-related proteins (FREPs) in the razor clam (Sinonovacula constricta) with a broad recognition spectrum and bacteria agglutination activity
Introduction
Invertebrates lack lymphocytes and an antibody-based humoral immune system and therefore have to rely on innate immune reactions to prevent pathogen invasion (Loker et al., 2004). When foreign microbes come into contact with a host, the recognition of pathogens by pattern recognition receptors (PRRs) initiates the first step of defense (Janeway and Medzhitov, 2002). These PRRs are important for non-self recognition to sense specific and conserved molecules called pathogen-associated molecular patterns (PAMPs), such as peptidoglycan (PGN) of Gram-positive bacteria and lipopolysaccharide (LPS) of Gram-negative bacteria. Although important advances have been made in understanding invertebrate innate immunity in recent years, there is still no comprehensive view of the immune mechanisms in invertebrate phyla (Akira et al., 2006).
In invertebrates, fibrinogen-related proteins (FREPs) act as PRRs to participate particularly in innate immunity (Hanington and Zhang, 2011). FREPs have been found in several animal species (Dai et al., 2017; Zhang et al., 2009). Their functions in invertebrates include participation in the defense process, such as pathogen recognition and bacterial defense. In the Mediterranean mussel (Mytilus galloprovincialis), expression of FREPs increased after bacterial infection or PAMP treatment, and they exhibited opsonic activities similar to mammalian ficolins (Romero et al., 2011). Furthermore, recombinant ficolin-like proteins of the freshwater crayfish Pacifastacus leniusculus helped the organism clear Gram-negative bacteria injected into the hemolymph (Wu et al., 2011).
The razor clam (Sinonovacula constricta), a member of the Solenidae family of bivalve molluscs, is widely distributed in muddy intertidal zones along the coasts of the western Pacific Ocean. Considering its relatively short production cycle and high production efficiency, the razor clam has become one of the most valuable economic species for bivalve fisheries in China. However, pathogenic diseases frequently occur, leading to reduced harvest yields of this clam. Thus, it is necessary to gain deeper insight into the molecular mechanisms underlying innate immunity in S. constricta. In this study, we cloned and characterized two FREPs from S. constricta and provided the first evidence of their potential roles in immune defense. The results of this study provided clues about the role of this kind of PRR in the molluscan immune response.
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Experimental samples
Adult razor clams were obtained from Donghang Farm, Sanmen County, Zhejiang Province, China. The average body weight and length of adult clams were 6.0 ± 0.4 g and 4.0 ± 0.4 cm, respectively. Before testing, clams were temporarily cultured in 20‰ salinity water under laboratory conditions.
To assess tissue expression of ScFREPs, samples were collected from seven different tissues (hemolymph, mantle, liver, foot, gill, gonad, and siphon) and then stored at −80 °C. Samples were used for total RNA
Characterization of the ScFREP sequences
The length of ScFREP-1 cDNA was 2065 nucleotides, with an 825 base pair (bp) ORF, a 361 bp 5′-untranslated region (UTR), and an 879 bp 3′-UTR (Genebank No: MW417224). The full length of ScFREP-2 cDNA was 1675 bp, and it contained an 882 bp ORF, a 316 bp 5′-UTR, and a 477 bp 3′-UTR (Genebank No: MW417225). ScFREP-1 had only one fibrinogen-related domain (residues 61–274), while ScFREP-2 contained a signal peptide (residues 1–18) and a fibrinogen-related domain (residues 81–265). The theoretical
Discussion
FREPs are a family of glycoproteins that contain fibrinogen-like domains in the C-terminal portion, but their N-terminal region is different. This family includes a variety of proteins, such as ficolins, tenascins, angiopoietins, and ixoderins (Gorbushin et al., 2010; Zhang et al., 2001). As a member of the complement system, ficolins function in recognition of the sugars presented on microorganisms and are responsible for complement activation through the lectin pathway (Garred et al., 2010;
Declaration of competing interest
The authors have no competing interests to declare.
Acknowledgments
This work was supported by a National Key R & D plan “Blue Granary Science and Technology Innovation” special project (2019YFD0900700) and the National Natural Science Foundation of China (31472278).
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