A double chitin catalytic domain-containing chitinase targeted by c-Jun is involved in immune responses in shrimp
Introduction
Chitin, a long-chain homopolymer of β-(1, 4)-N-acetyl-d-glucosamine (GlcNAc), is an essential component of a variety of organic structures in nature, including fish scales, nematode egg shells, cephalopod beaks, mollusca radula, fungal cell walls, worm and arthropod exoskeletons (Bowman and Free, 2006; Moussian et al., 2005; Naleway et al., 2016; Tharanathan and Kittur, 2003). Accumulating evidence has suggested that chitin is involved in regulation of multiple biological processes, especially immune response (Elieh et al., 2018; Koller et al., 2011). For instance, studies in mammals have demonstrated that through binding a series of cell surface receptors, including toll-like receptor 2 (TLR-2), Dectin-1 and macrophage mannose receptor, chitin from fungi, insects and parasites can recruit and activate immune functional cells in a size-dependent way via inducing production of multiple cytokines and chemokines (Da et al., 2008). Chitinases, belonging to the glycosyl hydrolase family, are a group of enzymes that hydrolyze glycosidic bonds in chitin (Arakane and Muthukrishnan, 2010). Chitinases contain a chitin-binding domain and a catalytic domain that serves to degrade chitin (Funkhouser and Aronson, 2007). Besides, there are other kinds of proteins called chitinase-like proteins (CLPs) that can only bind to chitin but have no chitin hydrolytic activity (Adrangi and Faramarzi, 2013; Rathore and Gupta, 2015). Chitinases and CLPs are widely distributed in organisms from bacteria to mammals and play important roles in multiple biological processes of various animals, including molting of arthropods, invading of pathogens into the host, and immunity of invertebrates and vertebrates (Arakane and Muthukrishnan, 2010; Chaudhuri et al., 2010; Pesch et al., 2016).
In crustaceans, chitin constitutes a key component of the peritrophic matrix (PM) and the inner layers of exoskeletons, and chitinases play roles in growth, molting, development and other biological processes (Martin et al., 2006; Zou and Bonvillain, 2004). A recent study showed that a novel chitinase (LvChi5) containing one catalytic domain is important for antiviral and antibacterial responses in Pacific white shrimp, Litopenaeus vannamei, a representative species of the class Crustacea with high economic value (Niu et al., 2018). This suggests that crustacean chitinase could also play a role in immunity. Most chitinases generally possess only one catalytic domain. Recently, a growing number of proteins that contain two chitin catalytic domains have been deposited in Genbank database but their functions have yet to be characterized. In the current study, a novel chitinase (dChi) with double catalytic domains was identified from L. vannamei. The role of dChi in immune responses was explored, which may enrich our knowledge on the function of double catalytic domain-containing chitinases.
Section snippets
Shrimp and pathogens
Pacific white shrimp (~10 g) were obtained from an aquaculture farm in Zhanjiang, Guangdong Province. Shrimp were randomly sampled to ensure free of Vibrio parahaemolyticus and WSSV by PCR following reported methods (Nunan and Lightner, 2011; Wei et al., 2014). Shrimp were acclimated at ~27 °C for at least 7 days in a recirculating water tank system filled with air-pumped seawater (2.0% salinity) before experiments. Preparation of the stocks of V. parahaemolyticus and WSSV was performed as
Sequence analysis of dChi
As Fig. 1A showed, the ORF of dChi is 2997 bp encoding a 998-amino acid protein with a calculated molecular weight of 122.21 kDa and a theoretical isoelectric point of 5.92. The dChi protein (GenBank Accession No. MN944099) contains two putative catalytic domains (Glyco_18) and one chitin-binding domain 2 (ChtB D2). Presence of the signal peptide in the N-terminus suggests that dChi is a secreted protein similar to other members of the chitinase family. A number of chitinases containing two
Discussion
It has long been known that as a common allergen and immunei-activating molecule, chitin plays complex roles in immune and allergic responses by interacting with a variety of immune cell receptors, resulting in production of a set of cytokines and chemokines and recruitment and activation of many immune functional cells (Da et al., 2008). As a group of proteins with activity to hydrolyze or bind chitin, the roles of chitinases and CLPs in immune responses have also been extensively concerned (
Acknowledgements
This work was supported by National Natural Science Foundation of China under grant No. 31772881, 31702371 and 31972823; National Key Research and Development Program of China 2018YFD0900505; Natural Science Foundation of Guangdong Province, China 2017A030313190; China Agriculture Research System CARS47.
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These authors contributed equally to this work.