Characterization and functional analysis of a clip domain serine protease (MncSP) and its alternative transcript (MncSP-isoform) from Macrobrachium nipponense

Highlights

• MncSP and MncSP-isoform were identified from M. nipponense by exon jumping.

• The expressions of MncSP and MncSP-isoform were increased after bacterial invasion.

• Silencing MncSP inhibited the up-regulation of MncSP-isoform caused by bacteria.

• Silencing MncSP significantly reduced the synthesis of some AMPs.

• Silencing of MncSP leads to a significant decrease in PO activity.

Abstract

Clip domain serine protease (cSPs) play an important role in the innate immune defense of crustaceans. In this study, a clip domain serine protease (MncSP) and its alternative transcript (MncSP-isoform) were identified from Macrobrachium nipponense. The full-length cDNA sequences of MncSP and MncSP-isoform were 2447 and 2351 bp with open reading frames comprising 1497 and 1401 bp nucleotides and encoding 498 and 466 amino acids, respectively. The genome of MncSP had 10 exons and 9 introns. MncSP contained all 10 exons, whereas MncSP-isoform lacked the second exon. MncSP and MncSP-isoform contained a signal peptide, a clip domain, and a Tryp_SPc domain. Phylogenetic tree analysis showed that MncSP and MncSP-isoform clustered with cSPs from Palaemonidae. MncSP and MncSP-isoform were widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine. The expression profiles of MncSP and MncSP-isoform in the hemocytes of M. nipponense changed after simulation by Vibrio parahaemolyticus or Staphylococcus aureus. The RNAi of MncSP could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. Phenoloxidase activity was also down-regulated in MncSP-silenced prawns. This study indicated that MncSP participated in the synthesis of AMPs and the activation of prophenoloxidase.

Introduction

The farming of freshwater prawns, including Macrobrachium nipponense, occupies a very important position in the global aquaculture industry. However, diseases frequently occur in prawn farms with the deterioration of the water environment and the invasion of a variety of pathogenic microorganisms, thus causing serious losses to the aquaculture industry. In contrast to vertebrates, invertebrates lack specific immunity. Crustaceans, such as shrimp and prawns, rely on the innate immune system to recognize and destroy microorganisms through cellular and humoral immune responses to protect themselves from harm (Bonami and Sri Widada, 2011; Chen and He, 2019; Rolff and Siva-Jothy, 2003; Vazquez et al., 2009). Cellular immunity, including phagocytosis and nodule formation, is used to resist bacterial invasion, and humoral immunity is mainly exerted through antimicrobial peptide (AMP) synthesis, the blood coagulation system, and melanization (Iwanaga and Lee, 2005; Jiravanichpaisal et al., 2006).

The prophenoloxidase activating system, which is similar to the complement system, was first proposed in 1982 (Söderhäll, 1982). Phenoloxidase (PO) is found in Pacifastacus leniusculus and can be cleaved by trypsin-like enzymes (Aspán et al., 1995). Later studies found that PO is involved in immune defense against Aeromonas hydrophila in P. leniusculus (Liu et al., 2007). In insects, the PO-catalyzed melanization reaction and the Spätzle-triggered Toll signal pathway are important innate immune responses for resisting bacterial challenges (Wang et al., 2020). The activation of prophenoloxidase (proPO) or Spätzle needs the participation of a series of serine protease (SP) cascades (Jiang and Kanost, 2000). SP cascades drive a variety of important biological processes, including embryonic development and immune response (Krem and Di Cera, 2002). Many of these SP cascades are completed by clip domain SPs (cSPs). The Spätzle-processing enzyme (SPE) is a cSP and is necessary for the activation of Spätzle (Jang et al., 2006). The proPO-activating proteinase (PAP), also known as the proPO-activating enzyme (PPAE), identified in Ostrinia furnacalis is also a cSP and is necessary for the activation of proPO (Feng et al., 2018). The cSP family comprises numerous members, and identifying unknown members of this family and investigating their biological function are greatly important.

The cSPs are important members of the SP family (Page and Di Cera, 2008). The first cSP was identified in Drosophila melanogaster and shown to be involved in dorso–ventral development (Anderson, 1998). The structure of cSP family members usually contains a regulatory clip domain at the N-terminal and a catalytic Tryp_SPc domain at the C-terminal (Jiang and Kanost, 2000; Piao et al., 2005). The Tryp_SPc domain contains a conserved catalytic triad structure that is usually composed of histidine, aspartic acid, and serine (Blow et al., 1969; Hedstrom, 2002). The cSP family plays an important role in arthropod hemolymph coagulation, AMP synthesis, cell adhesion, melanization, and other immune responses (Gorman and Paskewitz, 2001). cSPs have been found to participate in the immune response in insects. Hemolymph proteinase 6 (HP6) with a clip domain is involved in two immune pathways in Manduca sexta. In one pathway, HP6 can activate PAP1, leading to proPO activation and melanin synthesis; in another pathway, HP6 can activate HP8 and finally induce the Toll pathway to produce AMPs (An et al., 2009a). Several cSPs in Drosophila have been shown to participate in the Toll pathway to produce AMPs to resist pathogens (Jang et al., 2008). In Tenebrio molitor, SPE can also activate the proPO and Toll pathways simultaneously to kill bacteria (Kan et al., 2008). In crustaceans, such as Portunus trituberculatus (Cui et al., 2010), Limulus (Muta et al., 1990), Penaeus monodon (Amparyup et al., 2010; Charoensapsri et al., 2009; Sriphaijit et al., 2007), and Litopenaeus vannamei (In-Kwon et al., 2011), several cSPs have been identified as immune-related molecules. PPAE, as a cSP, has been found in P. leniusculus, and its clip domain may serve as an AMP (Wang et al., 2001). A clip-domain SP from P. leniusculus can interact with envelope protein VP28 of the white spot syndrome virus (Guo et al., 2017). PPAE-III identified from Macrobrachium rosenbergii plays an important role in immune defense (Arockiaraj et al., 2011). However, research on cSP in M. nipponense currently does not exist.

In this study, a clip domain SP gene (MncSP) and its alternative transcript (MncSP-isoform) were identified in M. nipponense. The expression pattern of MncSP and MncSP-isoform in the hemocytes of prawns after bacterial challenge was investigated. We also explored the effects of MncSP on the synthesis of AMPs and the activation of proPO.