Identification, functional characterization and the potential role of variable lymphocyte receptor EsVLRA from Eriocheir sinensis in response to secondary challenge after Vibrio parahaemolyticus vaccine

Highlights

• EsVLRA contained the conserved LRR_CT domain with a protruding loop at LRR_CT region.

• The highest transcript level of EsVLRA was detected in hepatopancreas.

• The recombinant EsVLRA exhibited strong microbial-binding activity.

• EsVLRA might be involved in immune priming via its immune priming.

Abstract

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

Introduction

Invertebrates are traditionally thought to solely rely on the innate immune system to resist infections [1]. They do not possess the ‘‘true’’ lymphocytes and any functional antibodies that are the crucial factors in vertebrates' adaptive immune system [2]. However, recent studies indicate that invertebrates have alternative adaptive immune, such as enhanced immune defenses or memory-like responses, which are referred to ‘immune priming’ or ‘specific immune priming’ [[3], [4], [5], [6], [7], [8]]. Research efforts have been made on the molecules that contribute to specific immune recognition and immune priming in invertebrates. A recent discovery suggests that many immune molecules may be involved in this mechanism, such as variable lymphocyte receptors (VLRs) in the jawless fish lampreys (Petromyzon marinus) [9].

VLRs were initially isolated from an enriched cDNA library of sea lampreys stimulated by antigen and mitogen [10]. The VLR germ-line genes usually consist of leader sequence, leucine-rich repeat (LRR), stalk region, incomplete N-terminal and C-terminal LRR subunits (LRRNT and LRRCT) [11]. LRR modules coding for the most abundant module types are located adjacent to the VLR germ-line genes with multiple copies [12]. Germ-line genes undergo the activation-induced cytosine deaminase (AID) mediated gene conversion–like process, which can generate a vast repertoire of immune receptors [[11], [12], [13], [14]]. Three mature VLR isotypes (VLRA, VLRB and VLRC) have been identified in lamprey and hagfish (Eptatretus stoutii) [13,[15], [16], [17]]. They are expressed as membrane-bound proteins and comprise an LRRNT, an LRR1, multiple LRRV modules, a connecting peptide (CP) LRR, an LRRCT and a stalk region [11].

In the lamprey adaptive immune system, VLRs play critical roles in the recognition of antigens [18]. The special recognition of diverse antigens is based on the concave surface, which assembled by the most highly variable LRR modules, along with the diversity in the sequence and length of the highly variable insert modules [9]. Moreover, the crystal structure of a VLR-antigen complex shows that the protruding loop at the base of the concave surface, which is coded by variable inserts in the C-terminal cap, also contributes directly to antigen binding [[18], [19], [20]]. It is reported that both VLRAs and VLRBs exhibit a long protrusion in the LRRCT region, but the lengths of LRRCT inserts in VLRC are much shorter than those in VLRAs and VLRBs. Thus VLRAs and VLRBs might possess a different antigen-binding way from VLRCs [16,21].

The Chinese mitten crab Eriocheir sinensis, (Crustacea: Decapoda: Grapsidae, Eriocheir) (Henri Milne Edwards, 1854), is very significant in evolutionary research and also a commercially important species in aquaculture. With the expansion of crab farming, E. sinensis often suffers various diseases caused by bacteria and viruses resulting in decreased production and enormous economic loss for E. sinensis [[22], [23], [24]]. Understanding the innate immunity may help in the discovery of new strategies for controlling the diseases in crab culture. Here, we reported the cDNA cloning, characterization and mRNA tissue-dependent expression pattern of a VLRA (EsVLRA) from E. sinensis. The functions of the recombinant EsVLRA protein were investigated by antimicrobial activity and binding activity. In addition, in order to know the possible involvement of EsVLRA in immune priming, the expression level of EsVLRA was detected after the “V. parahaemolyticus vaccine” stimulated. The obtained results suggest that EsVLRA is an important immune molecule and may play a role in immune priming.