Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus

Highlights

• Full-length cDNA of cathepsin B gene were firstly identified in Apostichopus japonicus.

• AjCTSB was ubiquitously expressed in all examined tissues with the larger magnitude in intestine.

• AjCTSB mRNA in coelomocytes could be significantly induced by Vibrio splendidus or LPS.

• Recombinant AjCTSB exhibited dose-dependent CTSB activities.

• Coelomocytes apoptosis was significantly inhibited in vivo after AjCTSB knock-down.

Abstract

Cathepsin B (CTSB), a member of lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and also plays crucial roles in host immune defense against pathogen infection in vertebrates. However, the function of CTSB within the innate immune system of invertebrates, particularly in marine echinoderms, has been poorly documented. In this study, the immune function of CTSB in Apostichopus japonicus (designated as AjCTSB), a commercially important and disease vulnerable aquaculture specie, was investigated by integrated molecular and protein approaches. A 2153 bp cDNA representing the full-length of AjCTSB was cloned via overlapping ESTs and RACE fragments. AjCTSB contained an open reading frame of 999 bp encoding a secreted protein of 332 amino acid residues with a predicted molecular mass of 36.8 kDa. The deduced amino acid of AjCTSB shared a typical activity center containing three conserved amino acid residues (Cys¹⁰⁸, His²⁷⁷ and Asn²⁹⁷). Phylogenetic tree analysis also supported that AjCTSB was a new member of CTSB family with clustering firstly with invertebrate CTSBs. Quantitative real time PCR analysis revealed that AjCTSB was ubiquitously expressed in all examined tissues with the highest levels in intestine. The Vibrio splendidus challenged sea cucumber and LPS-exposed coelomocytes could both significantly boost the expression of AjCTSB. Moreover, the purified recombinant AjCTSB exhibited dose-dependent CTSB activities at the concentration ranged from 0 to 0.24 μg μL⁻¹. Further functional analysis indicated that coelomocytes apoptosis was significantly inhibited by 0.16-fold in vivo and the apoptosis execution Ajcaspase 3 was extremely reduced in Apostichopus japonicus coelomocytes treated with specific AjCTSB siRNA. Collectively, all these results suggested that AjCTSB was an important immune factor and might be served as apoptosis enhancers in pathogen challenged sea cucumber.

Introduction

As a group of hydrolytic enzymes, lysosomal proteases were found ubiquitously expressed in most vertebrate and invertebrate species, which has major functions including protein degradation, antigen processing, protein precursor processing, and cell apoptotic processing [1]. Cathepsins, a group of intracellular hydrolytic proteases, were a class of lysosomal proteases with endopeptidases activities [2]. To date, three major types of cathepsins have been identified based on their targeting amino acid residues in their active sites, including serine protease (cathepsin A and G), aspartic protease (cathepsin D and E) and cysteine protease (cathepsin B, C, F, H, K, L, O, S, W and Z) [3]. Their functions have been intensively investigated in cathepsin-deficient mice [4], [5], [6]. One of the notable functions of cathepsins is the involvement in multiple immune response pathways [7], [8], [9].

As one of the major types of cathepsins, cyteine proteases, which are members of the papain family and belong to the C1 peptidase family, are ubiquitously produced in almost all organisms and responsible for intracellular and extracellular protein degradation and turnover via catalyzing protein hydrolysis [10], [11]. In addition, they are also be acting important regulators and signaling molecules in different levels of biological processes, such as antigen processing, hormone activation and inflammatory responses [12], [13]. Different than other cysteine proteases, cathepsin B (CTSB) was found to have double enzymatic activities of exopeptidase and endopeptidase [14]. In structure, CTSB is composed of two spherical domains with a cysteine residue C²⁵ and a histidine residue H¹⁵⁹ [15]. It exhibits peptidyl-dipeptidase, carboxypeptidase or endopeptidase activities depending on pH as well as substrate, such as the exopeptidase activity function mainly through two sequential histidines within the characteristic “occluding loop” at the “rear” of the cleft of substrate-binding pocket [16]. CTSB has been found to participate in cellular process during many disorder and diseases, such as immunological disorders [17], inflammatory response [18], apoptosis [19], Alzheimer's disease [20] and cancer [21]. Originally identified from rat [22], CTSB has been found and studied in a number of vertebrate and invertebrate species. All the previous studies on CTSB suggested that this molecule is structurally and functionally conserved across species [23], [24], [25]. Recently, increasing efforts were made in understanding the role of CTSB in the host defense systems of aquatic species, including orange-spotted grouper with a stimulation of Singapore grouper iridovirus (SGIV) [26],and UV-inactivated grass carp hemorrhage virus and LPS challenged flounder [27]. Despite the good amount of work on CTSB, actual functions of CTSB within the innate immune system of invertebrates remain largely unknown.

Considering the conservation of CTSB in the evolution, we are particularly interested in understanding the role of CTSB in echinoderma species since the immune systems of vertebrates and echinoderma species may be developed from common ancestor of the deuterostomes (PMID:10426433). Here we use sea cucumber A. japonicus as a model to investigate the role played by CTSB in echinoderm innate immunity. It is also noteworthy that A. japonicus is also an economically important aquaculture species in China with an annual production of around 200,969 tons in 2015 [28]. Recent outbreaks of infectious diseases, such as skin ulceration syndrome (SUS) severely impacted the production of A. japonicus [29]. Vibrio splendidus was believed to be the major cause of A. japonicus SUS [30]. And infection of V. spendidus leads to anorexia, shaking head and mouth tumidity at the early stage, flowed by tissue atrophy and skin ulceration in A. japonicus [31]. Degradation of infected tissues in organisms is primarily achieved by proteases as a self-protection mechanism to prevent the healthy tissue from further infections. CTSB has demonstrated the abilities in many organisms to cleave the basement membrane and degrade extracellular matrix proteins, such as collagen IV, fibronectin, elastin, etc [32], [33], [34]. However, the connection between SUS and CTSB activation in A. japonicus has not been studied. Here, we fully characterized the DNA and protein sequences of CTSB in A. japonicus (designated as AjCTSB and rAjCTSB). The spatial and time-course expression profiles of AjCTSB along the progress of V. splendidus challenge and LPS exposed were also tested in vivo and in vitro with A. japonicus individuals and coelomocytes primary culture model, respectively. Functional analyses of AjCTSB showed a close relationship between AjCTSB and cell apoptosis in A. japonicus. These findings highlighted the positive roles of AjCTSB in echinoderm host defense system and provided an important reference in the study of lysosomal proteases along the evolution of organisms. The fully characterized AjCTSB gene can be also used as a potential target in the development of therapeutic strategy for SUS in production of A. japonicus.