Cathepsin C (CTSC) contributes to the antibacterial immunity in golden pompano (Trachinotus ovatus)

Highlights

• Cathepsin C (TroCTSC) was identified and characterized from golden pompano (Trachinotus ovatus), and had hydrolase activity.

• TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome.

• The three conserved sites (Cys²⁵¹, His³⁹⁷, and Asn⁴¹⁹) were essential for hydrolase activity of TroCTSC.

• TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

Abstract

Cathepsins, as a class of protein hydrolases, are widely found in the lysosomes of many tissues and play an essential role in various physiological activities. Cathepsin C (CTSC), a lysosomal cysteine protease, is an essential component of the lysosomal hydrolase family. In this study, we identified a CTSC from Trachinotus ovatus (TroCTSC) and analyzed its function. TroCTSC contained an ORF of 1368 bp and encoded 455 amino acids, which included three conserved catalytically active sites (Cys²⁵¹, His³⁹⁷, and Asn⁴¹⁹). It shares high homology (69.47%–90.77%) with the other known CTSC sequences of teleosts, which was most closely related to Seriola dumerili. TroCTSC was most abundant in the muscle, liver, and head kidney. After Vibrio harveyi infection, the expression levels of TroCTSC in liver, spleen, and head kidney were significantly up-regulated. TroCTSC was found in the cytoplasm with some of which were co-located with the lysosome. After V. harveyi stimulation, TroCTSC was translocated to nucleus in golden pompano snout (GPS) cells. In vitro, results revealed that the optimal hydrolase activity of the recombinant protein, rTroCTSC, was at 40 °C and pH 5.5. The activity of rTroCTSC was promoted by Zn²⁺ and Ca²⁺ but inhibited by Fe²⁺ and Cu²⁺. However, three mutant proteins, rTroCTSC-C251A, rTroCTSC-H397A, rTroCTSC-N419A, were dramatically reduced the proteolytic activity. Furthermore, in vivo results showed that overexpression of TroCTSC could significantly enhance body's ability to resist V. harveyi and promote the expression of proinflammatory cytokines, including interleukin 1-beta (IL-1β), IL-6, IL-8, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). In contrast, the interference of TroCTSC expression induced a significant increase in the number of bacteria after V. harveyi infection. Our results suggested that TroCTSC was an essential effector of the innate immune system and played a pivotal role in antibacterial immunity.

Introduction

Cathepsins are a series of protein hydrolase mainly located in the lysosome, widely present in the animal and plant kingdoms, and have also been identified in viruses and bacteria, and play important roles in the physiological and pathological processes in various tissues of the organism [1,2]. Based on the amino acid residues of their active sites, cathepsins can be divided into three classes: serine proteases (cathepsin A and G), cysteine proteases (cathepsin B, C, F, H, K, L, O, S, T, U, V, W, and X), and aspartic proteases (cathepsin D and E) [3]. In addition to the key role of lysosomal cathepsins in autophagy, it is also involved in antigen processing and apoptosis [[4], [5], [6]].

Cathepsin C (CTSC), also known as dipeptidyl peptidase I (DPPI), is a lysosomal cysteine proteinase belonging to the papain superfamily [7]. In mammals, CTSC was expressed in a variety of inflammatory cells, including cytotoxic T cells (CTL cells), natural killer cells (NK cells), and epithelial cells [8,9]. In addition, CTSC deficiency in mammals causes autoimmune diseases, such as Papillon-Lefèvre syndrome (PLS), Halm-Munk syndrome (HMS), periapical periodontitis, and so on [[10], [11], [12], [13]]. On the other hand, excessive activity is related to different inflammatory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis [14]. As a multifunctional cysteine protease, CTSC has gradually become a research hotspot, which is a possible target for the treatment of immune disorders.

In recent years, CTSC had also been studied in aquatic animals and showed important roles in antibacterial and antiviral immune responses. For example, in Sinonovacula constricta and Eriocheir sinensis, the expression of CTSC was up-regulated in response to Vibrio. anguillarum stimulation [15,16]. And similarly, CTSC expression rose in Penaeus monodon after challenged with lipopolysaccharide (LPS) [17]. The expression of CTSC in F. chinensis and Epinephelus coioides was significantly increased after white spot syndrome virus (WSSV) and Singapore grouper iridovirus (SGIV) stimulation, respectively [18,19]. But the antibacterial immunity induced by CTSC of fish was not clearly so far.

Golden pompano (Trachinotus ovatus), belonging to the Carangidae family, is a popular marine fish, which has high economic value in the south sea of China [20,21]. With the large-scale development of intensive aquaculture, the environment is deteriorating and bacterial diseases are frequent, especially V. harveyi, causes serious losses in the golden pompano industry [22,23]. To prevent pathogenic infections, studies on the immune defense mechanisms of golden pompano have received more focus during these years [24,25]. In this study, a homolog of the CTSC gene from golden pompano, TroCTSC, was identified. The aim of our study was to analyze the enzyme activity and immune effects of TroCTSC through in vivo and in vitro assays. Our study enhanced the recognition of the biological activity of CTSC, and provided a valuable reference for the disease resistance of golden pompano.