Elsevier

Molecular Immunology

Volume 131, March 2021, Pages 78-88
Molecular Immunology

A hemocyte-specific cathepsin L-like cysteine protease is involved in response to 20-hydroxyecdysone and microbial pathogens stimulation in silkworm, Bombyx mori

https://doi.org/10.1016/j.molimm.2020.12.013Get rights and content

Highlights

  • A novel cathepsin L-like was cloned and identified in silkworm.

  • Cat L-like is highly and specifically expressed in hemocytes.

  • Cat L-like could be induced by 20-E.

  • Expression of Cat L-like as up-regulated during bacterial infection.

Abstract

Cathepsin L protease belongs to the papain-like cysteine proteases family, plays indispensable roles in animals' pathological and physiological processes. However, little is known about Cathepsin L in silkworm, Bombyx mori. Herein, a novel Cathepsin L-like (Cat L-like) was cloned and identified from silkworm by the rapid amplification of cDNA ends (RACE). Cat L-like contains an intact open reading frame (ORF) of 1 668 bp and encodes 556 amino acid residues, consisting of a signal peptide, typical cathepsins’ inhibitor_I29, and pept_C1 domain. Cat L-like is specifically and highly expressed in hemocytes. The cathepsin (including Cathepsin L, B, and H) crude extract from hemocytes had typical substrate specific catalytic activities and were sensitive to pH and temperature. Cat L-like up-regulated considerably after 20-hydroxyecdysone (20-E) administration, indicating that Cat L-like may be regulated by insect hormone. The responses of Cat L-like against bacterial infection suggest it may play essential roles in silkworm immunity. Overall, our studies provide a theoretical basis and insights to further investigate the functions of Cat L-like and in insects' innate immunity mechanisms.

Introduction

Cathepsins are lysosomal proteolytic enzymes belong to the cysteine proteases family. So far, more than 20 cathepsin members have been discovered, including cathepsin A, B, C, D, E, F, G, H, K, L, O, S, V, W and X (Turk et al., 2012; Yang et al., 2020a). According to the classification criteria of proteases, most cathepsins are cysteine proteases (Like Cathepsin B, C, F, H, K, L, O, S, V, X and W), which contain an active-site cysteine residue, a few of cathepsins are aspartic proteases (Like Cathepsin D and E) or serine proteases (Like Cathepsin A and G) (Rawlings et al., 2008; Zhang et al., 2015a). Cathepsins were originally viewed as a proteases, but recently have been considered the most important group within the papain superfamily for their functional roles in intracellular protein degradation (McDonald et al., 2020). Many studies suggested that cathepsin is closely involved in a variety of biological and pathologic processes, such as proenzyme activation (Haas et al., 1989), antigen presentation (Turk et al., 2002), tissue remodeling (Lu et al., 2020), blood glucose regulation, osteoporosis (Bromme et al., 2016; Drake et al., 2017), cancer (Mijanovic et al., 2019; Olson and Joyce, 2015), inflammatory response (Ni et al., 2019; Saluja et al., 2019) and immune response (Chisolm et al., 2019).

Cathepsin is also widely found in insects and associated with different functions. Cathepsin O is involved in innate immune response and metamorphosis in Antheraea pernyi (Sun et al., 2017). Cathepsin B from Sarcophaga peregrine is highly expressed in hemocytes during metamorphosis, and participates in the fat body dissociation (Yano et al., 1995b); In Helicoverpa armigera, Cathepsin L expressed by hemocytes play roles in fat body degradation (Zhai and Zhao, 2012). Cathepsin D from Dipetalogaster maxima is synthesized by fat body and ovary and functions as a yolk protein precursor (Leyria et al., 2018). These studies suggest that cathepsin is involved in growth, development, metamorphosis and metabolism of insects (Yang et al., 2020a), whereas, the underlying regulation mechanisms need to be further explored.

Several members of cathepsin have been identified and their functions were investigated in silkworm. Cathepsin B is reported to be associated with the programmed cell death (PCD) of the fat body cells, and both Cathepsin B and D contribute to metamorphosis (Lee et al., 2009). Cathepsin D expression could be induced by 20-E and other stimulations such as high temperature and H2O2 (Yu et al., 2012). Cathepsin O is specifically presented in hemocytes and also can be induced by 20-E, further studies suggested that it may be associated with innate immune in response to pathogens stimulation (Zhang et al., 2015a). Furthermore, several Cathepsin L homologues have been identified and characterized in silkworm. Yang and his colleagues reported that a homologue of Cathepsin L is involved in the degradation of the fat bodies through regulating the PCD process (Yang et al., 2020a). Both Cathepsin L-like 1 and 2 are involved in the process of fat body destruction (Guo et al., 2018).

It is well recognized that insect hemocytes regulate innate immunity via the phagocytosis of pathogens in insects (Zhai and Zhao, 2012), implying its potential roles in innate immunity. In our previous study, a novel hemocyte-specific Cathepsin L-like (Cat L-like) gene was identified through RNA-sequencing, however, its genetic information and potential functions remain unclear. Here, the full-length cDNA of a novel Cat L-like was cloned, and its temporal and spatial expression profiles were further investigated. The recombinant protein was also obtained through prokaryotic expression system and purified by Ni-affinity chromatography. The enzyme activity characteristics of Cat L-like were also detected and evaluated. Our results provide a theoretical basis and novel insights for further studying the functions of cathepsin in insects.

Section snippets

Experimental animals

The B.mori strain Dazao was maintained in the Silkworm Gene Bank of Southwest University, Chongqing, China (Li et al., 2020). The silkworms were reared in light and darkness for 12 h one day, respectively. All experimental animals were fed with fresh mulberry at 25–27 ℃, 60–80 % relative humidity. The silkworms were randomly selected for experiments in this study. The different tissues were extracted in DEPC-treated PBS and then stored at -80 ℃ after being flash-frozen.

RNA isolation and cDNA synthesis

Total RNA was extracted

Cloning and characterization of Cat L-like in silkworm

Gene expression profiles of different hematopoietic lineages in silkworm were analyzed by RNA-seq (Data were not shown). We found Cat L-like, a member of papain family in cysteine protease, was highly expressed in hemocytes. Cat L-like is clustered on nscaf 2860, located on chromosome 10, and the gene number in SilkDB is BGIBMGA006893. The full-length cDNA of Cat L-like contains an intact open reading frame (ORF) of 1 668 bp, a 5′-UTR with 54 bp and a 3′-UTR with 181 bp (Fig. 1A). Its ORF

Discussion

Affiliated to papain family, cathepsins are ubiquitously presented in almost all organisms including viruses, bacteria, plants, invertebrates and vertebrates (Zhang et al., 2015a). So far, several members belonging to Cathepsin B, D, O, and L have been identified in silkworm (Lee et al., 2009; Wang et al., 2008; Zhang et al., 2015a). In the present study, a novel cDNA of Cat L-like protease from hemocytes was identified and characterized from silkworm, and its sequence feature, evolution

Author statement

We have made substantial to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; and we have drafted the work or revised it carefully for important intellectual content; and we approved the final version to be published; and we agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

All persons who have made

Declaration of Competing Interest

The authors reported no declarations of interest.

Acknowledgements

The work was supported by National Natural Science Foundation of China (No. 31802142, and 31672496), China Postdoctoral Science Foundation grant (2017M620408, 2019T120801), the Fundamental Research Funds for Central Universities (XDJK2019C089), Natural Science Foundation of Chongqing (cstc2019jcyjzdxmX0033, cstc2019jcyj-bshX0020 and cstc2019jcyj-bshX0009), and Graduate Research Innovation Project of Chongqing (CYS18124 and CYB18105).

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