A hemocyte-specific cathepsin L-like cysteine protease is involved in response to 20-hydroxyecdysone and microbial pathogens stimulation in silkworm, Bombyx mori
Graphical abstract
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).
References (48)
- et al.
The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut
Insect Biochem. Mol. Biol.
(2012) - et al.
Characterization of the immune roles of cathepsin L in turbot (Scophthalmus maximus L.) mucosal immunity
Fish Shellfish Immunol.
(2020) - et al.
A role of cathepsin L gene in innate immune response of crayfish (Procambarus clarkii)
Fish Shellfish Immunol.
(2017) - et al.
Fibroinase and its physiological inhibitors involved in the regulation of silk gland development in the silkworm, Bombyx mori
Insect Biochem. Mol. Biol.
(2019) - et al.
The crustacean ecdysone cassette: a gatekeeper for molt and metamorphosis
J. Steroid Biochem. Mol. Biol.
(2019) - et al.
Expression of matrix metalloproteinase genes during basement membrane degradation in the metamorphosis of Bombyx mori
Gene.
(2018) - et al.
Expression profile of cathepsin B in the fat body of Bombyx mori during metamorphosis
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
(2009) - et al.
DmCatD, a cathepsin D-like peptidase of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae): purification, bioinformatic analyses and the significance of its interaction with lipophorin in the internalization by developing oocytes
J. Insect Physiol.
(2018) - et al.
Nitric oxide plays a crucial role in midgut immunity under microsporidian infection in Antheraea pernyi
Mol. Immunol.
(2020) - et al.
Angiotensin II-Induced vascular remodeling and hypertension involves cathepsin L/V- MEK/ERK mediated mechanism
Int. J. Cardiol.
(2020)
Cathepsin B: a sellsword of cancer progression
Cancer Lett.
Age- and morph-dependent activation of the lysosomal system and Buchnera degradation in aphid endosymbiosis
J. Insect Physiol.
Early intra-acinar events in pathogenesis of pancreatitis
Gastroenterology.
Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai) - Purification and characterization
Fish Shellfish Immunol.
Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori
Arch. Biochem. Biophys.
Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi
J. Invertebr. Pathol.
An update on ecdysone signaling during insect oogenesis
Curr. Opin. Insect Sci.
Lysosomal cathepsins: structure, role in antigen processing and presentation, and cancer
Adv. Enzyme Regul.
Cysteine cathepsins: from structure, function and regulation to new frontiers
Biochim. Biophys. Acta
Cathepsin-L is involved in degradation of fat body and programmed cell death in Bombyx mori
Gene.
Cathepsin-L is involved in degradation of fat body and programmed cell death in Bombyx mori
Gene.
Identification of ecdysone response elements (EcREs) in the Bombyx mori cathepsin D promoter
Biochem. Biophys. Res. Commun.
A novel granulocyte-specific alpha integrin is essential for cellular immunity in the silkworm Bombyx mori
J. Insect Physiol.
Molecular cloning, characterization and expression analysis of cathepsin O in silkworm Bombyx mori related to bacterial response
Mol. Immunol.
Cited by (11)
GATA binding protein 6 regulates apoptosis in silkworms through interaction with poly (ADP-ribose) polymerase
2024, International Journal of Biological MacromoleculesScavenger receptor C regulates antimicrobial peptide expression by activating toll signaling in silkworm, Bombyx mori
2021, International Journal of Biological MacromoleculesCitation Excerpt :Cellular immunity is primarily mediated by hemocytes, including phagocytosis, encapsulation, and node formation [1,2]. Humoral immunity mainly includes soluble effector molecules, such as lectins, prophenoloxidase, cathepsin [3,4], and antimicrobial peptides [5,6]. During pathogen invasion, the pattern-recognition receptors (PRRs) recognize the pathogen-associated molecular patterns (PAMPs)—conserved molecular structures present in pathogenic microorganisms.
Lineage-specific gene evolution of innate immunity in Bombyx mori to adapt to challenge by pathogens, especially entomopathogenic fungi
2021, Developmental and Comparative ImmunologyCitation Excerpt :Lepidoptera has life histories that generally differ from species of Diptera, Hymenoptera and Coleoptera (Xia et al., 2004; Dong et al., 2019). The silkworm (B. mori) is a model species of Lepidoptera, in which twenty-one gene families and 220 genes are involved in the innate immune system (Xia et al., 2009; Wang et al., 2021; Pan et al., 2021). Genes encoding pattern-recognition receptors (PRRs) and antimicrobial peptides (AMPs) show significant specificity and have multiple copies in the silkworm genome (Tanaka et al., 2008; Chen and Lu, 2018).
Group V Chitin Deacetylases Influence the Structure and Composition of the Midgut of Beet Armyworm, Spodoptera exigua
2023, International Journal of Molecular Sciences