Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

Fengli Zhang,Juan Hu,Yalin Yang,Zhigang Zhou,Rui Xia,Zhen Zhang,Chenchen Gao,Chao Ran,Yuanyuan Yao

doi:10.1186/s13568-020-01052-0

Fengli Zhang, Juan Hu + Show 7 more

Open Access

https://doi.org/10.1186/s13568-020-01052-0

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Abstract

Thelohanellus kitauei, is a member of obligate parasitic myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (− 13 KJ/mol) with cathepsin L whereas a higher binding energy (− 8.6 KJ/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-T. kitauei vaccine in carp culture in the future.

Highlights

Myxozoans, a group of obligate parasitic metazoans that are important for pathogenic effects on freshwater and marine fish, were reported in various countries of the world (Zhao et al 2016)
Cysteine protease inhibitors affected the connection between antigenic peptides and major histocompatibility complex class instability index (II) (MHC-II) in the host by inhibiting the corresponding protease, which prevented the host from presenting the parasite antigen (Turk et al 2001; Vray et al 2002; Wang et al 2017)
One potential N-glycosylation site (N68VKV71), one Casein kinase II phosphorylation site (S42FKD45) and three protein kinase C phosphorylation site (S42FK44、S52FK 54、T91AR93) in TK-cystatin were predicted by NetNGlyc 1.0 and PROSITE (Fig. 2b)

Summary

Introduction

A group of obligate parasitic metazoans that are important for pathogenic effects on freshwater and marine fish, were reported in various countries of the world (Zhao et al 2016). Known as cysteine protease inhibitor, was able to regulate many biological processes, and could suppress the host’s immune response to facilitate pathogen’s survival. OmC2 derived from soft tick could suppress the host’s adaptive immune response by reducing inflammatory cytokines and proliferation of antigen presenting cell (Salát et al 2010). Onchocystatin could significantly induce the production of anti-inflammatory cytokine IL-10, but could inhibit the synthesis of proinflammatory Th1-type cytokines and the differentiation and proliferation of regulatory Th1 cells, there by reducing the host’s immune killing effect on parasites (Schönemeyer et al 2001). Whether the structural features of a secretory cystatin (named as TK-cystatin, GenBank accession number: KII69890.1) in T. kitauei with high-expression level in the myxospore stage were similar to these inhibitors, and whether it could inhibit host cysteine proteases and plays similar roles?

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