Parasitology Research

, Volume 118, Issue 5, pp 1581–1592 | Cite as

Cathepsin L—a novel cysteine protease from Haemaphysalis flava Neumann, 1897

  • Yali Sun
  • Lan HeEmail author
  • Long Yu
  • Jiaying Guo
  • Zheng Nie
  • Qin Liu
  • Junlong ZhaoEmail author
Protozoology - Original Paper


Ixodid ticks are ectoparasites responsible for the transmission of a large number of bacterial, viral, and protozoan pathogens to animals and humans. As long-term blood-pool feeders, the digestion of host blood is critical to their development as well as to the establishment of the sexual cycle of hemoparasites such as Babesia parasites, the agents of human and animal babesiosis. Previous studies have demonstrated that cysteine proteases are involved in blood digestion, embryogenesis, and pathogen transmission in other species of ticks, but their characteristics and functions are still unidentified in Haemaphysalis flava. Here, we describe the characterization of a cysteine protease HfCL from H. flava. We show that HfCL belongs to the L-like papain family of proteases, exhibits high expression in nymphs and adults, and localizes to both the midgut and salivary glands. Biochemical assays using purified recombinant enzyme reveal that rHfCL can hydrolyze the fluorogenic substrate Z-phe-Arg-MCA with optimal activity detected at pH 6. Furthermore, the short-term growth assay indicates that rHfCL can inhibit the intraerythrocytic development of Babesia microti and Babesia gibsoni in vitro.


Cathepsin L Papain Haemaphysalis flava Babesia microti Babesia gibsoni 


H. flava

Haemaphysalis flava

B. microti

Babesia microti

B. gibsoni

Babesia gibsoni


Hf-cathepsin L


recombinant Hf-cathepsin


open reading frame


amino acid




tris base-boric acid-EDTA


three-dimensional structure


rapid-amplification of cDNA ends


coding sequence


quantitative real-time PCR


polymerase chain reaction




bull serum albumin


sodium dodecyl sulfate polyacrylamide gel electrophoresis


phosphate buffered solution


4′ 6-diamidino-2-phenylindole


enhanced chemiluminescent


tris buffer saline Tween 20


red blood cells


nitrocellulose membrane


citrate–sodium phosphate


l-trans-epoxysuccinyl-leucylamido 4-guanidino butane


optical density


indirect immunofluorescence assay



We thank all the research team members in our lab for assistance. The authors would like to thank Professor Choukri Ben Mamoun (Yale University, USA) for revising manuscript.

Author contributions

YS performed the study and wrote the manuscript. JZ and LH conceived the study. ZN, JG, and QL collected samples. LH critically revised the manuscript. YS and LY performed the experiments and data analyses. All authors have read and approved the final manuscript.


This study was supported by the National Basic Science Research Program (973 program) of China (Grant No. 2015CB150300), the National Key Research and Development Program of China (Grant No. 2017YFD0501201), the National Natural Science Foundation of China (Grant No. 31772729), and the Natural Science Foundation of Hubei Province (Grant No. 2017CFA020).

Compliance with ethical standards

Ethics approval and consent to participate

The care and maintenance of experimental rabbit, mice, and dog in this study was approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University, and all experiments were performed according to the regulations for the administration of affairs concerning experimental animals of Hubei Province, P.R. China consent for publication.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Data availability

HfCL mRNA sequence was available in GenBank (accession no. MG914066).

Supplementary material

436_2019_6271_MOESM1_ESM.docx (15 kb)
ESM 1 (DOCX 14 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Agricultural MicrobiologyCollege of Veterinary Medicine Huazhong Agricultural UniversityWuhanChina
  2. 2.Key Laboratory of Animal Epidemical Disease and Infectious Zoonoses, Ministry of AgriculturalHuazhong Agricultural UniversityWuhanChina

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