Elsevier

Biochimie

Volume 179, December 2020, Pages 127-134
Biochimie

Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design

https://doi.org/10.1016/j.biochi.2020.09.012Get rights and content

Abstract

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.

Introduction

The Babesia genus comprises a group of hemoparasites transmitted by ticks [1] and since Babesia vectors are distributed worldwide, these piroplasmids are commonly found in mammals’ bloodstream including, but not limited to, humans, horses, sheep, dogs, cats and cattle [2,3]. Bovine babesiosis caused by B. bovis induces severe fever, hemolytic anemia, hemoglobinuria, and hypotensive shock leading to high mortality rates in susceptible cattle [4]. Since more than half of the world cattle population is considered to be at risk of babesiosis infections [5], the Rhipicephalus microplus - B. bovis complex represents an important association from the economical perspective.

Babesiosis control is currently based on three different strategies, (i) vector control, (ii) cattle immunization and (iii) anti-Babesia drugs [6,7]. Vector control is mainly carried out by acaricides, although tick strains resistant to different compounds has already been described [8,9]. Cattle immunization has been proposed for both vector and Babesia sp. control. Tick immunological control studies resulted in the development of two commercially available vaccines, although the levels of protection vary largely between different regions [[10], [11], [12]]. Cattle immunization against B. bovis has been mainly carried out with live vaccines using attenuated parasites [13,14]; however, the use of recombinant vaccines based on B. bovis proteins has been proposed and is a subject of active investigation [15,16]. Currently the most commonly used drug to treat Babesia infections is imidocarb [17], but several other drugs are in the development stage [6,18].

In apicomplexan parasites, cysteine peptidases from the C1A subfamily have been identified and shown to be crucial for parasite survival and proliferation [[19], [20], [21], [22]]. Therefore, it was proposed that such enzymes could be explored as targets for drug development, in which enzyme specificity plays an important role [[23], [24], [25]]. Notably, B. bovis treatment with cysteine peptidase inhibitors reduces parasite growth [26], showing the contribution of cysteine peptidases for Babesia sp. life cycle. So far, a handful of C1A cysteine peptidases have been characterized [[27], [28], [29]]. One of them, ovipain-2 from B. ovis, displays a high degree of conservation with P. falciparum falcipain-2; ovipain-2 was found in merozoite stages and appears to be secreted into the erythrocyte cytoplasm, moreover, treatment of B. ovis in vitro cultures with anti-ovipain-2 antibodies resulted in a significant decrease of parasite proliferation [30], indicating the participation of ovipain-2 in the process of invasion of or egress from host red blood cells (RBCs). Similarly, bovipain-2, the falcipain-2 ortholog from B. bovis, was also found in merozoite stages scattered in the erythrocyte cytoplasm, suggesting its role during parasite egress [31]. Although these studies reinforce the role of cysteine peptidases for Babesia survival there is no information regarding their proteolytic specificity. So far only the BbiCPL1 from B. bigemina has been biochemically characterized [29], in which a preference for Val > Leu > Phe at the P2 position was found.

B. bovis genome is available [32] and 17 putative cysteine peptidases are currently annotated, from which four belong to the C1A subfamily [33]. Since C1A cysteine peptidases play an important role in apicomplexan life cycle and represent potential targets for anti-parasitic drug development, we present here the biochemical characterization of a putative C1A cysteine peptidase from B. bovis and its possible role during RBC invasion.

Section snippets

Bioinformatic analysis

The complete mRNA sequence of XP_001612131 was obtained from the B. bovis T2Bo strain deposited genome (http://protists.ensembl.org/). Domain analysis was conducted using PFAM (https://pfam.xfam.org/) [34] and detection of putative signal peptides was carried out with SignalP 5.0 [35]. The theoretical molecular weight and isoelectric point were estimated using the Compute pI/MW tool [36] and the topology analysis was carried out with TMHMM V.2.0 (http://www.cbs.dtu.dk/services/TMHMM/). An amino

Analysis of B. bovis cysteine peptidase (BbCp) primary structure

The deposited sequence XP_001612131 comprises a 49.1 kDa protein with a theoretical isoelectric point of 5.39 and no predicted signal peptide. Domain analysis of XP_001612131 revealed the presence of an inhibitory pro-domain I29 (F120 – F177) and a catalytic domain from the C1A family (I230 – A435). Topology prediction shows a short cytoplasmic stretch (M1 to S35), followed by a transmembrane domain (A36 to G58) in the N-terminal, while the rest of the protein is predicted as extracellular (K59

Discussion

Cattle babesiosis, caused by B. bovis, is a major concern for livestock production worldwide [2,5], yet knowledge regarding the molecular mechanisms of parasite survival is still sparse. Since cysteine peptidases play an important role for apicomplexan survival and proliferation [47] it was proposed that such molecules could be used as targets for parasite control, in which enzyme specificity is determinant [49]. Therefore, in this study, we carried out a kinetic characterization of a novel

Conclusion

In this study, we describe the biochemical characterization of a putative cysteine peptidase from B. bovis named BbCp. The active recombinant BbCp was obtained in bacteria after the refolding process and presented increased proteolytic activity in the presence of DTT and an optimum pH between 6.5 and 7.0. The rBpCp was inhibited by R. microplus Bmcystatin-1 and Rmcystatin-3 but not by Rmcystatin-4. Moreover, substrate profiling of rBbCp revealed preference for Val > Leu > Phe residues. Finally,

Declaration of competing interest

The authors declare there is no conflict of interest.

Acknowledgments

We are grateful to Jacilene Barbosa of Laboratório multiusuário 3 - INFAR, UNIFESP for performing the DNA sequencing. We also thank Dr. Luís Fernando Parizi and Dr. Itabajara da Silva Vaz Jr. from Universidade Federal do Rio Grande do Sul (UFRGS) for supplying the ticks used in this work.

References (65)

  • S.K. Chakka et al.

    Identification of novel class of falcipain-2 inhibitors as potential antimalarial agents

    Bioorg. Med. Chem.

    (2015)
  • K. Okubo et al.

    Babesia bovis: effects of cysteine protease inhibitors on in vitro growth

    Exp. Parasitol.

    (2007)
  • P.J. Holman et al.

    A cathepsin L-like cysteine protease is conserved among Babesia equi isolates

    Mol. Biochem. Parasitol.

    (2002)
  • T.M. Martins et al.

    Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases

    Exp. Parasitol.

    (2011)
  • T.M. Martins et al.

    Expression and characterization of the Babesia bigemina cysteine protease BbiCPL1

    Acta Trop.

    (2012)
  • T. Carletti et al.

    Characterization of a papain-like cysteine protease essential for the survival of Babesia ovis merozoites

    Ticks Tick Borne Dis

    (2016)
  • M. Mesplet et al.

    Genome-wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair

    Mol. Biochem. Parasitol.

    (2011)
  • B.R. Shenai et al.

    Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum

    J. Biol. Chem.

    (2000)
  • C.A. Lima et al.

    Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

    Biochem. Biophys. Res. Commun.

    (2006)
  • S. Lu et al.

    Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response

    Biochimie

    (2014)
  • T.H.S. Cardoso et al.

    Characterization of a novel cystatin type 2 from Rhipicephalus microplus midgut

    Biochimie

    (2017)
  • R.O. Clara et al.

    Boophilus microplus cathepsin L-like (BmCL1) cysteine protease: specificity study using a peptide phage display library

    Vet. Parasitol.

    (2011)
  • J.F. Morrison

    Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors

    Biochim. Biophys. Acta

    (1969)
  • R.W. Mason et al.

    Surface activation of pro-cathepsin L

    Biochem. Biophys. Res. Commun.

    (1992)
  • M. Hanspal et al.

    Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development

    Blood

    (2002)
  • I. Schechter et al.

    On the size of the active site in proteases. I. Papain. 1967

    Biochem. Biophys. Res. Commun.

    (2012)
  • M. Sajid et al.

    Cysteine proteases of parasitic organisms

    Mol. Biochem. Parasitol.

    (2002)
  • S.S. Cotrin et al.

    Substrate specificity studies of the cysteine peptidases falcipain-2 and falcipain-3 from Plasmodium falciparum and demonstration of their kininogenase activity

    Mol. Biochem. Parasitol.

    (2013)
  • V.K. Kolla et al.

    Independent amino acid residues in the S2 pocket of falcipain-3 determine its specificity for P2 residues in substrates

    Mol. Biochem. Parasitol.

    (2015)
  • S. Hasnain et al.

    Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization

    J. Biol. Chem.

    (1993)
  • C.E. Suarez et al.

    Advances in the application of genetic manipulation methods to apicomplexan parasites

    Int. J. Parasitol.

    (2017)
  • C. Rosa et al.

    Transient transfection of Babesia ovis using heterologous promoters

    Ticks Tick Borne Dis

    (2019)
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