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. 2024:31:39.
doi: 10.1051/parasite/2024036. Epub 2024 Jul 9.

Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis

Ariel Naidich  1 , Ariana M Gutierrez  1 , Federico Camicia  2
Affiliations

Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis

Ariel Naidich et al. Parasite. 2024.

Abstract
in English, French

Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.

Title: Caractérisation moléculaire d’EcCLP1, une nouvelle protéase putative de type cathepsine L d’Echinococcus canadensis.

Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l’échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l’arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l’évasion immunitaire et son établissement dans les tissus de l’hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d’Echinococcus canadensis (EcCLP1). L’analyse bioinformatique suggère qu’EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L’alignement de séquences multiples avec d’autres protéases de type cathepsine révèle d’importantes caractéristiques fonctionnelles conservées telles qu’un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L’analyse phylogénétique suggère qu’EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d’autres espèces de ce clade. Les études de modélisation suggèrent qu’EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d’occlusion pour le propeptide. L’analyse transcriptomique révèle différents niveaux d’expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L’immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d’expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.

Keywords: Cathepsin L proteases; Echinococcus canadensis; Echinococcus granulosus sensu lato; Propeptide.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Multiple sequence alignment of the deduced amino acid sequence of EcCLP1 from Echinococcus canadensis with the amino acid sequence of other cathepsin proteases from several species. In the upper part of the figure, the stretch of amino acids that spans the signal peptide region is indicated by a solid line. The propeptide region is indicated by a dotted line drawn above the alignment. A N-linked glycosylation site is indicated by an arrow above the alignment. The ERFNIN and the AXNXFXD conserved motifs are indicated by squares in the propeptide region. Important residues for propeptide inhibition are indicated in the propeptide region. The asparagine residues at the juncture of the propeptide and mature enzyme, which are important for trans-activation (and propeptide removal) by asparaginyl endopeptidase, are underlined. The S2 subsite conserved residues are marked by squares and numbered according to papain numbering. CT: catalytic triad. DB: cysteine involved in a disulfide bond. OAH: oxyanion hole. EmCLP1 and EmCLP2 refer to the cathepsins L proteases from E. multilocularis [34] (accession numbers BAF02516.1 and BAF02517.1, respectively). The amino acid sequences of TsCL_1 and TsolCL correspond to the cathepsins L from T. solium [20, 21] (accession numbers AAS00027.1 and AQQ11627.1, respectively). FhCL1, FhCL2, and FhCL3 correspond to the amino acid sequences of cathepsins L1, L2, and L3 from F. hepatica [11, 27, 31] (accession numbers AAB41670.2, AAC47721.1 and QPX50259.1 respectively). SsCLP corresponds to the amino acid sequence of cathepsin L from Sus scrofa [35] (accession number Q28944 ⸳ CATL1_PIG). HsCLP1 and HsCKP1 correspond to the cathepsins L and K from H. sapiens (accession numbers NP_001244900.1 and NP_000387.1, respectively). CpPAP corresponds to the papain from Carica papaya (accession number AAB02650.1).
Figure 2
Figure 2
Phylogenetic tree based on the deduced amino acid sequence of EcCLP1 and cathepsin proteases from other species. The unrooted maximum likelihood tree was constructed using the phylogeny program. The black arrow indicates the EcCLP1 protease reported in this study and is marked in red letters. The accession numbers in Uniprot or GenBank databases for the species represented in the tree were the following: A0A068WJU5, A0A068WM48, A0A068WG50, A0A068WF33, U6IZB4 for the E. granulosus s.s. EgCLP1a, EgCLP1b, EgCLP1c, EgCLP1d and EgCLP2, respectively. BAF02516.1 and BAF02517.1 for the E. multilocularis EmCLP1 and EmCLP2, respectively. AAS00027.1 for the T. solium TsCL_1. AAB41670.2, AAC47721.1, QPX50259.1, ABZ80400.1 and AAF76330.1 for the F. hepatica FhCL1, FhCL2, FhCL3, FhCL4 and FhCL5, respectively. NP_001244900.1 and NP_000387.1 for the Homo sapiens HsCLP1 and HsCKP, respectively. AAB02650.1 for the papain CpPAP. O45734-1 for the C. elegans CeCLP1. AAB18345.1 for DmCLP1 or cysteine proteinase 1 from Drosophila melanogaster. AAC46485.1, Q26564_SCHMA_CLP2, and AAA29865.1 for SmCLP1, SmCLP2, and SmCB1, respectively from Schistosoma mansoni. AAC48340.1 for Tc-Cpl-1 or cathepsin L-like cysteine proteinase from Toxocara canis. KRY31298.1 for TsCatL or cathepsin L cysteine proteinase from Trichinella spiralis.
Figure 3
Figure 3
Predicted three-dimensional structures of proEcCLP1 and mature EcCLP1 and comparison with the human cathepsin. (A) Topological representation of the mature EcCLP1 with the residues forming the S2 subsite marked in red. (B) proEcCLP1 showing the propeptide in cyan and blue. (C) Magnified image of the model shown in (A) showing the residues forming the S2 subsite in white with papain numbering in parentheses. (D) Superposition of the ribbon representation of the crystal structure of human cathepsin (brown) [6] and the modeled EcCLP1 (green). (E) Magnified image of the active site of the cathepsins shown in (D). Important S2 residues are marked in green in parasite numbering and red in human cathepsin. Differences between both sequences are marked with boxes.
Figure 4
Figure 4
Transcriptional levels of several cathepsin L proteases in some life cycle stages from Echinococcus. spp. (A) Transcriptional levels (in FPKM levels) of the E. granulosus s.s. L cathepsins EgCLP1a, EgCLP1b, EgCLP1c, EgCLP1d and EgCLP2 in the protoscolex stage. (B) Transcriptional levels (in FPKM levels) of the E. multilocularis L cathepsins EmCLP1a, EmCLP1b, EmCLP1c, EmCLP1d, and EmCLP2 in the metacestode (black balls), pre-gravid (black squares) and gravid stages (black triangles). The data were obtained from Tsai et al. [37].
Figure 5
Figure 5
Immunolocalization of EcCLP1 in protoscoleces of Echinococcus canadensis. Protoscoleces were probed with anti-EcCLP1 (green) and anti-Eca-5-HT1a (red) serum and visualized by confocal microscopy. (A) Phase contrast view of the protoscolex shown in (B). (B) Fluorescent image of the protoscolex labeled with anti-EcCLP1 hyperimmune serum. Intense signals like dots were found superficially in the body region. (C) Magnification of the panel (B) showing a dotted pattern of staining on the surface of the tegument. (D) Phase contrast view of the protoscolex shown in (E). (E) Fluorescent image of the protoscolex labeled with anti-Eca-5-HT1a hyperimmune serum. (F) Phase contrast view of the protoscolex shown in (G). (G) Fluorescent image of the protoscolex labeled with the preimmune rabbit serum. The white arrows show the localization of Eca-5-HT1a in the cerebral ganglia in panel E or the localization of EcCLP1 in small dots in panel (C), suggesting a secretory release of the protein. Abbreviations, Bo: body region, Ro: rostellum, Sc: scolex region, Su: sucker.
<i>Supplementary Figure 1</i>:
Supplementary Figure 1:
Polyacrylamide gel electrophoresis stained with Coomassie brilliant blue of bacterial cells transformed with pBAD/TOPO® ThiofusionTM Expression System (ThermoFisher™) which expresses part of the EcCLP1 as a protein fused to thioredoxin (Thio-EcCLP1-his). Lane 1. Non-induced E. coli bacterial cells at 37 °C. Lane 2. Bacterial cells induced with 0.2% of arabinose at 37 °C. Positions of the molecular marker proteins with the corresponding masses in kilodaltons (kDa) are indicated. The arrow shows the recombinant-induced protein.
<i>Supplementary Figure 2</i>:
Supplementary Figure 2:
Western-blot of recombinant Thio-EcCLP1-his protein stained with rabbit hyperimmune serum against Thio-EcCLP1-his. Lane 1. Preimmune rabbit serum, diluted 1/50 in PBS-milk 1.5%. Lane 2. Hyperimmune rabbit serum diluted 1/20. Lane 3. Same hyperimmune serum as in lane 2 but diluted 1/50. Lane 4. Same hyperimmune serum as in lane 2 but diluted 1/100.
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