Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis

Abstract

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

Keywords: Clonorchis sinensis; aspartic peptidase; cathepsin D; egg; intestine; serodiagnostic antigen.

Fig. 1

Multiple sequence alignment and phylogenetic analyses. (A) Multiple sequence analysis. The deduced amino acid sequences of 2 CsCatDs were aligned with those of Opisthorchis viverrini (AAZ39883), Fasciola hepatica (ABJ97284), Schistosoma mansoni (AAB63442), Schistosoma japonicum (AAC37302), Human (NP_001900), and Rat (NP_599161). Gaps are introduced into the sequences to maximize alignment. The asterisks indicate the catalytic aspartic acid residues. Black lines under the sequences are predicted signal peptide sequences. Potential pro-peptide regions are marked with a blue dotted box. The Y flap element is indicated by a bold red line. Predicted N-glycosylation sites in CsCatDs are represented by black lines. The shading represents the degree of identity among the sequences: black (100%), dark grey (50–92%), and light grey (15–49%). (B) Phylogenetic analysis. The phylogenetic tree was constructed with the neighbor-joining method using the MEGA4 program. Numbers on the branches indicate bootstrap proportions (1,000 replicates).

Fig. 2

Expression profiles of CsCatDs in different developmental stages of C. sinensis. Transcriptional profiles of CsCatDs at various developmental stages of C. sinensis were analyzed via semi-quantitative RT-PCR. Semi-quantitative RT-PCR was performed as described in Materials and Methods. The reaction was conducted in the presence (+) or absence (−) of reverse transcriptase (RTase) to verify DNA contamination. C. sinensis actin gene was amplified as an internal control. M, metacercariae; 2W, 2-week-old juvenile worms; 4W, 4-week-old adult worms; 6W, 6-week-old adult worms; 9W, 9-week-old adult worms.

Fig. 3

Expression and characterization of CsCatD2. (A) Productions of rCsCatD2 and anti-CsCatD2. The rCsCatD2 was produced as an insoluble protein in E. coli. Proteins were analyzed by SDS-PAGE and stained with Coomassie blue (left). Anti-CsCatD2 was produced by immunizing mice with purified rCsCatD2. The specificity of anti-CsCatD2 was confirmed by immunoblot (right). Lane 1, Escherichia coli lysate control; lane 2, IPTG-induced E. coli lysate; lane 3, affinity-purified rCsCatD2. (B) Localization of CsCatD2. IFA was performed with anti-CsCatD2 against sections of C. sinensis adult worms. The slide was observed with a confocal laser scanning microscope (×10 magnification). Scale bar indicated 100 mm. (C) Antigenic property of CsCatD2. The time course of antibodies targeting CsCatD2 in rats experimentally infected with C. sinensis was analyzed. Four rats were infected with 100 C. sinensis metacercariae and the sera from each rat were collected at 0 (0W), 2 (2W), 4 (4W), 6 (6W), 9 (9W), and 12 (12W) weeks post-infection. The purified rCsCatD2 was separated on 12% SDS–PAGE, transferred onto nitrocellulose membrane, cut into strips, and then probed with sera from four rats.

Fig. 4

Homodimeric structures of zymogen and mature CsCatD2. The 3D homology modeling analysis of zymogen (A) and mature form (B) of CsCatD2 predicted homodimeric structures of the proteins. The 3D structures of zymogen and mature form of CsCatD2 were predicted based on experimentally resolved structures of CatDs derived from tick Ixodes ricinus (PDB ID: 5N7N) and humans (PDB ID: 4OD9), respectively. The free energies of dissociation (ΔG^diss; kcal/mol), and interface area (Ų) were calculated using the PISA program [25]. The interfacial residues are shown in red sticks, computed using the ‘InterfaceResidues’ script in PyMOL v1.7.2.1 while the remaining structures of chain A (green) and chain B (cyan) are represented as a cartoon.

Fig. 5

Overall structural features of CsCatD2. (A) Overall 3D structure and secondary structural features of zymogen (A) and mature (B) forms of CsCatD2. The pro-peptide region in zymogen represented as conserved part A (light navy ribbon) and variable part B (green). The Y flap region (cyan) and the polyproline loop (purple) along with the 2 catalytic aspartic residues (red stick) are also marked. The flap tip residue and hinge residue of the polyproline loop are indicated by black and purple sticks, respectively. (B) Sausage representation of the CsCatD2 with sequence and structural conservation rendering. To visualize sequence conservation within the CaCatD2 and CatD family proteins (PDB entries 5UX4, 5N7N, 5N7Q, 5N70, 1TZS, 5MLG, 5MKT, 5NFG, 3D91, 2I4Q, 5T4S, 3PSG, 2PSG, 5PEP, 1PSA, 1F34, 4AA9, 3PEP, 4PEP, 1FLH, 1G0V, 1DP5, 2JXR, 1B5F, 3OAD, 3F9Q, 1M43, 5YIA, 5YID, 1SME, 1LF3, 5BWY, 1PFZ, 3QRV, 2BJU, 1QS8, 2ANL, 1LS5, 1MIQ, 1UH7, 3LIZ, 3FNS, 1YG9, 4RLD, 3QVC, 1LYW, 4OBZ, 3ZLQ, 2EWY, 3ZKM, 6FGY, 2QZL, 1TQF, 3EXO, 3QI1, 2Q11, 3TPJ, 1WKR, 1FKN, 2QK5, 3KMX, 3DM6, 2HM1, 2ZJN, 3IXK, 4DPF, 3L58, 2ZHR, 2FDP, 4TRZ, 4B1D, 1SGZ, 3UDH, 3U6A, 5HTZ, 2ZJK, 2OF0, 3VV6, 3CIB, 5MBW, 1YM2, 1W50, 4L7G, 2QU2, 3CKP, 3HVG, 5MXD, 5CLM, 1YM4, 2Q15, 2ZJI, 5EZX, 2VIE, 3LPI, 4YBI, 2WJO, 3TPR, 5V0N, 2HIZ, 6EJ2, 6EJ3, 2ZJJ, 2VA5, 4B78, 3BRA, 4B70, 4EWO, 2ZJH, 1MPP, 2QZX, 4YBF, 2RMP, 3R1G, 1HTR, 4OBZ, 1LYA, 3OAD, 3APR, 1PSO, and 1CZI), the sausage was colored from white (similarity score below 0.7) to red (strict identity). The degree of structural conservation is proportional to the thickness of the sausage, which corresponds to the mean root-mean-square deviation (RMSD) per residue between Cα pairs based on structural alignments. Putative disulfide bridges are indicated by a yellow line. All the graphics were prepared using ENDscript v2 [27] and PyMOL v1.7.2.1.