Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain)

Abstract

Background: Schistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1).

Results: Calpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host-parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion.

Conclusions: SmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Keywords: Calcium-dependent cysteine protease; Calpain; Drug and vaccine development; Schistosoma mekongi; Schistosomiasis.

Fig. 1

Gene expression level of calpain isoforms in adult male and female Schistosoma mekongi. a Gene expression level of calpain isoforms was determined using SYBR real-time RT-PCR, and the relative expression level of each isoform between adult males and females was calculated as Log₂ fold-change (Log₂FC). Comparison of gene expression level (in arbitrary units) of calpain isoform in b adult male and c adult female. Error bars show standard deviations

Fig. 2

Multiple sequence alignment of SmeCalp1 sequence with orthologs. The deduced amino acid sequence of calpain 1 homologs Schistosoma mekongi (SmeCalp1), S. japonicum-Chinese strain (Sj-CCalp1), S. mansoni (SmCalp1) and S. haematobium (ShCalp1) were aligned using Clustal Omega program. The identical and similar amino acids are shaded in black and gray, respectively. Gaps (−) are introduced to optimize homology. The arrows indicate the regions of domains I, II, III and IV. The cysteine protease active site is indicated by yellow dashed overline. The predicted catalytic triad cysteine (C154), histidine (H313) and asparagine (N337) is indicated by stars. Red box indicates the putative Ca²⁺-binding acidic loop (E-E/D-X-D-D/E-X-D-D/E-D-G-X). The four EF-hand motifs (EF1 to 4) are indicated by green overlines. The GenBank accession numbers of the sequences are provided in Additional file 2: Table S2

Fig. 3

Phylogenetic analysis of the calpains. a A rooted tree comparison of SmeCalp1 with other calpain 1 orthologs found in trematodes, cestodes, nematodes, protozoans, insects and vertebrates. b An unrooted tree comparison of calpain isoforms identified in human Schistosoma spp. The trees were constructed with maximum likelihood method using MEGA7 program, 1000 bootstrap replicates. Numbers at the nodes indicate the proportion of bootstraps. The abbreviations used and GenBank accession numbers are provided in Additional file 2: Table S2

Fig. 4

Expression of rSmeCalp1 and detection of native SmeCalp1 in parasite antigens. a rSmeCalp1 was successfully expressed in Escherichia coli. Lane M: PageRuler Prestained Protein Ladder (Thermo Fisher Scientific); Lane 1: non-induction; Lane 2: IPTG induction; Lane 3: soluble fraction; Lane 4: insoluble fraction; Lane 5: purified rSmeCalp1; Lane 6: refolded rSmeCalp1; Lane 7: western blot analysis of refolded rSmeCalp1 probe with anti-His tag antibody. Arrowhead indicates rSmeCalp1. b Western blot analysis detecting native SmeCalp1 in CWA of adult male and female and ES. Lane M: PageRuler Prestained Protein Ladder (Thermo Fisher Scientific Inc.); Lanes 1–4: mice number 1, 2, 3 and 4, respectively. Abbreviations: CWA, crude worm antigen; SmeES, S. mekongi excretory-secretory product

Fig. 5

Transcription level of SmeCalp1 in different developmental stages of Schistosoma mekongi. Transcription levels of SmeCalp1 in eggs (E), miracidia (Mi), schistosomula (Schis), adult male (AM) and adult female (AF) were determined using SYBR real-time RT-PCR and normalized with expression level of 18S RNA in each stage. The expression level is shown in arbitrary units (A.U.). Error bars show standard deviations. The experiments were performed in triplicate and repeated three times

Fig. 6

Localization of native SmeCalp1 in Schistosoma mekongi adult worm. a Immumohistochemical analysis of SmeCalp1 in adult worms. Arrowheads indicate the tegument of S. mekongi which reacted with mouse anti-rSmeCalp1 pAb. b Electron micrograph of the adult tegument showing immunogold labeling of SmeCalp1. Arrows indicate SmeCalp1 reacted with mouse anti-rSmeCalp1. Left, adult male; right, adult female. Abbreviations: m, adult male; f, adult female; e, eggs; T, tegumental surface; M, muscle; Lm, longitudinal muscle layers, Cm, circular muscle layers; Mv, membrane bound vesicle

Fig. 7

Proteolytic activity of rSmeCalp1. a rSmeCalp1 elicited proteolytic activity to hydrolyze the calpain fluorogenic substrate (N-succinyl-Leu-Leu-Val-Try-7-AMC), and irrelevant (rmDHFR) and negative controls could not be detected. b Proteolytic activity of rSmeCalp1 was inhibited by calpain inhibitors (MDL and E64c), broad cysteine protease inhibitor (E64) and metal chelating agent (EDTA) but not by other protease class inhibitors (PMSF, Peps A and 1,10-Phen). Statistical analysis was performed using Student’s t-test. **P < 0.01. c Proteolytic activity of rSmeCalp1 was dependent on concentration of Ca²⁺. d A wide pH range for proteolytic activity of rSmeCalp1 was observed (6.5–9.5) with an optimum pH at 8.5. Data are presented as means ± standard deviations. The experiments were performed in triplicate and repeated three times

Fig. 8

Degradation of biological substrates by rSmeCalp1. SmeCalp1 was incubated with substrates a albumin, b hemoglobin, c IgG, d complement C1q, e complement C3 and f fibronectin at different time points and then subjected to SDS-PAGE. rSmeCalp1 degraded only the α-subunit of complement C3 (115 kDa) (e) and fibronectin (f), and this activity was inhibited in the presence of cysteine protease inhibitor (E64) (data not shown)