Comparative Characterization of Four Calcium-Binding EF Hand Proteins from Opisthorchis viverrini

Abstract

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2-42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21-23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²⁺. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.

Keywords: EF hand motif; Opisthorchis viverrini; Platyhelminthes; calcium-binding; dynein light chain; tegument.

Fig. 1

Multiple sequence alignment of OvCaBP1–4 and OvCaBP22.8. The 2 EF hand motifs and single dynein light chain like domain are indicated. The 6 residues in each EF hand motif making contact to calcium are indicated by triangles (▼). OvCaBP1 is used as reference sequence. Positions with all identical residues are indicated as dots ( · ), positions with all similar residues are shown in lower letters. Gaps introduced for alignment are indicated by dashes (-).

Fig. 2

Phylogenetic tree based on maximum-likelihood analysis of characterized O. viverrini/C. sinensis CaBPs. The described OvCaBP1–4 are indicated in bold. CsTegu21.6, AEI69651, [18]; OvCaBP22.8, XP_009173200, [17]; CsABZ82044, ABZ82044, [27]; CsTg22.3, ABK60085, [28]; CsTP31.8, ABK60086, [29]; CsTegu20.6, GAA49981, [25]; CsTP20.8, ABC47326, [30]; CsTegu21.1, ADZ13689, [31]. The bootstrap support values are shown at the nodes, this is an unrooted tree, log likelihood: −3549.0.

Fig. 3

Stage-specific amplification of OvCaBPs transcripts by reverse transcriptase PCR. The total RNA of newly excysted juveniles (NEJ), 2-week juveniles (2 W), 4-week juveniles (4 W), and 8-week adult (8 W) O. viverrini were extracted in TRIzol and used as templates for RT-PCR with specific primers for each isoform. OvActin was used as standard. Lane M, 100 bp DNA ladder.

Fig. 4

Determination of ion-binding properties by mobility shift assays in non-denaturing gels. Five micrograms of rOvCaBP (A-D) were pre-incubated with 5 mM EDTA and post-incubated with 25 mM CaCl₂, MgCl₂, ZnSO₄, and CuSO₄. Minus (−) symbols indicate proteins only incubated with 5 mM EDTA.

Fig. 5

Western blot analysis of mature O. viverrini crude worm extracts (CW), ES product (ES), and recombinant OvCaBP1–4 (1, 2, 3, and 4) with mouse anti-rOvCaBP1–4 antisera at dilution 1:2,000. sCW, 20 μg soluble CW; iCW, 20 μg insoluble CW; ES, 20 μg ES product; lanes 1, 2, 3, and 4, 100 ng of rOvCaBP-1, -2, -3, and -4, respectively. Positions of 31.0, 21.5, and 14.4 kDa protein standards are indicated on the left.