Charlotte M. Thomas

Comparative biochemical analysis of three members of the Schistosoma mansoni TAL family: Differences in ion and drug binding properties.

The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms. These proteins have attracted interest as potential drug or vaccine targets, yet comparatively little is known about their biochemistry. Here, we compared the biochemical properties of three members of this family: SmTAL1 (Sm22.6), SmTAL2 (Sm21.7) and SmTAL3 (Sm20.8). Molecular modelling suggested that, despite similarities in domain organisation, there are differences in the three proteins’ structures. SmTAL1 was predicted to have two functional calcium binding sites and SmTAL2 was predicted to have one. Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional. These predictions were confirmed by native gel electrophoresis, intrinsic fluorescence and differential scanning fluorimetry: both SmTAL1 and SmTAL2 are able to bind calcium ions reversibly, but SmTAL3 is not. SmTAL1 is also able to interact with manganese, strontium, iron(II) and nickel ions. SmTAL2 has a different ion binding profile interacting with cadmium, manganese, magnesium, strontium and barium ions in addition to calcium. All three proteins form dimers and, in contrast to some Fasciola hepatica proteins from the same family; dimerization is not affected by calcium ions. SmTAL1 interacts with the anti-schistosomal drug praziquantel and the calmodulin antagonists trifluoperazine, chlorpromazine and W7. SmTAL2 interacts only with W7. SmTAL3 interacts with the aforementioned calmodulin antagonists and thiamylal, but not praziquantel. Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.

FhCaBP2: a Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains

FhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the proteins surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.

FhCaBP1 (FH22): A Fasciola hepatica calcium-binding protein with EF-hand and dynein light chain domains.

FH22 has been previously identified as a calcium-binding protein from the common liver fluke, Fasciola hepatica. It is part of a family of at least four proteins in this organism which combine an EF-hand containing N-terminal domain with a C-terminal dynein light chain-like domain. Here we report further biochemical properties of FH22, which we propose should be renamed FhCaBP1 for consistency with other family members. Molecular modelling predicted that the two domains are linked by a flexible region and that the second EF-hand in the N-terminal domain is most likely the calcium ion binding site. Native gel electrophoresis demonstrated that the protein binds both calcium and manganese ions, but not cadmium, magnesium, strontium, barium, cobalt, copper(II), iron (II), nickel, zinc, lead or potassium ions. Calcium ion binding alters the conformation of the protein and increases its stability towards thermal denaturation. FhCaBP1 is a dimer in solution and calcium ions have no detectable effect on the proteins ability to dimerise. FhCaBP1 binds to the calmodulin antagonists trifluoperazine and chlorpromazine. Overall, the FhCaBP1s biochemical properties are most similar to FhCaBP2 a fact consistent with the close sequence and predicted structural similarity between the two proteins.

The tegumental allergen-like proteins of Schistosoma mansoni: A biochemical study of SmTAL4-TAL13

Schistosoma mansoni, like other trematodes, expresses a number of unusual calcium binding proteins which consist of an EF-hand domain joined to a dynein light chain-like (DLC-like) domain by a flexible linker. These proteins have been implicated in host immune responses and drug binding. Three members of this protein family from S. mansoni (SmTAL1, SmTAL2 and SmTAL3) have been well characterised biochemically. Here we characterise the remaining family members from this species (SmTAL4-13). All of these proteins form homodimers and all except SmTAL5 bind to calcium and manganese ions. SmTAL9, 10 and 11 also bind to magnesium ions. The antischistosomal drug, praziquantel interacts with SmTAL4, 5 and 8. Some family members also bind to calmodulin antagonists such as chlorpromazine and trifluoperazine. Molecular modelling suggests that all ten proteins adopt similar overall folds with the EF-hand and DLC-like domains folding discretely. Bioinformatics analyses suggest that the proteins may fall into two main categories: (i) those which bind calcium ions reversibly at the second EF-hand and may play a role in signalling (SmTAL1, 2, 8 and 12) and (ii) those which bind calcium ions at the first EF-hand and may play either signalling or structural roles (SmTAL7, 9, 10 and 13). The remaining proteins include those which do not bind calcium ions (SmTAL3 and 5) and three other proteins (SmTAL4, 6 and 11). The roles of these proteins are less clear, but they may also have structural roles.

The mechanism of action of praziquantel: can new drugs exploit similar mechanisms?

Praziquantel (PZQ) is the drug of choice for treating infection with worms from the genus Schistosoma. The drug is effective, cheap and has few side-effects. However, despite its use in millions of patients for over 40 years its molecular mechanism of action remains elusive. Early studies demonstrated that PZQ disrupts calcium ion homeostasis in the worm and the current consensus is that it antagonises voltage-gated calcium channels. It is hypothesised that disruption of these channels results in uncontrolled calcium ion influx leading to uncontrolled muscle contraction and paralysis. However, other experimental studies have suggested a role for myosin regulatory light chains and adenosine uptake in the drugs mechanism of action. Assuming voltage-gated calcium channels do represent the main molecular target of PZQ, the precise binding site for the drug remains to be identified. Unlike other commonly used anti-parasitic drugs, there are few definitive reports of resistance to PZQ in the literature. The lack of knowledge about PZQs molecular mechanism(s) undermines our ability to predict how resistance might arise and also hinder our attempts to develop alternative antischistosomal drugs which exploit the same target(s). Some PZQ derivatives have been identified which also kill or paralyse schistosomes in culture. However, none of these are in widespread clinical use. There is a pressing need for fundamental research into the molecular mechanism(s) of action of PZQ. Such research would enable new avenues for antischsistosomal drug discovery.

Calmodulins from Schistosoma mansoni: Biochemical analysis and interaction with IQ-motifs from voltage-gated calcium channels

The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins - SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCav1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels.