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

Abstract

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 EFhand containing N-terminal domain with a C-terminal dynein light chainlike 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 protein's ability to dimerise. FhCaBP1 binds to the calmodulin antagonists trifluoperazine and chlorpromazine. Overall, the FhCaBP1's biochemical properties are most similar to FhCaBP2 a fact consistent with the close sequence and predicted structural similarity between the two proteins. properties are most similar to FhCaBP2 a fact consistent with the close sequence and predicted structural similarity between the two proteins.

The Schistosoma mansoni tegumental allergen protein, SmTAL1: Binding to an IQ-motif from a voltage-gated ion channel and effects of praziquantel

“…Full-length SmTAL1, a protein fragment corresponding to the EF-hand domain, four variant proteins (SmTAL1-E32A, EF-E32A, SmTAL1-D59A and EF-D59A) and a fragment corresponding to the DLC-like domain of the protein (which does not contain the EF-hand motifs required for calcium chelation), were expressed in, and purified from, E. coli (Supplementary Figure S2). These proteins were resolved under discontinuous native-PAGE and, in accordance with previous studies, the calcium-specific chelator EGTA was used to remove any endogenously bound calcium ions [10,[12][13][14][15][16]. Native-PAGE assays revealed that the electrophoretic mobility of full-length SmTAL1 was increased in the presence of EGTA (compared to untreated protein sample), whereas the addition of a molar excess of calcium ions to the EGTA-treated protein reduced the electrophoretic mobility ( Figure 2).…”

“…This is joined by a flexible linker to a C-terminal domain which has very high structural similarity to members of the DLC family ( Figure 1) [3][4][5]. Proteins from this family have been identified and characterised in a number of species including Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Fasciola hepatica, Fasciola gigantica, Opisthorchis viverrini and Clonorchis sinensis [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. In general, these proteins bind to calcium ions, although there are some exceptions, for example S. mansoni tegumental allergen-like (TAL) protein 3 (SmTAL3 or Sm20.8) [10,16].…”

“…We have previously demonstrated that, despite similarities in sequence and predicted structure, that members of this protein family have different biochemical properties. For example, of the four characterised family members from F. hepatica, one dimerises in response to the presence of calcium ions (FhCaBP4), one is converted from a dimer to a monomer by calcium ions (FhCaBP3) and the oligomeric states of the two are unaffected by this ion (FhCaBP1 and FhCaBP2) [12][13][14][15]. In the case of the 13 family members from S. mansoni, two do not appear to bind calcium ions at all (SmTAL3 and SmTAL5) [10,16].…”

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

“…In accordance with previous studies, the calcium chelator, EGTA, was routinely used to remove endogenously bound calcium ions from protein preparations: SmCaM proteins were then reconstituted with metal ions in a two-fold molar excess to ensure saturating binding conditions [54,[58][59][60][61][62]. For ion-binding assays, protein aliquots (10-40 µM) were incubated at 22 °C for 30 min in the presence of either EGTA, or EGTA and a two-molar excess of the appropriate ion solution, and diluted in Buffer R to a final volume of 10 µl.…”

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

“…One family member (SmTAL3) has been identified as part of a complex which also includes a component of the microbule motor, dynein [22]. In the liver flukes Fasciola hepatica and Facsiola gigantica four members of the family have been identified and characterised [23][24][25][26][27][28][29][30]. It seems likely that further family members will be revealed in these species once their genomes are fully annotated.…”