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

Thomas CM, Fitzsimmons CM, Dunne DW, Timson DJ - Biochimie (2014)

Bottom Line: The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms.Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional.Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK; Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast, BT9 5BN, UK.

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Drug binding by SmTAL proteins. (a) Limited proteolysis of SmTAL1 (14 μM), SmTAL2 (10 μM) and SmTAL3 (13 μM) with chymotrypsin (600 nM), trypsin (120 nM) and trypsin (650 nM) respectively. M, molecular mass markers (45, 35, 18, 14 kDa); U, untreated protein; +, protein in the presence of protease. The DMSO control and the reactions in the presence of drugs all contained 1% v/v DMSO and 0.8 mM calcium chloride. All drugs were present at a concentration of 250 μM and reactions were analysed by 15% SDS-PAGE. (b) First derivative curves for the thermal denaturation (“melting”) of SmTAL1 (5 μM) and SmTAL3 (7 μM) in the presence of drugs (each 250 μM in the presence of 1% v/v DMSO/0.8 mM calcium chloride). The vertical dotted lines on the graphs indicate the melting temperature of the protein in the presence of 1% v/v DMSO.
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fig3: Drug binding by SmTAL proteins. (a) Limited proteolysis of SmTAL1 (14 μM), SmTAL2 (10 μM) and SmTAL3 (13 μM) with chymotrypsin (600 nM), trypsin (120 nM) and trypsin (650 nM) respectively. M, molecular mass markers (45, 35, 18, 14 kDa); U, untreated protein; +, protein in the presence of protease. The DMSO control and the reactions in the presence of drugs all contained 1% v/v DMSO and 0.8 mM calcium chloride. All drugs were present at a concentration of 250 μM and reactions were analysed by 15% SDS-PAGE. (b) First derivative curves for the thermal denaturation (“melting”) of SmTAL1 (5 μM) and SmTAL3 (7 μM) in the presence of drugs (each 250 μM in the presence of 1% v/v DMSO/0.8 mM calcium chloride). The vertical dotted lines on the graphs indicate the melting temperature of the protein in the presence of 1% v/v DMSO.

Mentions: SmTAL1 was partially protected from limited proteolysis by chymotrypsin by PZQ, CPZ, W7, TFP and, possibly, thiamylal (Fig. 3). Similar results were observed with subtilisin (Supplementary Figure S2). This method has been previously used with calmodulin-like proteins from F. hepatica demonstrating that, as expected, TFP and W7 interacted with FhCaM1 (which differs from human calmodulin by just two amino acid residues) [55,56]. The same drugs, except thiamylal, caused a significant (p < 0.05; unpaired t-test with Welch's correction) increase in the melting temperature of the protein compared to the DMSO control (Fig. 3; Table 1). These data suggest that the drugs interact with SmTAL1 increasing its stability towards both proteolysis and thermal denaturation as a consequence. Of the drugs tested, only W7 protected SmTAL2 from limited proteolysis by trypsin or chymotrypsin (Fig. 3; Supplementary Figure S2). As noted above, it was not possible to carry out DSF analysis on this protein. Both TFP and thiamylal protected SmTAL3 from proteolysis by trypsin (Fig. 3). CPZ, TFP and W7 (but not PZQ or thiamylal) caused a significant (p < 0.05; unpaired t-test with Welch's correction) decrease in the melting temperature of SmTAL3 (Fig. 3; Table 1). A decrease in the melting temperature can arise from the drug binding to partially folded states and thus destabilising the overall population of protein molecules [57]. The situation with SmTAL3 is clearly more complex than with SmTAL1 where the results from limited proteolysis and DSF were broadly in agreement. However, the two techniques measure different consequences of ligand binding and a negative result does not provide evidence for a lack of a drug–protein interaction. Furthermore, it is possible for an interacting drug to show a positive result in only one of two experiments; indeed this has been observed previously with FhCaBP3 using limited proteolysis and fluorescence quenching [31]. While Tm values report on the protein's overall stability, proteolytic digestion reports on events in the immediate vicinity of the protease cleavage site. Therefore, it is not surprising to observe that TFP causes loss of overall stability as measured by DSF, while locally rigidifying the polypeptide backbone (or sterically hindering access to the protease) in limited proteolysis experiments.


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

Thomas CM, Fitzsimmons CM, Dunne DW, Timson DJ - Biochimie (2014)

Drug binding by SmTAL proteins. (a) Limited proteolysis of SmTAL1 (14 μM), SmTAL2 (10 μM) and SmTAL3 (13 μM) with chymotrypsin (600 nM), trypsin (120 nM) and trypsin (650 nM) respectively. M, molecular mass markers (45, 35, 18, 14 kDa); U, untreated protein; +, protein in the presence of protease. The DMSO control and the reactions in the presence of drugs all contained 1% v/v DMSO and 0.8 mM calcium chloride. All drugs were present at a concentration of 250 μM and reactions were analysed by 15% SDS-PAGE. (b) First derivative curves for the thermal denaturation (“melting”) of SmTAL1 (5 μM) and SmTAL3 (7 μM) in the presence of drugs (each 250 μM in the presence of 1% v/v DMSO/0.8 mM calcium chloride). The vertical dotted lines on the graphs indicate the melting temperature of the protein in the presence of 1% v/v DMSO.
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fig3: Drug binding by SmTAL proteins. (a) Limited proteolysis of SmTAL1 (14 μM), SmTAL2 (10 μM) and SmTAL3 (13 μM) with chymotrypsin (600 nM), trypsin (120 nM) and trypsin (650 nM) respectively. M, molecular mass markers (45, 35, 18, 14 kDa); U, untreated protein; +, protein in the presence of protease. The DMSO control and the reactions in the presence of drugs all contained 1% v/v DMSO and 0.8 mM calcium chloride. All drugs were present at a concentration of 250 μM and reactions were analysed by 15% SDS-PAGE. (b) First derivative curves for the thermal denaturation (“melting”) of SmTAL1 (5 μM) and SmTAL3 (7 μM) in the presence of drugs (each 250 μM in the presence of 1% v/v DMSO/0.8 mM calcium chloride). The vertical dotted lines on the graphs indicate the melting temperature of the protein in the presence of 1% v/v DMSO.
Mentions: SmTAL1 was partially protected from limited proteolysis by chymotrypsin by PZQ, CPZ, W7, TFP and, possibly, thiamylal (Fig. 3). Similar results were observed with subtilisin (Supplementary Figure S2). This method has been previously used with calmodulin-like proteins from F. hepatica demonstrating that, as expected, TFP and W7 interacted with FhCaM1 (which differs from human calmodulin by just two amino acid residues) [55,56]. The same drugs, except thiamylal, caused a significant (p < 0.05; unpaired t-test with Welch's correction) increase in the melting temperature of the protein compared to the DMSO control (Fig. 3; Table 1). These data suggest that the drugs interact with SmTAL1 increasing its stability towards both proteolysis and thermal denaturation as a consequence. Of the drugs tested, only W7 protected SmTAL2 from limited proteolysis by trypsin or chymotrypsin (Fig. 3; Supplementary Figure S2). As noted above, it was not possible to carry out DSF analysis on this protein. Both TFP and thiamylal protected SmTAL3 from proteolysis by trypsin (Fig. 3). CPZ, TFP and W7 (but not PZQ or thiamylal) caused a significant (p < 0.05; unpaired t-test with Welch's correction) decrease in the melting temperature of SmTAL3 (Fig. 3; Table 1). A decrease in the melting temperature can arise from the drug binding to partially folded states and thus destabilising the overall population of protein molecules [57]. The situation with SmTAL3 is clearly more complex than with SmTAL1 where the results from limited proteolysis and DSF were broadly in agreement. However, the two techniques measure different consequences of ligand binding and a negative result does not provide evidence for a lack of a drug–protein interaction. Furthermore, it is possible for an interacting drug to show a positive result in only one of two experiments; indeed this has been observed previously with FhCaBP3 using limited proteolysis and fluorescence quenching [31]. While Tm values report on the protein's overall stability, proteolytic digestion reports on events in the immediate vicinity of the protease cleavage site. Therefore, it is not surprising to observe that TFP causes loss of overall stability as measured by DSF, while locally rigidifying the polypeptide backbone (or sterically hindering access to the protease) in limited proteolysis experiments.

Bottom Line: The tegumental allergen-like (TAL) proteins from Schistosoma mansoni are part of a family of calcium binding proteins found only in parasitic flatworms.Despite the presence of two EF-hand-like structures in SmTAL3, neither was predicted to be functional.Overall, these data suggest that the proteins have different biochemical properties and thus, most likely, different in vivo functions.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK; Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast, BT9 5BN, UK.

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