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Proteolysis at a specific extracellular residue implicates integral membrane CLAG3 in malaria parasite nutrient channels.

Nguitragool W, Rayavara K, Desai SA - PLoS ONE (2014)

Bottom Line: Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone.These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function.They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

ABSTRACT
The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The clag3 genes of P. falciparum determine this channel's activity in human malaria, but how the encoded proteins contribute to transport is unknown. Here, we used proteases to examine the channel's composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake.

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Effects of proteases on CLAG3 and PSAC-mediated transport.(A) Immunoblots showing CLAG3 hydrolysis in HB3 and Dd2 parasites by chymotrypsin (“Ch”), trypsin (“Tr”), or pronase E (“PrE”); mouse anti-CLAG3 generated using a recombinant C-terminal fragment [23]. The band at ∼160 kDa reflects uncleaved CLAG3; a C-terminal proteolysis fragment at ∼35 kDa is visible upon protease treatment. Addition of 2 mM PMSF abolishes cleavage by chymotrypsin. (B) Osmotic lysis kinetics for HB3- and Dd2-infected cells in sorbitol. Control traces represent matched samples not exposed to proteases or PMSF (black traces). While pronase E retards PSAC-mediated osmotic lysis and trypsin is without effect in both parasites (green and blue traces, respectively), chymotrypsin inhibits transport in HB3- but not Dd2-infected cells (red solid traces). (C) Mean ± S.E.M. PSAC inhibition determined from osmotic lysis experiments, normalized to 0% for no protease controls. (D) Mean ± S.E.M. inhibition resulting from chymotrypsin treatment of erythrocytes infected with indicated parasites.
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pone-0093759-g001: Effects of proteases on CLAG3 and PSAC-mediated transport.(A) Immunoblots showing CLAG3 hydrolysis in HB3 and Dd2 parasites by chymotrypsin (“Ch”), trypsin (“Tr”), or pronase E (“PrE”); mouse anti-CLAG3 generated using a recombinant C-terminal fragment [23]. The band at ∼160 kDa reflects uncleaved CLAG3; a C-terminal proteolysis fragment at ∼35 kDa is visible upon protease treatment. Addition of 2 mM PMSF abolishes cleavage by chymotrypsin. (B) Osmotic lysis kinetics for HB3- and Dd2-infected cells in sorbitol. Control traces represent matched samples not exposed to proteases or PMSF (black traces). While pronase E retards PSAC-mediated osmotic lysis and trypsin is without effect in both parasites (green and blue traces, respectively), chymotrypsin inhibits transport in HB3- but not Dd2-infected cells (red solid traces). (C) Mean ± S.E.M. PSAC inhibition determined from osmotic lysis experiments, normalized to 0% for no protease controls. (D) Mean ± S.E.M. inhibition resulting from chymotrypsin treatment of erythrocytes infected with indicated parasites.

Mentions: Treatment of infected cells with chymotrypsin or pronase E reduces PSAC-mediated solute transport, but trypsin has negligible effect [23], [41]. We investigated the effects of these proteases on CLAG3 using intact infected erythrocytes and conditions that avoid protein cleavage at intracellular sites [23]. To explore possible effects of polymorphisms, we utilized the Dd2 and HB3 parasite clones, derived originally from patients in Indochina and Honduras respectively. After extracellular protease treatment, we performed denaturing gel electrophoresis and immunoblotting with an antibody that recognizes the CLAG3 C-terminus. With chymotrypsin, trypsin, or pronase E treatment, a single proteolytic fragment of approximately 35 kDa size was detected; 2 mM phenylmethanesulfonylfluoride (PMSF, a serine protease inhibitor) inhibited release of this fragment when added during chymotrypsin treatment (Fig. 1A). With each protease, semi-quantitative comparison to the retained 160 kDa full length CLAG3 indicated that a significant fraction of parasite CLAG3 is exposed and cleaved at the host cell membrane.


Proteolysis at a specific extracellular residue implicates integral membrane CLAG3 in malaria parasite nutrient channels.

Nguitragool W, Rayavara K, Desai SA - PLoS ONE (2014)

Effects of proteases on CLAG3 and PSAC-mediated transport.(A) Immunoblots showing CLAG3 hydrolysis in HB3 and Dd2 parasites by chymotrypsin (“Ch”), trypsin (“Tr”), or pronase E (“PrE”); mouse anti-CLAG3 generated using a recombinant C-terminal fragment [23]. The band at ∼160 kDa reflects uncleaved CLAG3; a C-terminal proteolysis fragment at ∼35 kDa is visible upon protease treatment. Addition of 2 mM PMSF abolishes cleavage by chymotrypsin. (B) Osmotic lysis kinetics for HB3- and Dd2-infected cells in sorbitol. Control traces represent matched samples not exposed to proteases or PMSF (black traces). While pronase E retards PSAC-mediated osmotic lysis and trypsin is without effect in both parasites (green and blue traces, respectively), chymotrypsin inhibits transport in HB3- but not Dd2-infected cells (red solid traces). (C) Mean ± S.E.M. PSAC inhibition determined from osmotic lysis experiments, normalized to 0% for no protease controls. (D) Mean ± S.E.M. inhibition resulting from chymotrypsin treatment of erythrocytes infected with indicated parasites.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3974804&req=5

pone-0093759-g001: Effects of proteases on CLAG3 and PSAC-mediated transport.(A) Immunoblots showing CLAG3 hydrolysis in HB3 and Dd2 parasites by chymotrypsin (“Ch”), trypsin (“Tr”), or pronase E (“PrE”); mouse anti-CLAG3 generated using a recombinant C-terminal fragment [23]. The band at ∼160 kDa reflects uncleaved CLAG3; a C-terminal proteolysis fragment at ∼35 kDa is visible upon protease treatment. Addition of 2 mM PMSF abolishes cleavage by chymotrypsin. (B) Osmotic lysis kinetics for HB3- and Dd2-infected cells in sorbitol. Control traces represent matched samples not exposed to proteases or PMSF (black traces). While pronase E retards PSAC-mediated osmotic lysis and trypsin is without effect in both parasites (green and blue traces, respectively), chymotrypsin inhibits transport in HB3- but not Dd2-infected cells (red solid traces). (C) Mean ± S.E.M. PSAC inhibition determined from osmotic lysis experiments, normalized to 0% for no protease controls. (D) Mean ± S.E.M. inhibition resulting from chymotrypsin treatment of erythrocytes infected with indicated parasites.
Mentions: Treatment of infected cells with chymotrypsin or pronase E reduces PSAC-mediated solute transport, but trypsin has negligible effect [23], [41]. We investigated the effects of these proteases on CLAG3 using intact infected erythrocytes and conditions that avoid protein cleavage at intracellular sites [23]. To explore possible effects of polymorphisms, we utilized the Dd2 and HB3 parasite clones, derived originally from patients in Indochina and Honduras respectively. After extracellular protease treatment, we performed denaturing gel electrophoresis and immunoblotting with an antibody that recognizes the CLAG3 C-terminus. With chymotrypsin, trypsin, or pronase E treatment, a single proteolytic fragment of approximately 35 kDa size was detected; 2 mM phenylmethanesulfonylfluoride (PMSF, a serine protease inhibitor) inhibited release of this fragment when added during chymotrypsin treatment (Fig. 1A). With each protease, semi-quantitative comparison to the retained 160 kDa full length CLAG3 indicated that a significant fraction of parasite CLAG3 is exposed and cleaved at the host cell membrane.

Bottom Line: Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone.These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function.They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

ABSTRACT
The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The clag3 genes of P. falciparum determine this channel's activity in human malaria, but how the encoded proteins contribute to transport is unknown. Here, we used proteases to examine the channel's composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake.

Show MeSH
Related in: MedlinePlus