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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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Schematic showing models for protease action on PSAC.(A) Functional channel with a variable extracellular loop and permeating solute (red circle and arrow). (B) Trypsin digestion of extracellular loop, yielding a positively charged end. Solute transport is preserved. (C-E) Possible models of chymotrypsin inhibition in sensitive clones. Panel C shows a collapsed pore due to proteolysis at a critical site in the extracellular loop. Panel D shows steric hindrance of the channel pore, which may prevent solute permeation. Panel E shows cleavage at additional site(s) exposed after cleavage of the extracellular loop.
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pone-0093759-g005: Schematic showing models for protease action on PSAC.(A) Functional channel with a variable extracellular loop and permeating solute (red circle and arrow). (B) Trypsin digestion of extracellular loop, yielding a positively charged end. Solute transport is preserved. (C-E) Possible models of chymotrypsin inhibition in sensitive clones. Panel C shows a collapsed pore due to proteolysis at a critical site in the extracellular loop. Panel D shows steric hindrance of the channel pore, which may prevent solute permeation. Panel E shows cleavage at additional site(s) exposed after cleavage of the extracellular loop.

Mentions: Our new biochemical studies should help clarify how CLAG3 contributes to transport. Because they demonstrate a role for integral membrane CLAG3, they exclude models with CLAG3 functioning as only a soluble enzymatic activator of channels. The critical L1115F polymorphism influences protease susceptibility and inhibitor efficacy, but does not affect transport rates in the absence of biochemical interventions. When combined with this residue’s localization to a variant extracellular loop, these findings are difficult to reconcile with an active site on an enzyme, and instead suggest that this protein loop is a stable component of the functional channel (Fig. 5A). Sequencing studies have determined that this extracellular protein loop is highly variable amongst parasite clones obtained from divergent locations [52]. Thus, this loop is probably unstructured; it appears to serve a scaffolding role as it holds functional domains that flank it together. Our findings provide experimental evidence as trypsin-mediated cleavage at one or more arginines and lysines within this extracellular loop does not interfere with solute transport (Fig. 4A). Because the trypsin-cleaved polypeptide transports solutes with unchanged rates, an intact extracellular loop at this site is not essential for preserving channel integrity (Fig. 5B). In contrast, CLAG3 sequences flanking this variable domain are conserved in geographically divergent parasite lines; because these flanking sequences are resistant to proteolysis, our biochemical studies suggest either a globular structure that conceals protease-susceptible residues or that the variable domain is bounded on both sides by transmembrane domains.


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

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

Schematic showing models for protease action on PSAC.(A) Functional channel with a variable extracellular loop and permeating solute (red circle and arrow). (B) Trypsin digestion of extracellular loop, yielding a positively charged end. Solute transport is preserved. (C-E) Possible models of chymotrypsin inhibition in sensitive clones. Panel C shows a collapsed pore due to proteolysis at a critical site in the extracellular loop. Panel D shows steric hindrance of the channel pore, which may prevent solute permeation. Panel E shows cleavage at additional site(s) exposed after cleavage of the extracellular loop.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3974804&req=5

pone-0093759-g005: Schematic showing models for protease action on PSAC.(A) Functional channel with a variable extracellular loop and permeating solute (red circle and arrow). (B) Trypsin digestion of extracellular loop, yielding a positively charged end. Solute transport is preserved. (C-E) Possible models of chymotrypsin inhibition in sensitive clones. Panel C shows a collapsed pore due to proteolysis at a critical site in the extracellular loop. Panel D shows steric hindrance of the channel pore, which may prevent solute permeation. Panel E shows cleavage at additional site(s) exposed after cleavage of the extracellular loop.
Mentions: Our new biochemical studies should help clarify how CLAG3 contributes to transport. Because they demonstrate a role for integral membrane CLAG3, they exclude models with CLAG3 functioning as only a soluble enzymatic activator of channels. The critical L1115F polymorphism influences protease susceptibility and inhibitor efficacy, but does not affect transport rates in the absence of biochemical interventions. When combined with this residue’s localization to a variant extracellular loop, these findings are difficult to reconcile with an active site on an enzyme, and instead suggest that this protein loop is a stable component of the functional channel (Fig. 5A). Sequencing studies have determined that this extracellular protein loop is highly variable amongst parasite clones obtained from divergent locations [52]. Thus, this loop is probably unstructured; it appears to serve a scaffolding role as it holds functional domains that flank it together. Our findings provide experimental evidence as trypsin-mediated cleavage at one or more arginines and lysines within this extracellular loop does not interfere with solute transport (Fig. 4A). Because the trypsin-cleaved polypeptide transports solutes with unchanged rates, an intact extracellular loop at this site is not essential for preserving channel integrity (Fig. 5B). In contrast, CLAG3 sequences flanking this variable domain are conserved in geographically divergent parasite lines; because these flanking sequences are resistant to proteolysis, our biochemical studies suggest either a globular structure that conceals protease-susceptible residues or that the variable domain is bounded on both sides by transmembrane domains.

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