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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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clag3 genes accounts for the differing sensitivities to chymotrypsin.(A) Mean ± S.E.M. block of sorbitol uptake by chymotrypsin treatment on indicated parental lines and progeny clones (black and gray bars, respectively). (B) Logarithm of odds (LOD) scores from a primary scan of QTL associated with PSAC inhibition. The peak at the 5′ end of chromosome 3 contains the two clag3 genes. The P = 0.05 significance threshold (dashed horizontal line) was calculated from 1000 permutations. Inset shows results from a secondary scan for additional QTL after controlling for the clag3 locus. No other loci reached the P = 0.05 threshold (dashed horizontal line). (C) Osmotic lysis kinetics for indicated parasites after selection for expression of a specific clag3 gene. Black and red traces represent no protease control and chymotrypsin-treated cells, respectively. The ribbon schematic at the top of each panel shows the gene structure for the two clag3 genes in each parasite with active transcription indicated by a bent arrow. The clag3 gene resulting from allelic exchange in HB33rec has a gray shaded 3′ end to indicate the fragment derived from Dd2 (“chimera”). For each parasite, relative expression of the two paralogs is shown with an ethidium-stained gel at the bottom right of each panel. (D) Mean ± S.E.M. chymotrypsin-induced inhibition for each selected parasite.
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pone-0093759-g002: clag3 genes accounts for the differing sensitivities to chymotrypsin.(A) Mean ± S.E.M. block of sorbitol uptake by chymotrypsin treatment on indicated parental lines and progeny clones (black and gray bars, respectively). (B) Logarithm of odds (LOD) scores from a primary scan of QTL associated with PSAC inhibition. The peak at the 5′ end of chromosome 3 contains the two clag3 genes. The P = 0.05 significance threshold (dashed horizontal line) was calculated from 1000 permutations. Inset shows results from a secondary scan for additional QTL after controlling for the clag3 locus. No other loci reached the P = 0.05 threshold (dashed horizontal line). (C) Osmotic lysis kinetics for indicated parasites after selection for expression of a specific clag3 gene. Black and red traces represent no protease control and chymotrypsin-treated cells, respectively. The ribbon schematic at the top of each panel shows the gene structure for the two clag3 genes in each parasite with active transcription indicated by a bent arrow. The clag3 gene resulting from allelic exchange in HB33rec has a gray shaded 3′ end to indicate the fragment derived from Dd2 (“chimera”). For each parasite, relative expression of the two paralogs is shown with an ethidium-stained gel at the bottom right of each panel. (D) Mean ± S.E.M. chymotrypsin-induced inhibition for each selected parasite.

Mentions: We next utilized an available HB3×Dd2 genetic cross to examine inheritance of chymotrypsin effect on transport. Erythrocytes infected with each progeny clone were treated with chymotrypsin under standardized conditions and used for sorbitol uptake measurements (Fig. 2A). To reduce possible contributions from switching between expression of the two clag3 genes, the 7C20, 7C12, and CH361 progeny clones were examined after selection for clag3.1 expression because Dd2, from which these daughters inherit both clag3 genes, is transcription-incompetent for clag3.2[23]. Under these conditions, there was negligible transport inhibition upon chymotrypsin treatment (progeny clones labeled 7C203.1, 7C123.1, and CH3613.1 in Fig. 2A). Because other daughters exhibited a range of chymotrypsin sensitivities, we used QTL analysis to search for contributing genomic loci. A primary scan identified a single significant locus at the 5′ end of chromosome 3 (LOD score of 12.1, Fig. 2B), which includes both clag3 genes and 40 additional genes. A secondary scan, performed after controlling for this locus, did not find additional loci reaching a P = 0.05 significance threshold (Fig. 2B, inset).


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

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

clag3 genes accounts for the differing sensitivities to chymotrypsin.(A) Mean ± S.E.M. block of sorbitol uptake by chymotrypsin treatment on indicated parental lines and progeny clones (black and gray bars, respectively). (B) Logarithm of odds (LOD) scores from a primary scan of QTL associated with PSAC inhibition. The peak at the 5′ end of chromosome 3 contains the two clag3 genes. The P = 0.05 significance threshold (dashed horizontal line) was calculated from 1000 permutations. Inset shows results from a secondary scan for additional QTL after controlling for the clag3 locus. No other loci reached the P = 0.05 threshold (dashed horizontal line). (C) Osmotic lysis kinetics for indicated parasites after selection for expression of a specific clag3 gene. Black and red traces represent no protease control and chymotrypsin-treated cells, respectively. The ribbon schematic at the top of each panel shows the gene structure for the two clag3 genes in each parasite with active transcription indicated by a bent arrow. The clag3 gene resulting from allelic exchange in HB33rec has a gray shaded 3′ end to indicate the fragment derived from Dd2 (“chimera”). For each parasite, relative expression of the two paralogs is shown with an ethidium-stained gel at the bottom right of each panel. (D) Mean ± S.E.M. chymotrypsin-induced inhibition for each selected parasite.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3974804&req=5

pone-0093759-g002: clag3 genes accounts for the differing sensitivities to chymotrypsin.(A) Mean ± S.E.M. block of sorbitol uptake by chymotrypsin treatment on indicated parental lines and progeny clones (black and gray bars, respectively). (B) Logarithm of odds (LOD) scores from a primary scan of QTL associated with PSAC inhibition. The peak at the 5′ end of chromosome 3 contains the two clag3 genes. The P = 0.05 significance threshold (dashed horizontal line) was calculated from 1000 permutations. Inset shows results from a secondary scan for additional QTL after controlling for the clag3 locus. No other loci reached the P = 0.05 threshold (dashed horizontal line). (C) Osmotic lysis kinetics for indicated parasites after selection for expression of a specific clag3 gene. Black and red traces represent no protease control and chymotrypsin-treated cells, respectively. The ribbon schematic at the top of each panel shows the gene structure for the two clag3 genes in each parasite with active transcription indicated by a bent arrow. The clag3 gene resulting from allelic exchange in HB33rec has a gray shaded 3′ end to indicate the fragment derived from Dd2 (“chimera”). For each parasite, relative expression of the two paralogs is shown with an ethidium-stained gel at the bottom right of each panel. (D) Mean ± S.E.M. chymotrypsin-induced inhibition for each selected parasite.
Mentions: We next utilized an available HB3×Dd2 genetic cross to examine inheritance of chymotrypsin effect on transport. Erythrocytes infected with each progeny clone were treated with chymotrypsin under standardized conditions and used for sorbitol uptake measurements (Fig. 2A). To reduce possible contributions from switching between expression of the two clag3 genes, the 7C20, 7C12, and CH361 progeny clones were examined after selection for clag3.1 expression because Dd2, from which these daughters inherit both clag3 genes, is transcription-incompetent for clag3.2[23]. Under these conditions, there was negligible transport inhibition upon chymotrypsin treatment (progeny clones labeled 7C203.1, 7C123.1, and CH3613.1 in Fig. 2A). Because other daughters exhibited a range of chymotrypsin sensitivities, we used QTL analysis to search for contributing genomic loci. A primary scan identified a single significant locus at the 5′ end of chromosome 3 (LOD score of 12.1, Fig. 2B), which includes both clag3 genes and 40 additional genes. A secondary scan, performed after controlling for this locus, did not find additional loci reaching a P = 0.05 significance threshold (Fig. 2B, inset).

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