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Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs.

Das S, Hertrich N, Perrin AJ, Withers-Martinez C, Collins CR, Jones ML, Watermeyer JM, Fobes ET, Martin SR, Saibil HR, Wright GJ, Treeck M, Epp C, Blackman MJ - Cell Host Microbe (2015)

Bottom Line: The function of MSP1 and its processing are unknown.Here we show that SUB1-mediated processing of MSP1 is important for parasite viability.Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect.

View Article: PubMed Central - PubMed

Affiliation: The Francis Crick Institute, Mill Hill Laboratory, Mill Hill, London, NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus

Processing Alters MSP1 Secondary Structure and Activates a Heparin and Spectrin-Binding Activity(A) Far-UV CD spectra of rMSP1-Dwt and rMSP1-Dmut as a function of molar absorptivity at 37°C. The vertical double arrow at 222 nm (negative minimum for the alpha helix spectrum) highlights the reduction in CD intensity following cleavage, which was 1.4-fold greater for rMSP1-Dwt than for rMSP1-Dmut. Below: secondary structure composition of the intact and processed proteins.(B) Processing enhances heparin binding. Intact or cleaved rMSP1-Dwt was incubated with heparin agarose ± soluble heparin (1 mg ml−1), then binding assessed by SDS PAGE. Quantification of band intensity (Image Lab) showed that 72.3% ± 6.5% of cleaved rMSP1-Dwt bound heparin agarose but only 18.3% ± 12.1% of intact rMSP1-Dwt (p = 0.006, Student’s t test).(C) Fixed, permeabilized erythrocytes probed with intact or rPfSUB1-cleaved rMSP1-Dwt. Binding was detected by IFA and imaged using equal exposure times. Mean pixel intensity (Adobe Photoshop Histogram tool) was 41.6 ± 4.7 (cleaved) and 12.6 ± 1.4 (uncleaved). Scale bar, 5 μm.(D) Processing enhances binding to IOVs. Vesicles (∼80 μg protein) incubated with intact or rPfSUB1-cleaved rMSP1-Dwt (4 μg) were washed then two different loadings analyzed by western blot in parallel with a 1:1 mixture of the starting protein preparations.(E) Processing enhances binding to the erythrocyte cytoskeleton. Mean density of bound gold beads following immunoEM of Triton X-100-treated erythrocyte ghosts incubated with intact or rPfSUB1-cleaved rMSP1-Dwt then probed with anti-MSP1 antibodies and 5 nm gold-conjugated secondary antibodies. Error bars, SD.(F) Overlay assay. Erythrocyte ghosts or Triton X-100-fractionated ghosts were separated by SDS PAGE, transferred to nitrocellulose, then probed with rPfSUB1-cleaved or intact rMSP1-Dwt and binding detected with anti-MSP1 antibodies.(G) Overlay assay. Erythrocyte ghosts, or purified erythrocyte spectrin (Sigma) mixed with molecular mass marker proteins (GE Healthcare), were subjected to SDS PAGE and either stained or transferred to nitrocellulose and probed as in (F) with rPfSUB1-cleaved rMSP1-Dwt.(H) Overlay assay. Erythrocyte ghosts probed as in (F) with intact or rPfSUB1-cleaved rMSP1-DCD4wt or rMSP1-DCD4mut. The latter, which is refractory to cleavage in the 38/42 region, did not bind spectrin. See also Figure S4.
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fig4: Processing Alters MSP1 Secondary Structure and Activates a Heparin and Spectrin-Binding Activity(A) Far-UV CD spectra of rMSP1-Dwt and rMSP1-Dmut as a function of molar absorptivity at 37°C. The vertical double arrow at 222 nm (negative minimum for the alpha helix spectrum) highlights the reduction in CD intensity following cleavage, which was 1.4-fold greater for rMSP1-Dwt than for rMSP1-Dmut. Below: secondary structure composition of the intact and processed proteins.(B) Processing enhances heparin binding. Intact or cleaved rMSP1-Dwt was incubated with heparin agarose ± soluble heparin (1 mg ml−1), then binding assessed by SDS PAGE. Quantification of band intensity (Image Lab) showed that 72.3% ± 6.5% of cleaved rMSP1-Dwt bound heparin agarose but only 18.3% ± 12.1% of intact rMSP1-Dwt (p = 0.006, Student’s t test).(C) Fixed, permeabilized erythrocytes probed with intact or rPfSUB1-cleaved rMSP1-Dwt. Binding was detected by IFA and imaged using equal exposure times. Mean pixel intensity (Adobe Photoshop Histogram tool) was 41.6 ± 4.7 (cleaved) and 12.6 ± 1.4 (uncleaved). Scale bar, 5 μm.(D) Processing enhances binding to IOVs. Vesicles (∼80 μg protein) incubated with intact or rPfSUB1-cleaved rMSP1-Dwt (4 μg) were washed then two different loadings analyzed by western blot in parallel with a 1:1 mixture of the starting protein preparations.(E) Processing enhances binding to the erythrocyte cytoskeleton. Mean density of bound gold beads following immunoEM of Triton X-100-treated erythrocyte ghosts incubated with intact or rPfSUB1-cleaved rMSP1-Dwt then probed with anti-MSP1 antibodies and 5 nm gold-conjugated secondary antibodies. Error bars, SD.(F) Overlay assay. Erythrocyte ghosts or Triton X-100-fractionated ghosts were separated by SDS PAGE, transferred to nitrocellulose, then probed with rPfSUB1-cleaved or intact rMSP1-Dwt and binding detected with anti-MSP1 antibodies.(G) Overlay assay. Erythrocyte ghosts, or purified erythrocyte spectrin (Sigma) mixed with molecular mass marker proteins (GE Healthcare), were subjected to SDS PAGE and either stained or transferred to nitrocellulose and probed as in (F) with rPfSUB1-cleaved rMSP1-Dwt.(H) Overlay assay. Erythrocyte ghosts probed as in (F) with intact or rPfSUB1-cleaved rMSP1-DCD4wt or rMSP1-DCD4mut. The latter, which is refractory to cleavage in the 38/42 region, did not bind spectrin. See also Figure S4.

Mentions: Size-exclusion chromatography of rPfSUB1-cleaved rMSP1-DCD4wt as well as a similar protein lacking the CD4 tag (rMSP1-Dwt; Figure S1E) showed that, like parasite MSP1 (McBride and Heidrich, 1987), the processed products remain associated under non-denaturing conditions (Figure S4A). This encouraged us to use the recombinant proteins to examine the structural consequences of cleavage. Circular dichroism (CD) of intact and rPfSUB1-processed rMSP1-Dwt, as well as of a mutant (rMSP1-Dmut) that was refractory to cleavage in the 38/42 region (Figure S1E), showed that processing altered the secondary structure of both proteins (Figure 4A). These changes were less extensive in rMSP1-Dmut, indicating that cleavage at the 38/42 region contributed to the conformational rearrangements.


Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs.

Das S, Hertrich N, Perrin AJ, Withers-Martinez C, Collins CR, Jones ML, Watermeyer JM, Fobes ET, Martin SR, Saibil HR, Wright GJ, Treeck M, Epp C, Blackman MJ - Cell Host Microbe (2015)

Processing Alters MSP1 Secondary Structure and Activates a Heparin and Spectrin-Binding Activity(A) Far-UV CD spectra of rMSP1-Dwt and rMSP1-Dmut as a function of molar absorptivity at 37°C. The vertical double arrow at 222 nm (negative minimum for the alpha helix spectrum) highlights the reduction in CD intensity following cleavage, which was 1.4-fold greater for rMSP1-Dwt than for rMSP1-Dmut. Below: secondary structure composition of the intact and processed proteins.(B) Processing enhances heparin binding. Intact or cleaved rMSP1-Dwt was incubated with heparin agarose ± soluble heparin (1 mg ml−1), then binding assessed by SDS PAGE. Quantification of band intensity (Image Lab) showed that 72.3% ± 6.5% of cleaved rMSP1-Dwt bound heparin agarose but only 18.3% ± 12.1% of intact rMSP1-Dwt (p = 0.006, Student’s t test).(C) Fixed, permeabilized erythrocytes probed with intact or rPfSUB1-cleaved rMSP1-Dwt. Binding was detected by IFA and imaged using equal exposure times. Mean pixel intensity (Adobe Photoshop Histogram tool) was 41.6 ± 4.7 (cleaved) and 12.6 ± 1.4 (uncleaved). Scale bar, 5 μm.(D) Processing enhances binding to IOVs. Vesicles (∼80 μg protein) incubated with intact or rPfSUB1-cleaved rMSP1-Dwt (4 μg) were washed then two different loadings analyzed by western blot in parallel with a 1:1 mixture of the starting protein preparations.(E) Processing enhances binding to the erythrocyte cytoskeleton. Mean density of bound gold beads following immunoEM of Triton X-100-treated erythrocyte ghosts incubated with intact or rPfSUB1-cleaved rMSP1-Dwt then probed with anti-MSP1 antibodies and 5 nm gold-conjugated secondary antibodies. Error bars, SD.(F) Overlay assay. Erythrocyte ghosts or Triton X-100-fractionated ghosts were separated by SDS PAGE, transferred to nitrocellulose, then probed with rPfSUB1-cleaved or intact rMSP1-Dwt and binding detected with anti-MSP1 antibodies.(G) Overlay assay. Erythrocyte ghosts, or purified erythrocyte spectrin (Sigma) mixed with molecular mass marker proteins (GE Healthcare), were subjected to SDS PAGE and either stained or transferred to nitrocellulose and probed as in (F) with rPfSUB1-cleaved rMSP1-Dwt.(H) Overlay assay. Erythrocyte ghosts probed as in (F) with intact or rPfSUB1-cleaved rMSP1-DCD4wt or rMSP1-DCD4mut. The latter, which is refractory to cleavage in the 38/42 region, did not bind spectrin. See also Figure S4.
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Related In: Results  -  Collection

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fig4: Processing Alters MSP1 Secondary Structure and Activates a Heparin and Spectrin-Binding Activity(A) Far-UV CD spectra of rMSP1-Dwt and rMSP1-Dmut as a function of molar absorptivity at 37°C. The vertical double arrow at 222 nm (negative minimum for the alpha helix spectrum) highlights the reduction in CD intensity following cleavage, which was 1.4-fold greater for rMSP1-Dwt than for rMSP1-Dmut. Below: secondary structure composition of the intact and processed proteins.(B) Processing enhances heparin binding. Intact or cleaved rMSP1-Dwt was incubated with heparin agarose ± soluble heparin (1 mg ml−1), then binding assessed by SDS PAGE. Quantification of band intensity (Image Lab) showed that 72.3% ± 6.5% of cleaved rMSP1-Dwt bound heparin agarose but only 18.3% ± 12.1% of intact rMSP1-Dwt (p = 0.006, Student’s t test).(C) Fixed, permeabilized erythrocytes probed with intact or rPfSUB1-cleaved rMSP1-Dwt. Binding was detected by IFA and imaged using equal exposure times. Mean pixel intensity (Adobe Photoshop Histogram tool) was 41.6 ± 4.7 (cleaved) and 12.6 ± 1.4 (uncleaved). Scale bar, 5 μm.(D) Processing enhances binding to IOVs. Vesicles (∼80 μg protein) incubated with intact or rPfSUB1-cleaved rMSP1-Dwt (4 μg) were washed then two different loadings analyzed by western blot in parallel with a 1:1 mixture of the starting protein preparations.(E) Processing enhances binding to the erythrocyte cytoskeleton. Mean density of bound gold beads following immunoEM of Triton X-100-treated erythrocyte ghosts incubated with intact or rPfSUB1-cleaved rMSP1-Dwt then probed with anti-MSP1 antibodies and 5 nm gold-conjugated secondary antibodies. Error bars, SD.(F) Overlay assay. Erythrocyte ghosts or Triton X-100-fractionated ghosts were separated by SDS PAGE, transferred to nitrocellulose, then probed with rPfSUB1-cleaved or intact rMSP1-Dwt and binding detected with anti-MSP1 antibodies.(G) Overlay assay. Erythrocyte ghosts, or purified erythrocyte spectrin (Sigma) mixed with molecular mass marker proteins (GE Healthcare), were subjected to SDS PAGE and either stained or transferred to nitrocellulose and probed as in (F) with rPfSUB1-cleaved rMSP1-Dwt.(H) Overlay assay. Erythrocyte ghosts probed as in (F) with intact or rPfSUB1-cleaved rMSP1-DCD4wt or rMSP1-DCD4mut. The latter, which is refractory to cleavage in the 38/42 region, did not bind spectrin. See also Figure S4.
Mentions: Size-exclusion chromatography of rPfSUB1-cleaved rMSP1-DCD4wt as well as a similar protein lacking the CD4 tag (rMSP1-Dwt; Figure S1E) showed that, like parasite MSP1 (McBride and Heidrich, 1987), the processed products remain associated under non-denaturing conditions (Figure S4A). This encouraged us to use the recombinant proteins to examine the structural consequences of cleavage. Circular dichroism (CD) of intact and rPfSUB1-processed rMSP1-Dwt, as well as of a mutant (rMSP1-Dmut) that was refractory to cleavage in the 38/42 region (Figure S1E), showed that processing altered the secondary structure of both proteins (Figure 4A). These changes were less extensive in rMSP1-Dmut, indicating that cleavage at the 38/42 region contributed to the conformational rearrangements.

Bottom Line: The function of MSP1 and its processing are unknown.Here we show that SUB1-mediated processing of MSP1 is important for parasite viability.Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect.

View Article: PubMed Central - PubMed

Affiliation: The Francis Crick Institute, Mill Hill Laboratory, Mill Hill, London, NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus