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Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

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PfA-M1 is minimally associated with FV fractions. A. P. falciparum food vacuole fractions were isolated as shown in the schematic diagram and in the Methods section. B. Fractions A to F were analysed for the presence of marker proteins. Equivalent amounts of proteins were loaded for each fraction (corresponding to 4 × 107 iRBC per lane), and immunoblotted with anti-MAP1 antibodies [6] to reveal PfA-M1, or antibodies against Plasmepsin 1 [23](Plas1), PfSERP[18] (SERP) and PfAldolase [15] (Ald) as controls. The numbers on the left indicate Mw in kDa. The densitometric analysis of these immunoblots is presented as Table 1.
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Figure 7: PfA-M1 is minimally associated with FV fractions. A. P. falciparum food vacuole fractions were isolated as shown in the schematic diagram and in the Methods section. B. Fractions A to F were analysed for the presence of marker proteins. Equivalent amounts of proteins were loaded for each fraction (corresponding to 4 × 107 iRBC per lane), and immunoblotted with anti-MAP1 antibodies [6] to reveal PfA-M1, or antibodies against Plasmepsin 1 [23](Plas1), PfSERP[18] (SERP) and PfAldolase [15] (Ald) as controls. The numbers on the left indicate Mw in kDa. The densitometric analysis of these immunoblots is presented as Table 1.

Mentions: PfA-M1 has been recently reported to be delivered to the food vacuole (FV) [26]. This result was obtained by fusing PfA-M1 to the Yellow Fluorescent Protein (YFP) in stably transfected parasites. Some yellow fluorescence was indeed somewhat associated with the FV in these transfected parasites and some fluorescence was also associated with nuclei [26]. Experiments were designed to test whether endogenous PfA-M1 was, indeed trafficked to the FV, and specifically in which form. Parasites were fractionated as previously described [3,20] (Figure 7A) and fractions were immuno-detected with antibodies specific for PfA-M1 and three control marker proteins (Figure 7B). In these analyses, the majority of PfPlasmepsin I, a specific marker of the FV [23] was indeed associated to the pure FV fraction (fraction E, Figure 7B) as well as some PfA-M1. Densitometric measurements indicated that only 16% of the p68 form of PfA-M1 was however associated with this FV fraction, together with traces of p120 and p96 forms, while 40% of Plasmepsin I was associated with this fraction (Table 1). In this assay, the majority of PfA-M1 was associated with the soluble fractions, mainly fraction A. PfAldolase, a cytoplasmic parasite marker [15], was also mainly associated with this soluble fractions, while PfSERP, a protease associated to the parasitophorous membrane [18], was as expected mainly associated with fraction C corresponding to membranes, vacuole, and debris. From this data, it was deduced that only a minority of PfA-M1, in the p68 form, was indeed targeted to the FV, with the majority of PfA-M1 being targeted to other locations inside the parasite. Interestingly these values are in close agreement with the data by Dalal and Klemba who reported a 20% enrichment of the enzymatic activity associated with PfA-M1 in enriched FV fractions [26].


Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole.

Azimzadeh O, Sow C, Gèze M, Nyalwidhe J, Florent I - Malar. J. (2010)

PfA-M1 is minimally associated with FV fractions. A. P. falciparum food vacuole fractions were isolated as shown in the schematic diagram and in the Methods section. B. Fractions A to F were analysed for the presence of marker proteins. Equivalent amounts of proteins were loaded for each fraction (corresponding to 4 × 107 iRBC per lane), and immunoblotted with anti-MAP1 antibodies [6] to reveal PfA-M1, or antibodies against Plasmepsin 1 [23](Plas1), PfSERP[18] (SERP) and PfAldolase [15] (Ald) as controls. The numbers on the left indicate Mw in kDa. The densitometric analysis of these immunoblots is presented as Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914058&req=5

Figure 7: PfA-M1 is minimally associated with FV fractions. A. P. falciparum food vacuole fractions were isolated as shown in the schematic diagram and in the Methods section. B. Fractions A to F were analysed for the presence of marker proteins. Equivalent amounts of proteins were loaded for each fraction (corresponding to 4 × 107 iRBC per lane), and immunoblotted with anti-MAP1 antibodies [6] to reveal PfA-M1, or antibodies against Plasmepsin 1 [23](Plas1), PfSERP[18] (SERP) and PfAldolase [15] (Ald) as controls. The numbers on the left indicate Mw in kDa. The densitometric analysis of these immunoblots is presented as Table 1.
Mentions: PfA-M1 has been recently reported to be delivered to the food vacuole (FV) [26]. This result was obtained by fusing PfA-M1 to the Yellow Fluorescent Protein (YFP) in stably transfected parasites. Some yellow fluorescence was indeed somewhat associated with the FV in these transfected parasites and some fluorescence was also associated with nuclei [26]. Experiments were designed to test whether endogenous PfA-M1 was, indeed trafficked to the FV, and specifically in which form. Parasites were fractionated as previously described [3,20] (Figure 7A) and fractions were immuno-detected with antibodies specific for PfA-M1 and three control marker proteins (Figure 7B). In these analyses, the majority of PfPlasmepsin I, a specific marker of the FV [23] was indeed associated to the pure FV fraction (fraction E, Figure 7B) as well as some PfA-M1. Densitometric measurements indicated that only 16% of the p68 form of PfA-M1 was however associated with this FV fraction, together with traces of p120 and p96 forms, while 40% of Plasmepsin I was associated with this fraction (Table 1). In this assay, the majority of PfA-M1 was associated with the soluble fractions, mainly fraction A. PfAldolase, a cytoplasmic parasite marker [15], was also mainly associated with this soluble fractions, while PfSERP, a protease associated to the parasitophorous membrane [18], was as expected mainly associated with fraction C corresponding to membranes, vacuole, and debris. From this data, it was deduced that only a minority of PfA-M1, in the p68 form, was indeed targeted to the FV, with the majority of PfA-M1 being targeted to other locations inside the parasite. Interestingly these values are in close agreement with the data by Dalal and Klemba who reported a 20% enrichment of the enzymatic activity associated with PfA-M1 in enriched FV fractions [26].

Bottom Line: Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole.This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole.These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: FRE3206 CNRS/MNHN, Department Regulations, Development, Molecular Diversity, CP52, 61 rue Buffon, F-75005 Paris, France.

ABSTRACT

Background: The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.

Methods: By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.

Results: The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.

Conclusions: The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Show MeSH
Related in: MedlinePlus