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The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains.

Dahlbäck M, Jørgensen LM, Nielsen MA, Clausen TM, Ditlev SB, Resende M, Pinto VV, Arnot DE, Theander TG, Salanti A - J. Biol. Chem. (2011)

Bottom Line: In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins.The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA.Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of International Health, Immunology, University of Copenhagen and the Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen K, Denmark. dahlback@sund.ku.dk

ABSTRACT
Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

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Levels of anti-VAR2CSA IgG reactivity with the native protein after immunizations with recombinant proteins. Bars indicate the relative reactivity against VAR2CSA expressed on the surface of erythrocytes infected with the parasite line FCR3 (IEs). The IEs were labeled by purified IgG (0.5 mg/ml) from rats immunized with recombinant VAR2CSA proteins. IgG specific for the CIDR1 domain from the VAR4 3D7 protein was used as a negative control. The mean fluorescence intensity (MFI) was recorded from 5000 IEs. The assay was performed twice with similar results.
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Figure 5: Levels of anti-VAR2CSA IgG reactivity with the native protein after immunizations with recombinant proteins. Bars indicate the relative reactivity against VAR2CSA expressed on the surface of erythrocytes infected with the parasite line FCR3 (IEs). The IEs were labeled by purified IgG (0.5 mg/ml) from rats immunized with recombinant VAR2CSA proteins. IgG specific for the CIDR1 domain from the VAR4 3D7 protein was used as a negative control. The mean fluorescence intensity (MFI) was recorded from 5000 IEs. The assay was performed twice with similar results.

Mentions: From the point of view of vaccine development it is important to analyze the ability of induced VAR2CSA-specific antibodies to block adhesion of IEs to CSA. Furthermore, such data may provide additional information about the receptor binding site. IgG induced by VAR2CSA constructs were analyzed by FCM for reactivity with native VAR2CSA protein on the surface of homologous FCR3 IEs. All the VAR2CSA-specific IgG purifications were positive in the FCM analysis although to a varying degree (Fig. 5). As expected, the multidomain proteins clearly induced a higher FCM antibody titer.


The chondroitin sulfate A-binding site of the VAR2CSA protein involves multiple N-terminal domains.

Dahlbäck M, Jørgensen LM, Nielsen MA, Clausen TM, Ditlev SB, Resende M, Pinto VV, Arnot DE, Theander TG, Salanti A - J. Biol. Chem. (2011)

Levels of anti-VAR2CSA IgG reactivity with the native protein after immunizations with recombinant proteins. Bars indicate the relative reactivity against VAR2CSA expressed on the surface of erythrocytes infected with the parasite line FCR3 (IEs). The IEs were labeled by purified IgG (0.5 mg/ml) from rats immunized with recombinant VAR2CSA proteins. IgG specific for the CIDR1 domain from the VAR4 3D7 protein was used as a negative control. The mean fluorescence intensity (MFI) was recorded from 5000 IEs. The assay was performed twice with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091200&req=5

Figure 5: Levels of anti-VAR2CSA IgG reactivity with the native protein after immunizations with recombinant proteins. Bars indicate the relative reactivity against VAR2CSA expressed on the surface of erythrocytes infected with the parasite line FCR3 (IEs). The IEs were labeled by purified IgG (0.5 mg/ml) from rats immunized with recombinant VAR2CSA proteins. IgG specific for the CIDR1 domain from the VAR4 3D7 protein was used as a negative control. The mean fluorescence intensity (MFI) was recorded from 5000 IEs. The assay was performed twice with similar results.
Mentions: From the point of view of vaccine development it is important to analyze the ability of induced VAR2CSA-specific antibodies to block adhesion of IEs to CSA. Furthermore, such data may provide additional information about the receptor binding site. IgG induced by VAR2CSA constructs were analyzed by FCM for reactivity with native VAR2CSA protein on the surface of homologous FCR3 IEs. All the VAR2CSA-specific IgG purifications were positive in the FCM analysis although to a varying degree (Fig. 5). As expected, the multidomain proteins clearly induced a higher FCM antibody titer.

Bottom Line: In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins.The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA.Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

View Article: PubMed Central - PubMed

Affiliation: Department of International Health, Immunology, University of Copenhagen and the Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen K, Denmark. dahlback@sund.ku.dk

ABSTRACT
Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.

Show MeSH
Related in: MedlinePlus