Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA.
BACKGROUND
Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.


METHODS
To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.


RESULTS
The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.


CONCLUSIONS
Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.
Centre for Medical Parasitology, Institute of International Health Immunology and Microbiology, University of Copenhagen, Department of Clinical Microbiology, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark. mafaldaresende@gmail.com
Mafalda
M
Resende
Morten A
MA
Nielsen
Madeleine
M
Dahlbäck
Sisse B
SB
Ditlev
Pernille
P
Andersen
Adam F
AF
Sander
Nicaise T
NT
Ndam
Thor G
TG
Theander
Ali
A
Salanti
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000378:metabolism*; D006801:Humans; D019151:Peptide Library; D010963:Plasmodium falciparum / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D019151:Peptide Library; C512601:VAR2CSA protein, Plasmodium falciparum; C038142:chondroitin sulfate glycosaminoglycan; D002809:Chondroitin Sulfates