Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding

Abstract

Malaria during pregnancy remains a major health problem in Plasmodium falciparum endemic areas. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Placental malaria can have severe consequences for both mother and child by causing maternal anemia, low birth weight and stillbirth. Several VAR2CSA-based vaccines have been developed and clinically tested but they have failed to induce an antibody response that effectively inhibits placental adhesion of different genetic variants of VAR2CSA. The interaction between VAR2CSA and CS represents a unique case of an evolution-driven high-affinity interaction between a protein and an oncofetal carbohydrate. Here, we report cryo-EM structures of the VAR2CSA ectodomain up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations enable rational design of second-generation placental malaria vaccines eliciting broadly VAR2CSA-reactive antibodies and novel cancer therapies.

Pregnancy and malaria: the perfect storm

“…They elicited antibody against the homologous VAR2CSA variant that blocked CSA binding but showed little or no recognition of other variants. Studies have shown how the VAR2CSA protein is folded to create one, or possibly two, conserved binding regions for CSA [27,28 ▪ ,29 ▪ ]. These binding regions are highly conserved, while adjacent polymorphic and flexible regions may be the main targets for antibodies.…”

Profound Impact of Decline in N-Acetylgalactosamine-4-Sulfatase (Arylsulfatase B) on Molecular Pathophysiology and Human Diseases

“…In human melanoma cell lines and melanoma tissue, decline in ARSB increased PD-L1 (programmed death ligand-1) expression [76]. Silencing ARSB by siRNA also increased PD-L1 expression in human prostatic and hepatic cell lines.…”

“…In experiments in prostate stromal and epithelial cells, when ARSB was silenced, binding of galectin-3 with C4S declined, nuclear galectin-3 increased and facilitated the binding of transcription factor AP-1 to the versican promoter [26]. Increased expression of CSPG4 (chondroitin sulfate proteoglycan 4) [32] and of PD-L1 [76] in human melanoma cells are also dependent on galectin-3. Expression of HIF-1α in human bronchial epithelial cells and in the colonic epithelial cell line NCM460 increased when ARSB was silenced and declined when ARSB was overexpressed [34].…”

Chondroitin Sulfate/Dermatan Sulfate-Protein Interactions and Their Biological Functions in Human Diseases: Implications and Analytical Tools

“…After treatment with chymotrypsin, LC-MS/MS was used to compare the oxidation differences of each peptide. Finally, it was found that the peptide with the largest redox is peptide 543-558, which proves that the surface of the DBL2 and Hb1 groove acts as a CS binding region (Wang K. et al, 2020). The irradiation conditions of HRF must be carefully controlled to avoid secondary modification of the protein.…”

“…Hydroxyl radical footprinting (HRF) uses hydroxyl radicals to rapidly oxidize amino acid side chains (Maleknia and Downard, 2014), which is a covalent labeling method for characterizing GAGprotein complexes. Wang K. et al (2020) used hydroxyl radical HRF to characterize the binding sites between CS and the protein VAR2CSA expressed by the parasite. Hydroxyl radicals were used to rapidly oxidize the recombinantly expressed DBL1-ID2 protein with and without CS.…”