Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x is identified as a minimal chondroitin sulfate A-binding region

Abstract

Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.

FIGURE 1.

Residues comprising the CSA-binding pocket located on the subdomain structure of DBL3x. A, DBL3x (Protein Data Bank code 3CPZ) is composed of subdomain 1 (S1, yellow), subdomain 2 (S2, blue), and subdomain 3 (S3, red). Indicated are the sulfate group (yellow, sulfur; red, oxygen) that was fitted to CSA electron density and the residues (green) located within 5 Å of the CSA electron density (see B) on S2 (Lys¹³²⁴, Lys¹³²⁷) and S3 (Arg¹⁴⁶⁷, Arg¹⁵⁰³, Arg¹⁵⁰⁴, Lys¹⁵⁰⁷, and Lys¹⁵¹⁰). Disulfide bonds are shown in cyan, and three of the four S3 disulfides are visible in this view. B, electron density for CSA (blue) was located across the two long helices of S3. Residues within 5 Å of the CSA density are labeled. Compared with A, this panel is rotated by ∼90° around the vertical axis in the page. Lys¹³²⁷ cannot be seen in this view.

FIGURE 2.

The residues of DBL3x important for binding CSA. Wild-type DBL3x (red peak) binds to CSA-expressing CHO K1 cells in a flow cytometry assay. DBL3x proteins bearing the subdomain 3 mutations R1467A and R1503A (blue and green peaks) show significant reductions in binding to CSA on CHO K1 cells. In contrast, the Subdomain 2 double mutation K1324A,K1327A (orange peak) has little effect, binding CSA almost like wild-type DBL3x (red peak). Anti-DBL3x antibody alone (black peak) is shown as a negative control.

FIGURE 3.

Subdomain 3 of DBL3x alone can bind CSA. S3 binds to CSA expressed on the surface of CHO-K1 cells (magenta peak). Preincubating S3 with bovine tracheal CSA (S3 + CSA, cyan peak) considerably reduces S3 binding to the cells. Anti-DBL3x antibody alone (black peak) is shown as a negative control.

FIGURE 4.

S3 and DBL3x binding to CSA oligosaccharide observed by STD NMR. A–C, STD spectrum (C) of 60 μm S3 protein without CSA oligosaccharide at 20 °C, 1024 scans, obtained by subtraction (C = B − A) of the on- (A) from the off- (B) saturation spectra. Present in solution are some low molecular weight contaminants (asterisk), but note that their signals are completely cancelled in the difference spectrum in C. D, 1D ¹H NMR spectrum of CSA oligosaccharide (20 °C, 16 scans, no water suppression). Residual signals from chondroitin-6-O-sulfate are labeled with x. Due to overlap, only selected resonances are labeled. E, STD spectrum of 1:100 molar ratio of S3 and CSA oligosaccharide. F, STD spectrum of 1:100 molar ratio of DBL3x and CSA oligosaccharide at 20 °C using 1024 scans each. G, same as E but the STD data were collected without water suppression at 10 °C to observe the STD signals for the GalNAc-H4, GalNAc-H1, and GlcA-H1 resonances that were obscured by water suppression in E. HDO, residual partially deuterated water.

FIGURE 5.

Varying the NaCl concentration does not affect CSA binding. A and B, STD NMR spectra of S3+CSA oligosaccharide (A) and DBL3x+CSA oligosaccharide (B) at 1:100 molar excess of CSA oligosaccharide in D₂O at 20 °C, 1024 scans each, collected in the presence of 50 mm NaCl (as in Fig. 4), 100 mm NaCl, and 150 mm NaCl.

FIGURE 6.

CSA oligosaccharide binds in the same manner to S3 and to DBL3x as revealed by 2D TrNOE NMR spectra. A, a 2D TrNOE spectrum of free CSA oligosaccharide. B and C, CSA oligosaccharide in the presence of either S3 (B) or DBL3x (C) protein at a molar ratio of 20:1 in D₂O at 30 °C. Note that the cross-peaks in B and in C are nearly identical, indicating that the conformations of CSA oligosaccharide that bind to S3 and to DBL3x are almost identical. Residual signals from chondroitin 6-O-sulfate are labeled with x. Cross-peaks due to low molecular weight contaminants are boxed in red. Due to overlap, only selected resonances are labeled.

FIGURE 7.

Female human plasma from malaria-endemic areas of Mali recognizes S3 and DBL3x. Plasma collected from Malian women show significantly higher reactivity to S3 (A) and DBL3x (B) compared with plasma from Malian men (for both antigens, p < 0.001 by exact Wilcoxon-Mann-Whitney tests). C, positive correlation between the reactivity of Malian female plasma to S3 and DBL3x (r = 0.78, by Spearman rank test, p < 0.0001).