Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

Abstract

Background: The HIV surface glycoprotein gp120 (SU, gp120) and the Plasmodium vivax Duffy binding protein (PvDBP) bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM). Infection by either pathogen has been found to be inhibited by polyanions.

Results: Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC). A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes.

Conclusion: The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

Figure 1

Design of peptides hbs-wt and hbs-kara. The consensus heparin binding motif in the DBP V3-like peptide of PvRII is contained in hbs-wt. The same alanine substitutions from a nonbinding mutant of PvRII (pv22KARA), tested in a previous study, were included in hbs-kara [16]. Alanine-substituted amino acids are in blue and red. The N-terminus is to the left and is FITC-conjugated. The final residues at the C-terminus, DYKDDDDK, represent the FLAG epitope and are in bold.

Figure 2

Polyanion inhibition of PvRII binding to DARC+ erythrocytes. Heparin, pentosan polysulfate, and the algal-derived sulfated polysaccharides Ca-spirulan, and Na-hornan (Na-HOR) have been shown to have potent inhibitory activity against HIV binding and infection. Chondroitin sulfate C does not and serves as a control. Inhibition of DARC+ erythrocytes binding to the DBP binding site was determined by comparing the number of COS-7 cells expressing pvRII with rosettes of polyanion-treated DARC+ human erythrocytes (per 20 fields at 200× magnification) with the number of rosettes of untreated erythrocytes.

Figure 3

Polyanion inhibition of P. knowlesi invasion of DARC+ erythrocytes. The same polyanions used to inhibit the pvRII binding assay shown in Figure 2 were used in the P. knowlesi invasion assay. The inhibition of invasion was determined by subtracting the number of chemokine-treated DARC+ human erythrocytes invaded by P. knowlesi merozoites (per 2000 erythrocytes) from the number of untreated DARC+ human erythrocytes invaded by P. knowlesi merozoites, and dividing by the number of untreated, invaded erythrocytes.

Figure 4

Heparin-Sepharose column fractions. Peptides in Table 1 were bound to a heparin-Sepharose column and eluted with NaCl at concentrations of 0.01, 0.15, 0.5, 1.0, and 2.0 M. The optical densities of the fractions of most peptides were determined in the at a wavelength of 280 nm. Representative examples of the optical densities of fractions from three peptides in Table 1 are shown in panels A-C in which the NaCl concentration at which the peak fraction eluted is written above the peak.

Figure 5

Polyanion inhibition of the hbs-wt peptide binding to heparin in the BSA-heparin ELISA. An ELISA was used to determine the inhibition of the hbs-wt peptide binding to heparin by various polyanions. The hbs-wt peptide was added to BSA-heparin coated plates and detected by a horse-radish peroxidase conjugated antibody that recognizes the FLAG epitope. Polyanions used to inhibit hbs-wt peptide binding to heparin included those used in the pvRII binding and P. knowlesi invasion assays. Also included were dextran sulfate and sodium spirulan which have also been shown to be potent inhibitors of X4 HIV strains. Percentage inhibition of binding was determined by dividing the absorbance at each polyanion concentration by the absorbance at 0 μg/ml of the polyanion.

Figure 6

Competitive Inhibition of RANTES-ELISA with V3 loop and hbs-wt peptides. An ELISA format similar to that in Figure 5 was used to measure RANTES binding to heparin and compare the ability of various peptides to compete with this binding. A fixed concentration of 5 nM RANTES was mixed with various concentrations of competitors before addition to BSA-heparin coated plates. RANTES was then detected by an anti-RANTES mAb, a biotinylated secondary antibody and streptavidin conjugated to horseradish peroxidase. Percentage inhibition of binding was determined by dividing the absorbance at each peptide concentration by the absorbance at 0 nM of the peptide.

Figure 7

Heparin inhibition of region II of P. knowlesi EBPs binding to rhesus erythrocytes. The P. knowlesi alpha, beta and gamma proteins are members of the DBP family, bind rhesus erythrocytes by region II, and contain a consensus HBM. Only the alpha protein binds DARC. The PvRII binding assay shown in Figure 2 was repeated using plasmids to express the P. knowlesi EBPs, and using rhesus erythrocytes. Inhibition was determined in the same manner as the PvRII binding assay.

Figure 8

PvRII binding to heparitinase treated DARC+ erythrocytes. DARC+ human erythrocytes were digested with heparitinase I, an endoglycosidase specific for heparan sulfate. The number of rosettes of heparitinase-treated erythrocytes on COS-7 cells transfected with the pvRII expression vector, pHVDR22, is shown as the mean of three separate treatments.