Heparitinase digestion
Red blood cells were washed 3 times in PBS and resuspended at a hematocrit of 10%. For the PvRII erythrocyte binding assay, 1 ml of 10% hct blood was used. To each 1 ml of red blood cells, 0, 0.001, 0.002 or 0.01 International Units (which correspond to 0.6, 1.2 and 6 Sigma Units, respectively) of Heparitinase (EC 4.2.2.8, Seikagaku America, Falmouth, MA) was added. The cells were incubated at 43°C for 90 min. with intermittent agitation. An aliquot of 30 μl of the cells was taken for analysis by flow cytometry (data not shown). The remaining cells were then centrifuged at 3000 RPM for 5 min., and resuspended in 1 ml of complete for use in the PvRII erythrocyte binding assay.
Region II of the P. Vivax DBP is blocked from binding to DARC by the same polyanions that inhibit X4 HIV strains
Within the DBP V3-like peptide is a site that conforms to the consensus heparin binding sequences BBXB and BBBXXB, where B represents a basic amino acid and X represents any amino acid including basic amino acids. Some strains of HIV, such as MN, contain a consensus heparin binding motif in the V3 loop, and many X4 strains can be inhibited from infecting target cells by polyanions which may bind to the V3 loop. Polyanions that have been shown to inhibit HIV infection include pentosan polysulfate, heparin, and the algal-derived sulfated polysaccharides Na-spirulan, Ca-spirulan and Na-hornan (Na-HOR). These polyanions also inhibited the binding of DARC+ erythrocytes to PvRII in a dose-dependent manner. They also block P. knowlesi invasion of DARC+ erythrocytes. Chondroitin sulfate C represents a polyanion with similar charge to heparin, but differs in the conformational placement of those charges. Chondroitin sulfate C does not block PvRII binding to DARC, or P. knowlesi invasion of DARC+ erythrocytes, suggesting that the interaction between PvRII and polyanions is related to conformation as well as charge. The same is true for inhibition of the V3 loop by polyanions.
A peptide based on the consensus heparin binding motif in the DBP V3-like peptide binds to heparin with the same affinity as V3 loop peptides of X4 HIV strains and recapitulates polyanion inhibition of PvRII binding to DARC
A peptide based on the consensus heparin binding motif in the DBP V3-like peptide was designed to test the affinity of this site for heparin and compare it to V3 loop peptides and RANTES. Based on results with an alanine substitution mutant of the consensus heparin binding motif in the DBP V3-like peptide, two peptides were designed; one contains the wild type heparin binding site (hbs-wt), and the other contains the same alanine substitutions as the pv22KARA construct (hbs-kara) found in our previous work to abrogate binding to DARC [16]. The peptides are FITC conjugated at the N-terminus and terminate at the carboxyl end with the DYKDDDDK “flag epitope” sequence for fluorescence or antigenic detection, respectively. They are identical with the exception of the alanine substitutions.