Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein. Part 2
One possible function of the HBM in chemokines, HIV and DBPs is to associate with cell surface proteoglycans. Alternatively, HBMs could participate in binding to negatively charged amino acid side chains on the chemokine receptors. RANTES is known to bind to sulfated polysaccharides as part of its processing and function, but tyrosine sulfation of CCR5 is also important for binding of chemokines and HIV, and sulfation of Tyr 41 on DARC is important for DBP binding. Here we designed a peptide from PvDBP subdomain 1 that contains the HBM, tested its ability to bind sulfated polysaccharides, and compared it to the binding of the PvDBP, PkDBP, P. knowlesi β and γ proteins, HIV V3 loop peptides and RANTES to see if they shared similar binding specificities.
Polyanions
Ca-spirulan, Na-spirulan, and Na-hornan (Na-HOR) were kindly provided by Toshimutsu Hayashi, Department of Virology, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan. Heparin, dextran sulfate, and pentosan polysulfide were obtained from Sigma-Aldrich (St. Louis, MO).
Peptide preparation
Peptides based on the wild type (wt) putative polyamine binding site of the PvDBP and a non-binding mutant, pvR22KARA (Figure 1) were obtained from Gene med Synthesis, Inc. (San Francisco, CA). The synthesis included N-terminal fluoresce in conjugation and HULK purification to greater than 80%. Peptides of the V3 loop were obtained from the NHI AIDS Reagent Program (NHI AIDS Reagent Program, Rockville, Md.)
Heparin-sepharose columns
The binding affinity of the PvDBP HBM and V3 loop peptides for heparin was determined by chromatography on a heparin-Sepharose column. Heparin-Sepharose CL-6B beads (Pharmacia Biotech) were swollen in 50 mM Tris-HCl pH 7.5 (column buffer), degassed for 1 h, and 1 ml of slurry was added to a 10 ml column. The column was equilibrated with 10 volumes of column buffer. Peptides were added at 1 mg/ml in 300 μl and allowed to enter the column. The column was washed with 3 ml of column buffer. The peptide was eluted with 3 ml volumes of increasing NaCl concentrations of 0.01, 0.15, 0.5, 1.0 and 2.0 M, and 0.5 ml fractions were collected. The column was regenerated between peptides by adding alternating 3 ml volumes of 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5 and 0.1 M NaOAc, 0.5 M NaCl, pH 5.0 for three cycles. The column was re-equilibrated with 10 vol. of column buffer before adding the next peptide. Fractions were measured for absorbance at 280 nm on a spectrophotometer.
P. Knowlesi in vitro culture
Whole blood from rhesus macaques was collected in 10% CPD and allowed to separate overnight at 4°C. The erythrocyte phase was washed in RPMI with L-glutamine and supplemented with 25 mM HEPES, 300 μM hypoxanthine, 10 μM thymidine, 1.0 mM sodium pyruvate, and 11 mM glucose. This RPMI with malaria supplements was then used to prepare malaria culture medium by adding to a final concentration of 0.24% sodium bicarbonate and 0.2% Albumax-I (Life Tech, Gibco BRL). Cultures were maintained at a hematocrit of 10% in malaria culture medium under an atmosphere of 5% O2, 5% CO2, balanced N2 (Air Liquide, Houston, TX) at 38°C.