This project elucidates the actions of heparin and heparan sulfate (H/HS) and heparin-mimetic sulfated xylan oligosacchrides in malarial infection and pathogenicity. Heparan sulfate proteoglycan is believed to be an hepatocyte receptor for malarial sporozoite invasion through the binding of the circumsporozoite membrane protein (Nussenzweig & coworkers,1993). In addition, erythocyte membrane H/HS might be a receptor for the invasion of normal RBC by newly released merozoites, and it is involved as a receptor in the complex reactions of sequestration of parasitized RBC to the microvasculature in severe malaria. Heparin has been shown to inhibit the parasite invasion of hepatocytes and RBC in vitro, and it also rapidly disolves rosettes in vitro and in vivo. Heparin was tried as a treatment for cerebral malaria but was abandoned because of bleeding toxicity. The molecular details of these functional reactions of H/HS remain to be clarified and applied. This year, to study whether there is a degree of structural specificity in the H/HS hepatocyte receptor function, we conducted a preliminary study of the capacity of eight S-oligoS, range of concentration 2.5 ug - 25 ug, to inhibit hepatocyte invasion by malaria sporozoites in vitro, following our macro combinatorial strategy [HD 01315-07]. The S-oligoS Components were representative of the size and anionic distribution range of our complete series. The target test was an Inhibition of Liver Stage Development Assay (ILSDA) using Plasmodium yoelii (J. Sacci, 2001). Briefly, sporozoites are prepared from the heads and thorax of infected mosquitoes by the Ozaki method. ~24h murine hepatocyte monolayer cultures in multi-well plates are infected and incubated for 3 h, washed and incubated for 48 h. Schizonts are visualized by immunoflourescent staining and counted under an epifluorescent microscope to measure the percent inhibition. Results showed that only two of the 8 S-oligoS exhibited significantly high and concentration-dependent capacity to inhibit the schizont development . One Component was relatively small, ~ 3500 in mass (~ a dodecamer which would be devoid of anti-thrombin capacity). The other Component was ~ 7000 in mass (~22 saccharides). Complete dose-response curves in the ILSDA assays were not measured in this study, but an estimate of the IC50s for these S-oligoS was ~1 uM. A similar prliminary study was conducted on the capacity of these S-oligoS, concentration 0.5 ng - 5000 ng, to inhibit the invasion of erythrocytes by plasmodium parasite clones D6 and W2. Only one Component displayed inhibitory capacity at these low concentrations. This S-oligoS was ~12000 in mass and exhibited an IC50 against W2 comparable to that of chloroquine (0.26 nM vs 0.41 nM, resp.) but less active against D6 (0.21 nM vs 0.01 nM). The mefloquine standard was ~10 fold more potent in both clones (W2, 0.26 nM vs 0.01 nM; D6, 0.17 nM vs 0.02 nM). These assays for the IC50s will be repeated at a higher concentration range. We express the relative activities on the basis of molarity rather than ug because the mass of the S-oligoS varies and all are higher than that of the standards. The above results will be reported at the November Glycobiology meeting and the abstract published: AL Stone """"""""Structure-Function Relations of heparin-mimetic sulfated oligoxylans in the inhibition of the invasion of hepatocytes by malaria parasites in vitro"""""""" Glycobiology Vol. 11. 2001. The above data indicate that S-oligoS may have differential capacities to inhibit sporozoite invasion of hepatocytes. Thus, further study of possible structural specificity in this H/HS function is warranted. We will repeat the study using complete dose-response curves. In addition,. a new approach utilizing sensitive binding techniques and varuius potential protein partners will be employed to discriminate between inhibitory and inactive S-oligoS. Such studies would have immediate application to research on the H/HS family.
