PfEMP1 binding to recombinant human P-selectin (rhP-selectin) is inhibited by neuraminidase and fucoidin.

(A) PfEMP1 immobilized on anti-ATS beads was digested with neuraminidase (0.5 U/mL) from V cholerae for 1 hour at 37°C prior to incubation with the soluble receptor. The enzyme treatment completely abrogated its interaction with P-selectin (lane 1). Binding of rhP-selectin was seen when PfEMP1 was treated with the neuraminidase buffer alone (lane 2), and the solubilized receptor protein comigrated with rhP-selectin (lane 3). (B) PfEMP1 immobilized on ATS beads were incubated with rhP-selectin in the absence (lane 1) or presence (lane 2) of fucoidin 100 μg/mL. Fucoidin competitively inhibited the binding of rhP-selectin to PfEMP1. An unrelated sugar dextran had no effect on the binding of rhP-selectin (lane 3). The solubilized receptor protein comigrated with rhP-selectin (lane 4). (C) PfEMP1 immobilized on anti-ATS beads were incubated with 0.5 U/mL neuraminidase (lane 2) or with the buffer alone (lane 1) for 1 hour at 37°C. Treated beads were extensively washed and run on an SDS-PAGE gel for 30 minutes and blotted as described. The blot was probed using anti-ATS. There was no significant difference in the molecular size or thickness of the bands in the 2 lanes by densitometry.