Pf332 is not exposed at the red blood cell surface.

Abstract

<p>(<b>A</b>) Surface expression of Pf332 was studied in live intact (left panel) or RBC plasma membrane permeabilized (EqtII-treated, right panel) schizont-stage FCR3S1.2 pRBC by flow cytometry. To detect Pf332, monoclonal mouse anti-Pf332-DBL, polyclonal rat anti-Pf332-DBL (N-terminus of Pf332), and polyclonal rabbit anti-Pf332-EB200 (C-terminus of Pf332) were used. As a marker for a positive surface expression, a monoclonal mouse antibody towards the ectodomain of the PfEMP1 variant expressed by the FCR3S1.2 parasite strain was used (anti-PfEMP1-NTSDBL1α). As a marker for an intracellular localization, a mouse monoclonal antibody towards the intracellular acidic terminal segment (ATS) of PfEMP1 was used. Non-immune mouse immunoglobulin G (IgG) and pre-immune rabbit/rat sera were used as negative controls and are displayed in red. Specific anti-Pf332 and anti-PfEMP1 antibodies are displayed in blue. (<b>B</b>) Intact schizont-stage HB3 pRBC were treated with (+) or without (−) trypsin, and whole cell lysates were analyzed by Western blotting using monoclonal anti-PfEMP1-ATS (left) or polyclonal anti-Pf332 antibodies (C-terminus, right). Full-length PfEMP1 and Pf332 are indicated with asterisks. Surface-exposed PfEMP1 is cleaved by trypsin, resulting in a truncated polypeptide migrating at approximately 80 kDa (black arrow head). The blot was also probed with monoclonal mouse anti-spectrin antibodies to confirm equal loading and to show that the anti-PfEMP1-ATS antibody cross-reacts with spectrin (middle).</p