Ectophosphorylation of CD36 regulates cytoadherence of Plasmodium falciparum to microvascular endothelium under flow conditions

Abstract

The adhesion of Plasmodium falciparum-infected erythrocytes (IRBCs) to human dermal microvascular endothelial cells (HDMECs) under flow conditions is regulated by a Src family kinase- and alkaline phosphatase (AP)-dependent mechanism. In this study, we showed that the target of the phosphatase activity is the ectodomain of CD36 at threonine-92 (Thr92). Mouse fibroblasts (NIH 3T3 cells) transfected with wild-type CD36 or a mutant protein in which Thr92 was substituted by Ala supported the rolling and adhesion of IRBCs. However, while the Src family kinase inhibitors PP1 and PP2 and the specific AP inhibitor levamisole significantly reduced IRBC adhesion to wild-type CD36 transfectants as with HDMECs, the inhibitors had no effect on IRBC adhesion to the mutant cells. Using a phosphospecific antibody directed at a 12-amino-acid peptide spanning Thr92, we demonstrated directly that CD36 was constitutively phosphorylated and could be dephosphorylated by exogenous AP. Endothelial CD36 was likewise constitutively phosphorylated. The phosphospecific antibody inhibited IRBC adhesion to HDMECs that could be reversed by preincubating the antibody with the phosphorylated but not the nonphosphorylated peptide. Pretreatment of HDMECs with AP abrogated the effect of PP1 on IRBC adhesion. Collectively, these results are consistent with a critical role for CD36 dephosphorylation through Src family kinase activation in regulating IRBC adhesion to vascular endothelium.

FIG. 1.

Flow cytometric and Western blot analyses of NIH 3T3 transfectants. Cells expressing (A) wild-type human CD36 (hCD36) (clone 1-10) or (B) mutant protein CD36ala92 (clone 22-2-F1) were stained with MAb OKM5 (5 μg/ml) for flow cytometric analysis. Dashed curves represent cells stained with secondary antibody alone. FL1-H, fluorescent intensity at 530 nm. For Western blot analysis, CD36 was immunoprecipitated from 1-10, 22-2-F1, and untransfected 3T3 cells. After electrophoresis, the blots were first probed with (D) a phosphospecific Ab (pCD36) (10 μg/ml) raised to a 12-amino-acid peptide flanking Thr⁹² and then reprobed with (C) a polyclonal anti-CD36 Ab (1 μg/ml).

FIG. 2.

Effect of exogenous AP on CD36 phosphorylation in transfectants expressing wild-type CD36. (A) For flow cytometry, untreated and AP-treated cells were stained with OKM5 (5 μg/ml) and the phosphospecific Ab (pCD36) (10 μg/ml). Normal mouse and sheep IgG were used as controls. Dashed curves represent cells stained with secondary antibody alone. FL1-H, Fluorescent intensity at 530 nm. (B) For Western blotting, untreated and AP-treated immunoprecipitates were first probed with a phosphospecific Ab (pCD36) and then reprobed with a polyclonal anti-CD36 Ab.

FIG. 3.

Effects of the Src family kinase inhibitors PP1 and PP2 and the specific AP inhibitor levamisole on IRBC adhesion to NIH 3T3 transfectants expressing wild-type CD36 under flow conditions. Confluent monolayers of transfectants were pretreated with 10 μM of (A) PP1, (B) PP2, (C) the inactive analog PP3, or (D) levamisole, 1 mM, for 30 min at 37°C. IRBCs were infused at 1 dyne/cm² (n = 9 for PP1 and n = 6 for PP2, PP3, and levamisole). The total numbers of adherent cells at the end of the 7-min infusion for inhibitors versus controls, respectively, were (A) 105 ± 21 versus 184 ± 37 IRBCs/mm², (B) 110 ± 30 versus 179 ± 31 IRBCs/mm², (C) 159 ± 43 versus 179 ± 31, and (D) 81 ± 19 versus 179 ± 31 IRBCs/mm². P values shown are for paired comparison of each treatment to the corresponding controls.

FIG. 4.

Effects of the Src family kinase inhibitors PP1 and PP2 and the specific AP inhibitor levamisole on IRBC adhesion under flow conditions to (top) 3T3 transfectants expressing mutant CD36 (CD36ala92) or (bottom) transfectants expressing wild-type CD36. Bars represent the total numbers of adherent IRBCs at the end of the 7-minute infusion (control, 177 ± 28 IRBCs/mm²; PP1, 163 ± 27 IRBCs/mm²; PP2, 160 ± 29 IRBCs/mm²; PP3, 178 ± 29 IRBCs/mm²; and levamisole, 157 ± 30 IRBCs/mm²; n = 6 [n = 9 for PP1, bottom panel]). P values shown are for paired comparison of each treatment to the corresponding control.

FIG. 5.

Phosphospecific Ab and HDMECs. (A) Western blot analysis of CD36 immunoprecipitated from HDMECs. The blots were first probed with a phosphospecific CD36 Ab (pCD36) and then reprobed with a polyclonal anti-CD36 Ab. (B to D) Effect of the phosphospecific Ab on IRBC adhesion to HDMECs under flow conditions. Confluent monolayers were pretreated for 30 min at 37°C with (B) Ab at 10 μg/ml (n = 7), (C) Ab and 2 μg of phosphorylated (Phos) peptide (n = 4), or (D) Ab and 2 μg of nonphosphorylated (Non-phos) peptide (n = 4). The total numbers of adherent cells at the end of the 7-min infusion for inhibitors versus controls, respectively, were (B) 88 ± 19 versus 161 ± 24 IRBC/mm², (C) 145 ± 25 versus 169 ± 21 IRBCs/mm², and (D) 92 ± 22 versus 153 ± 19 IRBCs/mm².