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 (Thr 92 ). Mouse fibroblasts (NIH 3T3 cells) transfected with wild-type CD36 or a mutant protein in which Thr 92 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 Thr 92 , 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.The scavenger receptor CD36 expressed on erythroblasts, platelets, monocytes/macrophages, dendritic cells, microvascular endothelial cells, striated muscle cells, adipocytes, and mammary epithelial cells is increasingly recognized as a signaling molecule and/or coreceptor for diverse ligands that are implicated in the pathogenesis of major inflammatory diseases. Specifically, CD36 has been shown to elicit a proinflammatory response in microglial cells in the brain (3, 22) and macrophages in an atheroma (21) through its interaction with fibrillar ␤-amyloid. In binding to CD36, ␤-amyloid inhibits CD36-mediated clearance of oxidized lipoproteins and thus promotes accumulation of lipid peroxidases and accelerated atherogenesis (19). Evidence is also emerging that CD36 acts as a coreceptor for some but not all bacterial ligands for Toll-like receptor 2 activation (17). As a result, CD36-deficient mice were hypersusceptible to the gram-positive microbe Staphylococcus aureus.In Plasmodium falciparum malaria, CD36 has long been considered a major contributor to pathogenesis by acting as a vascular receptor for the adhesion of infected erythrocytes (IRBCs) (16). The deleterious effect of cytoadherence has been attributed to impairment of microcirculatory blood flow, with subsequent tissue hypoxia and organ dysfunction. There is strong clinical evidence to support this mechanical mechanism, such as the demonstration of lower-than-expected oxygen tension and reduced perfusion pressure in the cerebral circulation of patients with cerebral malaria (23,28). IRBC adhesion may a...

Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

“…CD36 acts in cooperation with other host receptors that tether and capture pRBCs from blood flow (14,54). Adhesion of pRBCs to CD36 on endothelial cells has been shown to induce postadhesion signaling events that further strengthen binding by causing CD36 receptor clustering and cytoskeletal rearrangement (18,19). Here, we used concentrations of recombinant CD36 that have previously been shown to support static adhesion of the same parasite clones (31).…”

“…Perhaps the changes in membrane stiffness help stabilize the rolling velocities of pRBCs by increasing the energy needed to peel the cell membrane away from the substrate. If true, then the development of a stiffer membrane and stabilizing of rolling velocity under microvascular shear stresses represent adaptations that increase the tendency of pRBCs to slow on endothelial cells, followed by postadhesive CD36 signaling events that lead to stable binding (18,19).…”

“…CD36 mediates a strong binding interaction and acts in cooperation with intercellular adhesion molecule 1 (ICAM-1) or other upstream rolling receptor interactions to firmly anchor and immobilize pRBCs to endothelial cells (14)(15)(16)(17). The adhesion of pRBCs to CD36 on endothelial cells induces receptor clustering and dephosphorylation of an external threonine residue on CD36, which further strengthen the binding interaction and allow adherent pRBCs to withstand higher shear stress (18,19). During adhesion, leukocytes deform from a spherical to a tear-drop shape (20).…”

CD36: Implications in cardiovascular disease

“…& McGregor, 1995) has also been implicated in binding apoptotic cells (Navazo, Daviet, Savill, Ren, Leung, & McGregor, 1996) and modified LDL (Puente Navazo, Daviet, Ninio, & McGregor, 1996), but because antibody binding to this domain disrupts all CD36 functions, it may relate more to conformational change of the receptor than actual binding site for these ligands. An interesting property of CD36 is the ectophosphorylation of threonine 92 which has been shown to impact binding of certain ligands (Asch, et al, 1993,Hatmi, Gavaret, Elalamy, Vargaftig, & Jacquemin, 1996,Ho, et al, 2005. The intracellular domains of CD36 are located on very short cytoplasmic tails: the N-terminal tail is probably the result of an uncleaved signal peptide.…”

“…Modulation of the binding of platelets to collagen and thrombospondin-1, and the binding of Plasmodium falciparum infected erythrocytes to microvascular endothelium has been shown to be regulated by phosphorylation/dephosphorylation of the ectodomain of CD36, specifically, threonine 92 (Asch, et al 1993,Ho, et al, 2005. Asch et al found that resting platelets constitutively express phosphorylated CD36, and have greater affinity for collagen compared to TSP-1.…”