Plasmodium falciparum-Infected Erythrocytes Adhere Both in the Intervillous Space and on the Villous Surface of Human Placenta by Binding to the Low-Sulfated Chondroitin Sulfate Proteoglycan Receptor

Abstract

In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies.

Structural Basis for the Adherence of Plasmodium falciparum-infected Erythrocytes to Chondroitin 4-Sulfate and Design of Novel Photoactivable Reagents for the Identification of Parasite Adhesive Proteins

“…Particularly we showed that an unusually low sulfated CSPG localized predominantly in the intervillous space is the natural receptor for the placental IRBC adherence (14). Further we and others demonstrated that optimal IRBC binding requires the participation of both 4-sulfated and 4-nonsulfated disaccharide moieties (15)(16)(17)(18).…”

Chondroitin Sulfate Proteoglycan but Not Hyaluronic Acid Is the Receptor for the Adherence of Plasmodium falciparum-Infected Erythrocytes in Human Placenta, and Infected Red Blood Cell Adherence Up-Regulates the Receptor Expression

“…19 -21 A previous study from our laboratory has shown that a uniquely low-sulfated chondroitin sulfate proteoglycan (CSPG) mediates IRBC adherence, and that the CSPG is localized predominantly in the intervillous space and to a lesser extent on the surface of the syncytiotrophoblast cells. 22 We and others have also demonstrated that a dodecasaccharide motif of C4S consisting of two foursulfated and four-nonsulfated disaccharide moieties comprise the minimum motif for optimal IRBC binding. [23][24][25][26] Thus, C4S has been shown to be the receptor for placental IRBC adherence.…”

“…22 The fixed tissues were transferred to ice-cold PBS and stored at 4°C. The tissues were paraffin-embedded, cut into 5-m-thick sections onto glass slides, deparaffinized with xylene, and rehydrated by sequential immersion in 100, 95, and 70% ethanol and then in Tris-buffered saline, pH 8.0 (TBS).…”

“…For immunohistochemical analysis using antibodies against the core proteins of placental CSPG receptor, the tissue sections were treated with protease-free chondroitinase ABC (50 mU/ml) in 100 mmol/L Tris-HCl and 30 mmol/L NaOAc, pH 8.0, at 37°C for 3 hours and washed with TBS. 22 Before staining, all tissue sections were pretreated with 3% H 2 O 2 for 4 minutes and washed with TBS. The tissue sections were blocked with 1:60 diluted normal goat serum in TBS for 3 hours followed by incubation with 10 g/ml of the purified rabbit antibodies against the core proteins of low-sulfated placental CSPG.…”

“…The tissue sections were blocked with 1:60 diluted normal goat serum in TBS for 3 hours followed by incubation with 10 g/ml of the purified rabbit antibodies against the core proteins of low-sulfated placental CSPG. 22 The slides were washed and incubated for 30 minutes with 1:60 diluted biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). For analysis of HA, the sections were incubated with 4-g/ml solutions of HABP.…”