Chondroitin sulfate proteoglycan expression and binding of Plasmodium falciparum-infected erythrocytes in the human placenta during pregnancy

Abstract

A characteristic feature of malaria during pregnancy is the sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the intervillous spaces of the placenta. We have recently shown that unusually low-sulfated chondroitin sulfate proteoglycans (CSPGs) present in the intervillous spaces mediate the adherence of IRBCs in the placenta. In areas of endemicity, the prevalence of P. falciparum infection in pregnant women peaks during weeks 13 to 20 and then gradually declines, implying that the placental CSPGs are available for IRBC adhesion early during the pregnancy. However, there is no information on the expression and composition of CSPGs during pregnancy. In this study, the expression pattern of CSPGs during the course of pregnancy was investigated. The CSPGs were purified from placentas of various gestational ages, characterized, and tested for the ability to bind IRBCs. The data demonstrate that the CSPGs are present in the intervillous spaces throughout the second and third trimesters. The levels of CSPGs expressed per unit tissue weight were similar in placentas of various gestational ages. However, the structures of the intervillous-space CSPGs changed considerably during the course of pregnancy. In particular, the molecular weight was decreased, with an accompanying gradual increase in the CSPG size polydispersity, from 16 weeks until 38 weeks. The sulfate content was increased considerably after 24 weeks. Despite these structural changes, the CSPGs of placentas of various gestational ages efficiently supported the binding of IRBCs. These results demonstrate that CSPGs can mediate the sequestration of IRBCs in the intervillous spaces of the placenta during the entire second and third trimesters and possibly during the later part of the first trimester as well.

FIG. 1.

Isolation of CSPGs from extracts of human placentas by DEAE-Sephacel chromatography. The extracts of human placentas were passed through columns of DEAE-Sephacel (3.5 by 12 cm) and eluted with a linear gradient of 0.15 to 0.8 M NaCl. Shown is the elution pattern of proteoglycans from the extract of a 16-week-old placenta. The extracts of 24-, 28-, 33-, and 38-week-old placentas showed similar elution patterns (not shown). Note that the uronic acid levels in fractions 23 to 32 are significantly lower than indicated by absorption at 530 nm because of interference by the high contents of coeluted proteins and nucleic acids. Fractions containing CSPGs were pooled as indicated by the horizontal bars. Fraction I, containing the CSPGs of the placental intervillous spaces, was studied further.

FIG. 2.

Sepharose CL-6B chromatography of placental intervillous space CSPGs. The CSPGs from placentas of various gestational ages, after purification by CsBr density gradient centrifugation as described previously (2), were chromatographed on Sepharose CL-6B columns (2 by 65 cm) in 50 mM sodium acetate-150 mM NaCl (pH 6.0) containing 4 M guanidine-HCl. The column was calibrated with standard molecular weight marker proteins. The elution position of blue dextran (BD), BSA, and glucose (Glc) are indicated. The CSPG-containing fractions were pooled as indicated by the horizontal bars, dialyzed, and lyophilized.

FIG. 3.

Adherence of IRBCs to the placental CSPGs. (A) CSPGs purified from placentas of various gestational ages were applied to plastic petri dishes at the indicated concentrations, blocked with BSA, and then overlaid with IRBCs. The unbound cells were washed, and bound cells were fixed, stained, and counted under a light microscope. Shown is the dose-dependent binding of IRBCs to CSPGs from 16 (○)- and 33 (•)-week-old placentas. The IRBC binding patterns of CSPGs from 24-, 28-, and 38-week-old placentas were similar to those of CSPGs from 16- and 33-week-old placentas (not shown). (B) CSPGs previously purified from a full-term placenta, i.e., BCSPG-2 fraction (2), were applied at 200 ng/ml and blocked with BSA. IRBCs, incubated with the CSPGs at the indicated concentrations (expressed with respect to their CS chain contents) of various placentas, were layered on CSPG-coated spots. The unbound cells were washed, and bound cells were fixed, stained with Giemsa stain, and counted under a light microscope. •, CSPGs from 16-week-old placentas; ○, CSPGs from 24-week-old placentas; ▴, CSPGs from 28-week-old placentas; ▪, CSPGs from 33-week-old placentas; □, CSPGs from 38-week-old placentas.