Plasmodium falciparum: Assessment of parasite-infected red blood cell binding to placental chondroitin proteoglycan and bovine tracheal chondroitin sulfate A
Introduction
In endemic areas where people are constantly infected with Plasmodium falciparum, immunity to clinical malaria is acquired during childhood and therefore people, irrespective of gender, are resistant to malaria pathogenesis by their adolescent years (Trape et al., 1994). However, while men are protected by acquired immunity throughout their lives, women become susceptible to malaria during pregnancy (Brabin, 1983, Brabin and Rogerson, 2001, Steketee et al., 2001, Gamain et al., 2007, Hviid and Salanti, 2007). This is because, in pregnant women, P. falciparum infected erythrocytes sequester in the placenta by binding to chondroitin 4-sulfate (C4S) (Fried and Duffy, 1996, Achur et al., 2000, Fried et al., 2006), resulting in the selection of an antigenically distinct variant form of parasite to which the individuals have no immunity. Multiplication of the selected parasites leads to accumulation of IRBCs at high density in the placenta, initiating infiltration of inflammatory cells and inflammatory responses (Ismail et al., 2000, Miller et al., 2002, Rogerson et al., 2007). These processes affect placental physiology, resulting in poor pregnancy outcomes, including low birth weight, spontaneous abortion, and anemia and death in the mother and fetuses (McGregor et al., 1983, Duffy and Fried, 2003). Since the first discovery by Fried and Duffy (Fried and Duffy, 1996), a number of studies have shown that C4S mediates adherence of IRBCs in the placenta (Rogerson and Brown, 1997; Gysin et al., 1999, Achur et al., 2000, Maubert et al., 2000).
Previous studies from our laboratory have shown that a very low sulfated chondroitin sulfate proteoglycans (CSPGs) present predominantly in the intervillous space and at lower levels on the syncytiotrophoblast surface is the receptor of IRBC adherence (Achur et al., 2000, Muthusamy et al., 2004a). The CSPG purified from human placental intervillous space has been used for studying C4S–IRBC interactions (Achur et al., 2000). Alternatively, the commercially available bovine tracheal cartilage chondroitin sulfate A (bCSA) has been used in many studies for studying C4S–IRBC adhesion interactions and adhesion inhibitory immune responses (Chai et al., 2002; Lekana Douki et al., 2002, Muthusamy et al., 2004b, Salanti et al., 2004, Oleinikov et al., 2008). bCSA is a copolymer consisting of 4- and 6-sulfated chondroitin disaccharide moieties (Alkhalil et al., 2000, Muthusamy et al., 2004b). Since bCSA isolation procedure involves extensive proteolysis of cartilage tissue, the core protein of bovine cartilage proteoglycan has been lost and bCSA thus prepared contains short peptides consisting of only a few amino acids residues to which the CSA chains are attached. Moreover, bCSA, in addition to 4-sulfate, contains 39% of 6-sulfate, which interferes with IRBC binding (Alkhalil et al., 2000, Fried et al., 2000, Muthusamy et al., 2004b).
In the present study, to determine the validity of bCSA as an alternative ligand for studying C4S–IRBC adhesion, we evaluated in parallel the characteristics of IRBC binding to the CSPG purified from human placenta and the commercial bCSA using adherent IRBCs selected from three commonly used parasite strains. The data demonstrate that the binding capacity of IRBCs to bCSA is significantly lower than that of placental CSPG. These results should be considered in interpreting data obtained from C4S–IRBC adhesion studies using bCSA.
Section snippets
Placental CSPG and bovine tracheal CSA
The CSPG was isolated from the isotonic buffer extracts of freshly delivered placentas, collected from the Hershey Medical Center Maternity Ward. The placental extracts were applied onto DEAE-Sepharose columns and the bound CSPG was eluted using NaCl gradient as described previously (Achur et al., 2000). The CSPG was then purified by successive CsBr density gradient centrifugation and gel filtration on Sepharose CL-6B (Achur et al., 2000). The final purified CSPG preparation was dialyzed,
Results
To determine the concentration required for effective coating of bCSA onto plastic plates for maximal IRBC binding, we coated plates with bCSA at concentrations ranging from 0.75 to 400 μg/ml, and measured the IRBC binding using three C4S-adherent parasite strains, FCR3-CSPG, 3D7-CSPG and CS2-CSPG. In parallel, we also measured IRBC binding to plastic plates coated with 1.5 to 1600 ng/ml solutions of CSPG purified from human placenta. The binding capacity of IRBCs freshly selected for maximal
Discussion
Subpopulations of P. falciparum parasites, expressing placental adherence-specific PfEMP1 called Var2CSA protein on the IRBC surface, have been shown to bind to C4S. The results presented here show that these C4S-adherent parasites bind well to placental CSPG, the natural receptor for IRBC adherence in human placenta. The results also show that parasites could bind to bCSA, even though it is substantially different in its structural features (consisting of 9% non-sulfated, 52% 4-sulfated and
Acknowledgment
This work was supported by Grant AI45086 from National Institute of Allergy and Infectious Diseases, NIH.
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How specific is Plasmodium falciparum adherence to chondroitin 4-sulfate?
2011, Trends in ParasitologyCitation Excerpt :A bovine tracheal chondroitin sulfate A (bCSA; copolymer of C4S and C6S), consisting of 53% 4-sulfated, 39% 6-sulfated and 8% nonsulfated disaccharide moieties, attached to short peptides formed by the degradation of the core protein of bovine tracheal CSPG has been widely used for the selection of C4S-adherent IRBCs and for evaluating IRBC binding specificity. However, the strength of IRBC binding to bCSA is considerably lower than that to the C4S chains of placental CSPGs [43,44]. Some studies have used structurally undefined decorin to study VAR2CSA–C4S interactions [33,34,45,46].