INHIBITORY ACTIVITIES OF SULFATED PROTEOGLYCANS ON CHONDROITIN SULFATE A-MEDIATED CYTOADHERENCE OF PLASMODIUM FALCIPARUM ISOLATES FROM THAILAND SUJITTRA CHAISAVANEEYAKORN, PRACHYA KONGTAWELERT, PORNPIMON ANGKASEKWINAI, ROSE McGREADY, FRANC ¸ OIS NOSTEN, AND SANSANEE C. CHAIYAROJ Department of Microbiology, Faculty of Science, and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Biochemistry, Chiang Mai University, Chiang Mai, Thailand; Shoklo Malaria Research Unit, Mae Sot, Tak, Thailand; Nuffield Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom Abstract. Chondroitin sulfate A (CSA) is an important receptor for Plasmodium falciparum-infected erythrocytes in the placenta. To study the molecular interaction between parasitized erythrocytes (PE) to CSA, we performed in vitro cytoadherence inhibition assays of PE infected with wild and laboratory isolates of P. falciparum to CSA using various glycosaminoglycans (GAGs). Marked decrease in PE adhesion to immobilized CSA and CSA-expressed cells was achieved with soluble chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE) at low concentrations. The effect was dose dependent with the degree of inhibition exceeded that of soluble CSA in certain clinical isolates. The results suggested the influence of oversulfation of CS variant chains on PE adherence to CSA. Interestingly, PE of the tested wild isolates could adhere to immobilized CSD and CSE at different levels while PE of CSA-selected laboratory lines could not. Partial inhibitory activity was observed when chondroitin sulfate C (CSC), chondroitin sulfate B (CSB), and polyolpolysulfate were used even at high concentrations. Keratan sulfate, colominic acid, and Suramine were unable to inhibit PE adherence. Taken together, the results confirm that the 4-sulfate amino sugar moiety, as well as the basic disaccharide structure of N-acetylgalactosamine linked to glucuronic acid, may influence the degree of this molecular interaction. However, other sulfation patterns that could influence the interaction could not be overlooked, as in the case of CSD which contains 2-O-sulfation at glucuronic acid. Studies using pentosan polysulfate, an oversulfated molecule with a xylan backbone, as an inhibitor also showed a reduction of PE adherence of most isolates tested. Thus, only the sulfate content and pattern of this molecule could affect the adhesive interactions. In addition, difference in capacity of low molecular weight heparins to inhibit CSA-mediated PE cytoadherence of clinical isolates was also observed, thereby providing evidence on the heterogeneity in cytoadherence characteristics of maternal parasite isolates as well as their therapeutic potentials. INTRODUCTION In areas where malaria is endemic, pregnancy is often com- plicated by Plasmodium falciparum infection. Maternal ane- mia and low birth weight of newborns as well as increased maternal morbidity and mortality are associated with the se- questration of P. falciparum malarial parasites in the intervil- lous spaces of the placenta. 1,2 This process appears to be me- diated by adhesive interactions between parasite ligands on the surface of parasitized erythrocytes (PE) and host mol- ecules. It has been demonstrated that a high-molecular weight galactosaminoglycan, chondroitin sulfate A (CSA), on syncy- tiotrophoblasts that line placenta is a receptor for PE. 3 An- other study suggested that intercellular adhesion molecule-1 (ICAM-1) on the syncytiotrophoblast surface can act as a receptor for parasite adhesion in placental sequestration. 4 Recently, Beeson and others 5 identified a new placental re- ceptor, hyaluronic acid (HA) and demonstrated that some isolates adhered to either CSA or HA, while others demon- strated binding to both receptors. The same study also indi- cated that other unknown receptor(s) may be involved in PE adherence. Apart from the direct binding of PE to the adher- ence receptors, non-immune immunoglobulins bound to the surface of PE were subsequently proposed to act as a bridge to Fc-receptors present in the placenta. 6 It has become apparent that glycosaminoglycan (GAG) oli- gosaccharide sequences, which occur in free forms or as com- ponents of proteoglycans, participate in a number of impor- tant biologic processes. 7,8 In the case of blood stage malaria, CSA was the first GAG to be identified as a cell adhesion molecule for PE in both static and flow-based assays. 9–12 Fur- thermore, CSA side chains of thrombomodulin, a type I trans- membrane glycoprotein expressed by vascular endothelial cells, have also been implicated as a mediator of PE adher- ence. 13–15 The CS chains comprise a repeating disaccharide unit of N-acetyl-galactosamine (GalNAc) and hexuronic acid, both monosaccharides being linked with each other -glyco- sidically (1-3). 16 Non-sulfated sequences with this structure are named chondroitin. Heterogeneity in sulfation pattern and hexuronic acid compositions differentiates CS into vari- ous types. CSA contains a GalNAc-4-sulfate linked to gluc- uronic acid (GlcA). Chondroitin sulfate C (CSC) is distin- guished from CSA by a 6-sulfation amino sugar moiety. Chondroitin sulfate B (CSB) is similar in sulfation to CSA but the uronic acid is predominantly iduronic acid (IdoA). Chon- droitin sulfate D (CSD) and chondroitin sulfate E (CSE) con- tain repeating units of GlcA(2SO 3 - )1-3GalNAc(6SO 3 - ) and GlcA1-3GalNAc (4SO 3 - , 6SO 3 - ), respectively. 17 Biochemical characterization of the molecular interaction between parasite molecule(s) on the surface of PE and CSA of placental tissue has been performed. In a study by Beeson and others, investigators found that inhibition of adherence of a laboratory-selected isolate, CS2, was dependent on the minimum chain length of CSA. 18 The CS chain of 12-14 monosaccharide residues and under sulfation was the impor- tant structural feature of active CSA. Alkhalil and others later demonstrated that placental intervillous spaces contain high levels of CS proteoglycans. 19 Of the CS isolated from the placenta, the low-sulfated chondroitin sulfates most effi- ciently bind PE. 19 This study conclusively established that the minimum CS chain length required for effective inhibition of PE binding to the placental CS is a dodecasaccharide. In this study, we have investigated specific characteristics of PE and CSA interaction using various GAGs together with Am. J. Trop. Med. Hyg., 70(2), 2004, pp. 149–157 Copyright © 2004 by The American Society of Tropical Medicine and Hygiene 149