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    Inhibition of adherences to immobilized chondroitin sulfate A (CSA) of maternal isolates (SML1-4) and a laboratory isolate (SL83-S6) of Plasmodium falciparum by glycosaminoglycans at various concentrations. Values are means for quadruplicate experiments. Bars represent standard errors. CSA = chondroitin sulfate A; CSD = chondroitin sulfate D; CSE = chondroitin sulfate E; SP54 = pentosan polysulfate; POPs = polyolpolysulfate; CSB = chondroitin sulfate B, CSC = chondroitin sulfate C.

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    Inhibition of adherence to immobilized CSD and CSE of maternal isolate SML3 of Plasmodium falciparum by CSA at various concentrations. Values are means for quadruplet experiments. Bars represent standard errors. For definitions of abbreviations, see Figure 1.

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    Cleavage of surface proteins on intact parasitized erythrocytes (PE) before adhesion to immobilized CSA (A) and CSE (B). All data represent the proportion of bound PE expressed as percentage of control. All assays were performed in triplicate or quadruplicate. Bars represent standard errors. For definitions of abbreviations, see Figure 1.

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    Inhibition of adherence to immobilized chondroitin sulfate A at increasing concentrations of heparins and Suramine. Values are means for quadruplicate experiments. Bars represent standard errors.

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INHIBITORY ACTIVITIES OF SULFATED PROTEOGLYCANS ON CHONDROITIN SULFATE A-MEDIATED CYTOADHERENCE OF PLASMODIUM FALCIPARUM ISOLATES FROM THAILAND

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  • 1 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

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 complicated by Plasmodium falciparum infection. Maternal anemia and low birth weight of newborns as well as increased maternal morbidity and mortality are associated with the sequestration of P. falciparum malarial parasites in the intervillous spaces of the placenta.1,2 This process appears to be mediated by adhesive interactions between parasite ligands on the surface of parasitized erythrocytes (PE) and host molecules. It has been demonstrated that a high-molecular weight galactosaminoglycan, chondroitin sulfate A (CSA), on syncytiotrophoblasts that line placenta is a receptor for PE.3 Another 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 others5 identified a new placental receptor, hyaluronic acid (HA) and demonstrated that some isolates adhered to either CSA or HA, while others demonstrated binding to both receptors. The same study also indicated that other unknown receptor(s) may be involved in PE adherence. Apart from the direct binding of PE to the adherence receptors, nonimmune 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) oligosaccharide sequences, which occur in free forms or as components of proteoglycans, participate in a number of important 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 Furthermore, 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 adherence.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 β-glycosidically (β1-3).16 Non-sulfated sequences with this structure are named chondroitin. Heterogeneity in sulfation pattern and hexuronic acid compositions differentiates CS into various types. CSA contains a GalNAc-4-sulfate linked to glucuronic acid (GlcA). Chondroitin sulfate C (CSC) is distinguished 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). Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE) contain repeating units of GlcA(2SO3)β1-3GalNAc(6SO3) and GlcAβ1-3GalNAc (4SO3, 6SO3), 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 important 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 efficiently 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 certain drugs to inhibit clinical and laboratory P. falciparum PE adherence to immobilized CSA and CSA-expressed cells. There were differences in the profile of binding to CS proteoglycans among wild isolates tested. We found that not only specific disaccharide structure and sulfation pattern, but also oversulfation of CS variant chains may exert inhibitory activity on the binding of PE to CSA. We also demonstrated specific adherence of PE to CSD, as well as the interference of heparin on CSA-mediated cytoadherence of PE.

MATERIALS AND METHODS

Plasmodium falciparum isolates and culture.

Clinical P. falciparum isolates were established from peripheral blood or delivered infected placentae of pregnant women from the Shoklo Malaria Research Unit in Mae Sot, Thailand. Parasite clone SL83 was generated from an isolate obtained from Professor Sornchai Looareesuwan (Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand) and maintained as previously described.20 The study received ethical approval from the Ethics Committee of the Faculty of Tropical Medicine of Mahidol University in Bangkok, Thailand. All cultures were maintained using standard in vitro cultivation.21 Parasite line T4 was generated from FAF-EA8 by repeatedly selecting for trypsin-treated PE.9 This parasite line adhered specifically to CSA.

Glycosaminoglycans and cell line.

The following chemicals were used in cytoadherence assays: chondroitin sulfate A (Sigma-Aldrich, St. Louis, MO) from bovine trachea; chondroitin sulfate B (Sigma-Aldrich) from bovine mucosa; chondroitin sulfate C (Seikagaku Kogyo, Tokyo, Japan) from shark cartilage; chondroitin sulfate D (Seikagaku Kogyo) from shark cartilage; chondroitin sulfate E (Seikagaku Kogyo) from squid cartilage; keratan sulfate (Seikagaku Kogyo); colominic acid (Sigma-Aldrich), a sialic acid polymer from Escherichia coli; pentosan polysulfate (SP54; Arthropharm Laboratories, Sydney, Australia); polyolpolysulfate (POPS; Luitpold-Werk, Munich, Germany); Suramine (Germanin; Bayer AG, Leverkusen, Germany); Fragmin (a low molecular weight [LMW] heparin; from Dr. Chalotte Bryno, Pharmacia Upjohn, Uppsala, Sweden); heparin (H-9399; Sigma-Aldrich); and heparin (H-5284; Sigma-Aldrich) from porcine intestinal mucosa. Chondroitin sulfates and heparan sulfate (HS) were linked to dipalmitoyl phosphatidylethanolamine through the reducing terminus, using the method of Sugiura and others.22

Human lung carcinoma cells (ATCC CCL-185, A549) obtained from the American Type Culture Collection (Manassas, VA) were cultured in Ham F12 (Gibco-BRL, Gaithersburg, MD) with 10% fetal bovine serum (Flow Laboratories, McLean, VA). This cell line was used in the SL83 parasite clone selection experiment, for higher levels of adherence to CSA. Cells were seeded at a concentration of 1 × 105 cells/mL into Petri dishes and incubated for at least 24 hours before cytoadherence assays.

Cytoadherence assays.

Cytoadherence assays were performed as described9 with some modifications. Adhesion molecules were preadsorbed to plastic Petri dishes (Nalge Nunc International, Naperville, IL) at 37°C for four hours. We found that preadsorption with 50–100 μg/mL of chondroitin sulfates supported high levels of adhesion. Subsequently, dishes were blocked with 1% bovine serum albumin in phosphate-buffered saline. The immobilized chondroitin sulfates were overlaid with 3% or more trophozoite-infected erythrocytes and adhesion was allowed to occur for 45 minutes at 37°C with occasional gentle agitation. Non-adherent erythrocytes were removed by washing with RPMI 1640 medium-HEPES, pH 6.8. Bound cells were fixed with 2% glutaraldehyde and stained with Giemsa. Results were expressed as the number of parasitized erythrocytes bound per 100 cells or per mm2. For inhibition assays, GAGs were tested for inhibition of PE adhesion to the cell line or to purified receptors by adding GAGs at various concentrations to a PE suspension prior to cytoadherence assays.

Selection for parasite cytoadherence to cell line.

Cytoadherence of clone SL83 was enhanced by performing cytoadherence assays on lung carcinoma cells (A549). After the unattached cells were removed by gentle washing with RPMI 1640 medium-HEPES, pH 6.8, the attached cells were flushed with RPMI 1640 medium-HEPES supplemented with 10% human sera and transferred to new plastic Petri dishes. Fresh erythrocytes were added to a hematocrit of 4%. Parasites were repeatedly selected on five additional occasions until they adhered strongly to the target cells as well as to the immobilized CSA. This selected parasite line was designated SL83-S6.

Treatment of PE with trypsin.

The trypsin sensitivity of PE was determined using the method previously described.9 Briefly, parasite cultures were washed three times with RPMI 1640 medium-HEPES and subsequently treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Biochemicals, Lakewood, NJ) suspended in RPMI 1640 medium-HEPES at final concentrations of 100, 10, and 1 μg/mL. After incubation for 10 minutes at 37°C, soybean trypsin inhibitor (Worthington Biochemicals) was added to concentrations 10 times that of the trypsin. The tubes were incubated at 37°C for five minutes and parasitized cells were washed twice with RPMI 1640 medium-HEPES supplemented with 10% human sera. Cell pellets were resuspended in RPMI 1640 medium-HEPES with 10% serum for cytoadherence assays. Results were expressed as the percent inhibition of binding to target cells or adhesion molecules induced by trypsin treatment compared with a control.

Enzymatic digestion of GAGs.

Chondroitin sulfates immobilized on Petri dishes, as well as A549 cells, were treated with 0.5 units/mL of chondroitinase ABC (Sigma-Aldrich) and chondroitinase ACII (Seikagaku Kogyo) in 0.1 M Tris-HCl, 0.1 M sodium acetate, pH 8.0, for 30 minutes at 37°C. The dishes were then washed with RPMI 1640 medium-HEPES, pH 6.8, blocked, and adhesion of PE was tested as described earlier in this report.

Determination of the 50% inhibitory concentration (IC50).

Determination of the IC50 was done using the closely spaced concentrations near the value of the 50% inhibitory reaction from the semi-log sigmoidal plots.23 Extra cytoadherence inhibition assays needed to be performed using inhibitors at those concentrations. The equation used for the calculation was

IC50=10log(conc<50conc>50)×(%inh>50%50%%inh>50%%inh50%+logconc.>50)

RESULTS

Cytoadherence of PE to purified receptors and CSA-expressed cells.

Fifteen (75%) of 20 maternal isolates of P. falciparum demonstrated PE adhesion to immobilized CSA. The levels of adhesion ranged between 2 and 1,864 PE/mm2 with a mean ± SEM binding level of 250.83 ± 442.18 PE/mm2. Only seven isolates adhered to CD36 and none bound to HS. No difference was found in the levels of PE adherence to CSA when compared between peripheral and placental lines. To assess further the ability of GAGs to inhibit cytoadherence, three peripheral (SML1-3) and one placental (SML4) isolates of P. falciparum with different levels of adhesion were chosen and their adhesion characteristics to purified CSA and HS are shown in Table 1. SL83 was subjected to six repeated rounds of selection for binding to CSA. Its levels of adhesion increased approximately 10-fold from that of the original (Table 1). T4, another laboratory line, bound specifically to the immobilized CSA at high levels.

Inhibition of PE adherence to immobilized CSA by GAGs and polysulfated compounds.

The GAGs and polysulfated compounds at various concentrations were assessed for inhibition of PE adhesion to immobilized CSA. Figure 1 shows that complete inhibition of PE adherence was found with CSA polysaccharides in most isolates tested. Significant inhibition of CSD and CSE polysaccharides was observed in all isolates. The IC50 derived for CSE was lower than those of CSD polysaccharides. Interestingly, as with erythrocytes infected with SML3, 96% inhibition was seen when using CSE polysaccharides at a concentration of 10 μg/mL, while CSA at the same concentration demonstrated only 59% inhibition. With SML2-infected PE, a similar observation found that IC50 values derived for CSE and CSA on PE adhesion to CSA were 0.04 μg/mL and 0.2 μg/mL, respectively.

Polyolpolysulfate and SP54 were used as controls for sulfate contents and patterns. Only partial inhibitory activity was observed when POPs was used even at high concentrations. Conversely, SP54 demonstrated similar inhibitory effects on SML2, SML4, and SL83-S6 adherence to CSA, with IC50 values of 2.4, 1.8, and 3.4 μg/mL, respectively.

Little or no inhibitory activity of colominic acid and keratan sulfate (for SL83-S6) was observed. CSB and CSC exerted varying levels of inhibitory effects among all isolates. The IC50 values derived for CSB on adhesion of peripheral maternal isolates SML1 and SML3 were similar: 19.9 and 20.6 μg/mL, respectively. For the peripheral isolate (SML2) and the placental isolate (SML4), CSB at a concentration of 100 μg/mL exerted nearly complete inhibition of binding. The IC50 values of CSB on CSA-mediated adhesion of SML2 and SML4 were 1.6 and 2.8 μg/mL, respectively. In contrast, CSB polysaccharides had little or no effect on inhibition of SL83-S6 adherence to immobilized CSA.

Assessment of PE binding to immobilized chondroitin sulfates.

We assessed PE adherence to CSD and CSE and the results are shown in Table 1. All four maternal isolates were able to bind to both CSD and CSE at different levels. The ability of PE adherence of each isolate to CSE appeared to correlate with their levels of adherence to CSA, except for the adhesion profiles of SML4, a placental isolate. To our surprise, no binding to immobilized CSD and CSE was observed for PE of the laboratory isolates SL83-S6 and T4, which bound specifically to CSA. In addition, although CSB showed inhibitory effects on adhesion of SML4 PE, little cytoadherence of SML4 PE to immobilized CSB was observed.

Sensitivity of PE adherence to chondroitnase ABC and ACII treatments was also determined. Little or no adhesion of PE of all isolates was observed when A549 cells were treated with chondroitinases. This lack of cytoadherence revealed adhesion specificity of PE for chondroitin sulfates (Table 1).

Inhibition of PE adherence to immobilized CSE and CSD by CSA.

The reverse effects of CSA on adherence of PE to immobilized CSE and CSD are shown in Figure 2. Similarly, CSA demonstrated inhibitory activities to SML3 adhesion to CSD and CSE. The binding levels to CSE reduced sharply when free CSA was added to a final concentration of 1 μg/mL. The inhibitory effect of CSA was less in the case of PE adherence to CSD. However, the levels of adherence to both CSD and CSE were reduced to approximately 10% when 50 μg/mL of CSA were used.

Treatment of PE with trypsin.

Cleavage of SML3-and SL83-S6-infected PE surface proteins by treatment with trypsin (10 μg/mL) reduced binding to CSA by more than 90% and 80%, respectively (Figure 3A). In contrast, adhesion of SML4 PE to CSA was not affected when treating PE with the same concentration of trypsin. Approximately 40–50% reduction in PE adherence was observed for SML1 while SML2 displayed 35% reduction after treatment. The results of SML3 implied that adhesion to CSA and CSE might be mediated by different domains or adhesion molecules. With SML3 PE adhesion to immobilized CSE (Figure 3B), treatment with trypsin at a very low concentration (1 μg/mL) abolished more than 90% of the binding.

Inhibition of PE adherence to immobilized CSA by heparins.

The effects of different heparins on binding of PE to immobilized CSA are shown in Figure 4. With the laboratory line SL83-S6, the inhibition effect of LMW heparins, i.e., Fragmin and H-5284, was approximately 75–80%, but the effect was significantly increased when higher molecular weight heparin (H-9399) was used under the same experimental conditions. Suramine, a compound with a high magnitude and complexity of sulfation, as well as no carbohydrate backbone, was also used. It was found that there was no inhibitory activity of Suramine on PE adherence to all isolates tested.

DISCUSSION

We have examined the interaction of PE adherence to immobilized CSA polysaccharides using competitive inhibition assays with polysaccharides of different chondroitin sulfates. The data presented indicate that CSD and CSE specifically cause complete inhibition of PE adherence to CSA in all isolates tested. In most wild isolates and a CSA-upselected isolate, the inhibitions occurred in a dose-dependent manner, with derived IC50 values of CSD and CSE similar to those of CSA polysaccharides. With the placental isolate SML4, CSE exerted even higher inhibitory activity when compared with that of CSA. Our results suggest that the interaction between PE and CSA is highly dependent on disaccharide structure and sulfation pattern. CSD and CSE, which possess similar disaccharide units as CSA, showed comparable IC50 values to those of CSA for each isolate tested, while keratan sulfate (Gal(6SO3)β1-3GlcNAc(6SO3)) and colominic acid (poly-2, 8-N-acetyl neuraminic acid), which contain a different oligosaccharide backbone with no sulfation, demonstrated little or no inhibition. The dependency of the GlcA structure was also apparent when chondroitin sulfate B (IdoAβ1-3GalNAc(4SO3)) was used since only a partial inhibitory effect was observed. The results supported the previous findings18,19,24 that PE adherence to immobilized CSA is dependent on oligosaccharide sequence(s) with a backbone of-4GlcAβ1-3GalNAc-1.

The comparable levels of inhibitory activities of CSA and CSE also reflected the effect of sulfation pattern. Both polysaccharides contained sulfate groups at position 4 of Gal-NAc. Conversely, chondroitin sulfate C, which comprises the same disaccharide unit as CSA and CSE but differed in sulfation pattern of GalNAc (6-O-sulfated GalNAc), weakly inhibited PE adherence to CSA. The importance of 4-O-sulfation of CS for the adhesion with PE has been previously demonstrated.5,11,15,25 Our CSE results were consistent with the previous finding of Chai and others24 that extra sulfate at a position other than 4 of GalNAc might have less role in the interaction. By selectively removal of 6-O-sulfate from oligosaccharides and polysaccharides to increase the proportion of non-sulfated disaccharides, the inhibitory activity of PE binding to CSA could be enhanced. The results indicated that 6-O-sulfation interferes with the interaction of CSA with PE.24

In this study, the important role of oversulfation of CS variant chains was also observed since CSE gave higher inhibitory capability than CSA in some clinical isolates. For instance, almost complete inhibition of SML3 PE adherence was detected with CSE at a concentration of 10 μg/mL, whereas partial inhibition was seen with CSA at the same concentration. Studies on the structure of CSE derived from squid cartilage previously demonstrated that CSE contains extra disulfated and trisulfated disaccharide units including GlcA(3SO3)-GalNAc(4SO3), GlcA(3SO3)-GalNAc(6SO3) and GlcA(3SO3)-GalNAc(4SO3, 6SO3), in addition to the conventional E unit GlcA-GalNAc(4SO3, 6SO3).26 Such heavily sulfated structural features of CSE may therefore contribute to the low IC50 observed in SML2, SML3, and SML4. Further investigation is necessary to verify whether the strong inhibitory effect of CSE was specific or was caused merely by electrostatic interaction. Extra sulfation property of CSE was reported elsewhere to promote specific inhibition of cortical neuronal cell adhesion mediated by a neuroregulatory mid-kine.27

Additionally, to provide further evidence on extra sulfation, we also assessed the inhibitory activities of polysulfated molecules with different sugar backbone such as POPs and SP54.28,29 As predicted, SP54, which is a semisynthetic compound derived from oversulfation of xylan, caused a complete inhibition of PE adherence to CSA in certain strains such as SL83-S6 and the maternal placental isolate (SML4). Conversely, POPs, which is a synthetic sulfated bislactobiotic acid amide, did not result in significant levels of inhibition in most isolates tested. These results confirmed the significance of the specific configuration and pattern of sulfation on PE adherence. The view that oversulfation probably has certain influence on cytoadherence of clinical isolates to CSA contrasts with earlier findings stating the importance of low-sulfated disaccharide units for the interaction between CSA and PE.19 Alkahlil and others19 demonstrated that PE adherence of a laboratory isolate (3D7) to chondroitin sulfate proteoglycans of placental intervillous spaces is mediated by low-sulfated CS chains whereas highly sulfated GAGs such as SP54 were non-inhibitory to the adherence.

As for clinical samples, PE adherence to immobilized CSD and CSE was observed. The PE of every wild isolate tested also bound to immobilized CSA. The results indicate that these isolates co-express parasite molecules for more than one adhesion receptor. The inability of PE of the laboratory-selected isolates SL83-S6 and T4 to bind with CSD and CSE confirmed the specificity of the interaction. The variability in the level of adhesion to these three chondroitin sulfates of clinical isolates may be due to the existence of mixed parasite populations. However, multiple interaction of an adhesive ligand to several CS-types has been speculated. Beeson and others previously observed different levels of PE binding to hyarulonate and CSA among parasite isolates.5 L-selectin, a lymphocyte homing receptor, also bound to several types of GAGs, as demonstrated by the ability of CSB and CSE to block the binding of versican to L-selectin.30 More importantly, our results suggest that in vitro assessment of adherence may not fully represent the complexity of adherence in vivo and demonstrate the importance of analyzing isolates taken from patients.

Despite the relevance of the combination of 4-O-sulfated and non-sulfated disaccharide units for the PE-CSA interaction,24 we have observed that wild isolates (e.g., SML1) were able to adhere to CSD at relatively high levels. CSD, which contains repeating units of GlcA(2SO3)β1-3GalNAc(6SO3), significantly inhibited PE adhesion at levels comparable to CSA. Notably, due to the heterogeneous composition of CS chains used in this study, particularly the sulfate content and pattern, the relevance of the 2-O-sulfation of GlcA by assaying PE of laboratory as well as P. falciparum isolates from patients against different size-homogeneous fractions isolated from CSD remains to be investigated.

Since LMW heparins, which are sulfated GAGs with distinct disaccharide compositions, have been used successfully in the prevention and treatment of venous thromboembo-lism,31 we evaluated the potential of LMW heparins as inhibitors for CSA-mediated adherence of P. falciparum. The results gave additional evidence of the adhesive heterogeneity of PE. We observed a dose-dependent inhibition ability of H-5284, H-9399, and Fragmin (LMW heparins) on adherence of SML2 and the laboratory-selected isolate SL83-S6. Notably, both isolates bound predominantly to CSA. Lower cy-toadherence inhibitory levels were seen in other isolates tested. This incomplete inhibition of binding of LMW heparins may imply that other adhesion molecules were co-expressed in maternally derived parasites. The LMW heparins are prepared from the unfractionated normal heparin (usually of porcine origin) by controlled depolymerization and purified to the lower degree of heterogeneity.32 As for inhibition, LMW heparins comprise GAGs and possess a more restricted configuration of sulfation pattern when compared with unfractionated heparin, thereby leading to the inhibitory capacity.

The observation that CSB exerted significant inhibition on CSA-mediated cytoadherence in some maternal isolates tested (SML2 and SML4) might be explained by the presence of small proportion of GlcA residues linked to GalNAc-4-sulfate residues in the CSB chain. Incomplete epimerization at the C-5 position of GlcA residues to yield IdoA residues, as well as incomplete sulfation of GalNAc residues, were previously suggested to confer this heterogeneity in the CSB chain structure.31 Moreover, it was previously shown that the existence of IdoA could give flexibility to the CSB chain by the characteristics of conformation that consequently contributes to the interaction of CSB with several molecules such as fibroblast growth factor-2, fibronectin, and platelet factor 4.32 Thus, the interaction of CSB with some protein molecules on the surface of PE might give rise to steric hindrance of PE adhesion. It is important to note that although CSB could inhibit CSA-PE adhesion, no appreciable adhesion level of any isolates tested with immobilized CSB was observed. The particular phenomenon was noticeable in LMW heparins used in this study. One probable interpretation could be that the structural requirement of CS for PE adhesion, with respect to both disaccharide structure and sulfation pattern, is different from that for effective inhibition.

The different effects of trypsin on adhesion of PE have been previously reported.5,12,20 CS2 PE12 and T4 PE20 demonstrated minimal reduction in adherence to immobilized CSA after trypsin digestion at high concentrations, in contrast to PE of parasite lines that adhered to CD36 or ICAM-1.5,12,20 In this study, PE of trypsin-sensitive and-insensitive variants were able to adhere to CSA, implying that not only trypsin-resistant domains are responsible for CSA-mediated cytoadherence, but that trypsin-sensitive domains can interact with CSA as well. Moreover, the difference in trypsin sensitivity of adhesive ligand(s) of SML3 PE, which mediated binding to CSA and CSE, suggested that the process of adherence involved more than one adhesion molecule or domain on PE. Partial unfolding of P. falciparum erythrocyte membrane protein-1 adhesive domain after digestion with trypsin, resulting in a molecular conformational change, could also be an alternative explanation.

In conclusion, our data have clearly demonstrated that apart from CSA, certain maternally derived parasites could bind specifically to CSE and CSD. The adherence of PE to chondroitin sulfate chains depends on the carbohydrate backbone as well as the specific sulfation pattern. However, certain semisynthetic compounds with oversulfation and without the specific sugar backbone can cause inhibition of adherence in some isolates. Furthermore, LMW heparins exhibit their capacity to inhibit PE adherence to CSA. Studies on the compositional analysis of LMW heparins and work evaluating the potential of these compounds as therapeutic agents are in progress.

Table 1

Adhesion of parasitized erythrocytes (PE) of wild and laboratory Plasmodium falciparum isolates to purified receptors* and chondroitinase sensitivity

Adhesion receptorChondroitinase†
ParasiteCSAHSCSDCSECSBABCACII
* Data represent mean number of bound PE/mm2. All assays were performed in duplicate. CSA = chondroitin sulfate A; HS = heparan sulfate; CSD = chondroitin sulfate D; CSE = chondroitin sulfate E; CSB = chondroitin sulfate B; NA = not assessed.
† Percent inhibition of cytoadherence by 0.5 units/mL of chondroitinase ABC and ACII treatments.
Maternal peripheral isolates
    SML11,252, 1,6350, 0648, 7341,091, 1,094NA99.9599.8
    SML2165, 1710, 02, 35, 8NA100100
    SML3938, 1,0110, 061, 64961, 1,0390, 0100100
Maternal placental isolates
    SML4370, 5230, 05, 625, 173, 610099.95
Laboratory isolates
    SL8350, 600, 0NANANA100NA
    SL83-S6517, 5560, 00, 00, 00,0100100
    T41,548, 1,7050, 00, 00, 0NANANA
Figure 1.
Figure 1.

Inhibition of adherences to immobilized chondroitin sulfate A (CSA) of maternal isolates (SML1-4) and a laboratory isolate (SL83-S6) of Plasmodium falciparum by glycosaminoglycans at various concentrations. Values are means for quadruplicate experiments. Bars represent standard errors. CSA = chondroitin sulfate A; CSD = chondroitin sulfate D; CSE = chondroitin sulfate E; SP54 = pentosan polysulfate; POPs = polyolpolysulfate; CSB = chondroitin sulfate B, CSC = chondroitin sulfate C.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 2; 10.4269/ajtmh.2004.70.149

Figure 2.
Figure 2.

Inhibition of adherence to immobilized CSD and CSE of maternal isolate SML3 of Plasmodium falciparum by CSA at various concentrations. Values are means for quadruplet experiments. Bars represent standard errors. For definitions of abbreviations, see Figure 1.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 2; 10.4269/ajtmh.2004.70.149

Figure 3.
Figure 3.

Cleavage of surface proteins on intact parasitized erythrocytes (PE) before adhesion to immobilized CSA (A) and CSE (B). All data represent the proportion of bound PE expressed as percentage of control. All assays were performed in triplicate or quadruplicate. Bars represent standard errors. For definitions of abbreviations, see Figure 1.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 2; 10.4269/ajtmh.2004.70.149

Figure 4.
Figure 4.

Inhibition of adherence to immobilized chondroitin sulfate A at increasing concentrations of heparins and Suramine. Values are means for quadruplicate experiments. Bars represent standard errors.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 2; 10.4269/ajtmh.2004.70.149

Authors’ addresses: Sujittra Chaisavaneeyakorn, Pornpimon Angkasekwinai, and Sansanee C. Chaiyaroj, Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand, Telephone: 66-2-201-5689, Fax: 66-2-644-5411, E-mail: scscy@mahidol.ac.th. Prachya Kongtawelert, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand, Telephone: 66-1-784-6907. Alan Brockman and François Nosten, Shoklo Malaria Research Unit, Intarakiri Road, Mae Sot, Tak 63110, Thailand, Telephone and Fax: 66-55-531-531.

Acknowledgments: We thank Professor N. J. White and his colleagues for their assistance on the field trip to Mae Sot, Tak, and Sumalee Kamchonwongpaisan for her excellent mathematical and statistical advice, including the determination of 50% inhibitory concentrations. We also express our sincere appreciation to Maurice Broughton for suggestions during manuscript preparation.

Financial support: This work was supported by the Thailand Research Fund (TRF). Sansanee C. Chaiyaroj is a TRF scholar. Sujittra Chaisavaneeyakorn is supported by the Medical Scholars Program of Mahidol University. François Nosten and Rose McGready are supported by the Wellcome Trust of Great Britain.

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