Plasmodium falciparum Cytoadherence to Human Placenta: Evaluation of Hyaluronic Acid and Chondroitin 4-Sulfate for Binding of Infected Erythrocytes

Experimental Parasitology 99, 57–65 (2001) doi:10.1006/expr.2001.4642, available online at http://www.idealibrary.com on

Plasmodium falciparum Cytoadherence to Human Placenta: Evaluation of Hyaluronic Acid and Chondroitin 4-Sulfate for Binding of Infected Erythrocytes

Manojkumar Valiyaveettil,* Rajeshwara N. Achur,* Abdulnaser Alkhalil,* Christian F. Ockenhouse,† and D. Channe Gowda*,1 *Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, U.S.A.; and †Department of Immunology, Walter Reed Army Institute of Research, Maryland 20910, U.S.A.

Valiyaveettil, M., Achur, R. N., Alkhalil, A., Ockenhouse, C. F., and Gowda, D. C. 2001. Plasmodium falciparum cytoadherence to human placenta: Evaluation of hyaluronic acid and chondroitin 4-sulfate for binding of infected erythrocytes. Experimental Parasitology 99, 57–65. Chondroitin 4-sulfate (C4S) is known to mediate the adherence of Plasmodium falciparum infected red blood cells (IRBCs) to human placenta. Recently, hyaluronic acid (HA) has also been reported to bind IRBCs, and HA has been suggested as an additional receptor for the sequestration of IRBCs in the placenta. In this study, we assessed the adherence of 3D7 parasite strain, which has been reported to bind both C4S and HA, using highly purified clinical grade rooster comb HA, Streptococcus HA, several preparations of human umbilical cord HA (hucHA), and bovine vitreous humor HA (bvhHA). While all hucHA preparations and bvhHA bound with moderate to high density to IRBCs, the rooster comb and bacterial HAs did not bind IRBCs. IRBCs binding to the hucHA and bvhHA could be abolished by pretreatment with testicular hyaluronidase but not with Streptomyces hyalurolyticus hyaluronidase, suggesting that IRBC binding to hucHA and bvhHA was due to chondroitin sulfate (CS) contaminants in HAs. Compositional analysis confirmed the presence of CS in both hucHA and bvhHA. The CSs present in these commercial hucHA and bvhHA samples were isolated, characterized, and studied for their ability to bind IRBCs. The data suggested that IRBC adherence to hucHA and bvhHA was mediated by the CS present in these samples. However, our data did not exclude the possibility of a minor population of distinct parasite subtype adhering to HA and further studies using pure HA conjugated to proteins or lipids and placental parasite isolates should

clarify whether HA is an in vivo receptor for IRBC adherence. 䉷 2001 Elsevier Science

Index Descriptors and Abbreviations: Plasmodium falciparum; chondroitin 4-sulfate; hyaluronic acid; placental adherence; IRBCs, infected red blood cells; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; PG, proteoglycan; C4S, chondroitin 4-sulfate; GAG, glycosaminoglycan; HA, hyaluronic acid; huc, human umbilical cord; bvh, bovine vitreous humor; BSA, bovine serum albumin; GlcN, glucosamine; GalN, galactosamine; PVDF, polyvinylidene difluoride; TLCK, N-␣-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-Lphenylalanine chloromethyl ketone; PMSF, phenylmethylsulfonyl fluoride; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis.

INTRODUCTION

Plasmodium falciparum is distinguished from the other three human malaria parasites by the ability of its infected red blood cells (IRBCs) to accumulate in the microvascular capillaries of vital organs (Pasloske and Howard 1994). This adherence property of P. falciparum is believed to contribute to the virulence of the parasite (Newbold et al. 1997). A number of studies have shown that IRBC sequestration is mediated by endothelial cell adhesion molecules including CD36, ICAM-1, VCAM-1, E-selectin, platelet endothelial cell adhesion molecule-1/CD31, and thrombospondin as well

1

To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007. Fax: (202) 687-7186 E-mail: [email protected].

0014-4894/01 $35.00 䉷 2001 Elsevier Science All rights reserved.

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58 as chondroitin 4-sulfate (C4S) (Ockenhouse et al. 1989, 1992; Berendt et al. 1989; Chaiyaroj et al. 1994; Rogerson et al. 1995; Robert et al. 1995; McCormick et al. 1997; Udomsangpetch et al. 1997; Treutiger et al. 1997). These receptor molecules interact with plasmodium falciparum erythrocyte membrane protein-1, the parasite var-gene product found on the surface of IRBCs (Smith et al. 1995; Su et al. 1995; Baruch et al. 1996; Gardner et al. 1996; Newbold et al. 1997; Buffet et al. 1999; Reeder et al. 1999; Smith et al. 2000). While sequestration of IRBCs in various organs including the brain is mediated by endothelial cell adhesion molecules, IRBC adherence to human placenta is selectively mediated by C4S (Fried and Duffy 1996; Rogerson and Brown 1997; Maubert et al. 1997, 2000; Gysin et al. 1997, 1999; Pouvelle et al. 1997, 2000). In the placenta, IRBCs predominantly accumulate in the intervillous spaces (Yamada et al. 1989; Miller and Smith 1998); some IRBCs may attach to syncytiotrophoblast cell lining (Fried and Duffy 1996; Hofman et al. 1998). This is consistent with the presence of high levels of uniquely low sulfated, extracellular chondroitin sulfate proteoglycans (CSPGs) in the placental intervillous spaces that efficiently bind IRBCs and low levels of IRBC-binding, cell-associated CSPGs (Achur et al. 2000). Based on the binding of P. falciparum IRBCs to human umbilical cord hyaluronic acid (hucHA) and bovine vitreous humor hyaluronic acid (bvhHA) samples (supplied by Sigma) coated onto plastic, hyaluronic acid (HA) has been recently reported to be an additional receptor for placental cytoadherence (Beeson et al. 2000; Chai et al. 2001). In these studies, it was presumed that HA was coated onto a plastic surface when incubated with HA solutions. However, it is known that pure HA and other anionic polysaccharides including C4S cannot be coated onto a plastic surface unless conjugated to proteins or lipids (Yamagata et al. 1989; Sugiura et al. 1993; Underhill and Zhang 2000; Valiyaveettil et al. unpublished results). Previous studies have reported high levels of HA on the surfaces of the syncytiotrophoblast lining of intervillous spaces (Sunderland et al. 1985). However, by detailed structural analysis of glycosaminoglycans purified from human placenta, we found that the placenta contains very low levels of HA: 1–2% compared with CSPGs of the intervillous spaces (Achur et al. 2000). The criteria used in the previous studies that concluded the abundant presence of HA in human placenta would likely not distinguish HA from very low sulfated CSPGs (Lee et al. 1973; Ponting and Kumar 1995). Furthermore, a recent study, using placental P. falciparum isolates, found that IRBCs bound to hucHA

VALIYAVEETTIL ET AL.

containing 3–7% chondroitin sulfate (CS), but not to Streptococcus HA (Fried et al. 2000), the latter HA being completely devoid of CS. These considerations raise a strong possibility that the reported adhesion of IRBCs to HA was predominantly due to the presence of CS. The above study, based on differential adherence of trypsin-treated IRBCs to C4S with and without 6-sulfate groups, raised the possibility of polyvalent binding or unusual structural features within C4S. These observations as well as our finding that human placenta contains negligible amounts of HA (Achur et al. 2000) prompted us to evaluate IRBC adhesion to HA and determine the structural features of CS present in commercial sources of hucHAs and bvhHA. The results presented here indicate that the studies reporting IRBC binding to commercial HA samples did not exclude the possibility of IRBC binding to CSs.

MATERIALS AND METHODS Materials. C4S (sturgeon notochord), Proteus vulgaris chondroitinase ABC (5 units/vial), Streptomyces hyalurolyticus hyaluronidase (2000 turbidity reducing units/mg), and Flavobacterium heparinum heparitinase (113 units/mg) were purchased from Seikagaku America (Falmouth, MA, U.S.A.). Ovine testicular hyaluronidase (2160 units/ mg) was from ICN Biomedicals; C4S (bovine trachea), hucHAs, bvhHA, gelatin (300 bloom), PMSF, NEM, and BSA were from Sigma Chemical Co. (St. Louis, MO, U.S.A.); pharmaceutical grade rooster comb HA was from Anika Therapeutics (Woburn, MA, U.S.A.); Streptococcus species HA was from Calbiochem (La Jolla, CA, U.S.A.); RPMI 1640 medium and other cell culture reagents were from Life Technologies (Rockville, MD, U.S.A.); human blood and serum were from Interstate Blood Bank (Memphis, TN, U.S.A.); polyacrylamide minigels and protein molecular weight standards were from Bio-Rad Laboratories (Richmond, CA, U.S.A.); 1,9-dimethylmethylene blue was from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.); TLCK and TPCK were from Boehringer Mannheim (Indianapolis, IN, U.S.A.); Sepharose CL-6B and blue dextran were from Amersham Pharmacia Biotech (Piscataway, NJ, U.S.A.); HPLC-grade 6 N HCl and the micro BCA protein assay kit were from Pierce Chemical Co. (Rockford, IL, U.S.A.); polystyrene petri dishes (Falcon 1058) were from Becton– Dickinson Labware (Lincoln Park, NJ, U.S.A.). C4S adherent IRBCs and parasite culturing. The C4S-adherent parasites (3D7 clone) were selected from the NF-54 laboratory strain by several rounds of panning on bovine trachea C4S-coated plastic dishes (Ockenhouse et al. 1991). These IRBCs bound CSPGs of the placental intervillous spaces in high density. Since the adherent property of IRBCs decreases significantly over a period of continuous culture, the parasites were panned every 6–8 weeks on placental CSPGcoated plates. The parasite was cultured in RPMI 1640 medium containing 10% O+ human serum using O+ human red blood cells at 3% hematocrit (Naik et al. 2000). Cultures with ⬎20% parasitemia were washed with RPMI 1640 medium or PBS, pH 7.2, and used for adhesion assay

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without further enrichment of IRBCs, and cultures with lower parasitemia were enriched to 50–60% parasitemia by gelatin flotation as reported previously (Alkhalil et al. 2000). IRBC adherence assay. The adherence assay was performed as described previously (Achur et al. 2000). HAs and CSPGs were coated onto plastic petri dishes as circular spots, blocked with BSA, and then overlaid with a 2% suspension of IRBCs in RPMI 1640 medium or PBS, pH 7.2 (Achur et al. 2000). After 40 min, the dishes were washed with PBS, and bound IRBCs were fixed with 2% glutaraldehyde, stained with 1% Giemsa dye, and counted under light microscopy and photographed. Enzyme treatment studies. HAs and CSPGs were coated as above on plastic petri dishes (35 ⫻ 10 mm), and the whole dish was blocked with 2% BSA and incubated with 1 ml of chondroitinase ABC (50 munits/ml), testicular hyaluronidase (50 units/ml), S. hyalurolyticus hyaluronidase (40 turbidity reducing units/ml), or heparitinase (20 munits/ml) (Alkhalil et al. 2000). The plates were washed with PBS, pH 7.2, and IRBC adhesion was measured as described above. IRBC adherence inhibition assay. The IRBC suspensions (3–4% hematocrit in PBS, pH 7.2) were mixed with the polysaccharides, incubated at room temperature for 30 min with intermittent mixing, and then layered on HA- or CSPG-coated spots (Alkhalil et al. 2000). After 40 min, the unbound cells were washed, and the bound cells were fixed with 2% glutaraldehyde, stained with Giemsa. Isolation and Purification of CSPGs from commercial HA samples. The hucHA (20 mg, Sigma Cat. No. H1504) or bvhHA (20 mg, Sigma Cat. No. H7630) was dissolved in 10 ml of 20 mM NaOAc, 150 mM NaCl, pH 6.0. The solution was incubated with S. hyalurolyticus hyaluronidase (150 turbidity reducing units) in the presence of 0.1 mM PMSF, 0.1 mM TLCK, 0.25 mM TPCK, and 0.1 mM NEM at 60⬚C overnight (Hatae and Makita 1975) and then lyophilized. The enzyme digest was dissolved in 0.5 ml of 0.2 M NaCl and chromatographed on a Sepharose CL-6B column (2 ⫻ 65 cm) using 0.2 M NaCl. Fractions (3.7 ml) were collected and monitored for protein (280 nm) and uronic acid content (Dische 1947). CsBr density gradient centrifugation. The solutions (2 mg/ml) of the enzyme-resistant uronic-containing polymer fraction, in 25 mM sodium phosphate, pH 7.2, containing 50 mM NaCl, 0.02% NaN3, 4 M

guanidine hydrochloride, and 42% (w/w) CsBr, were centrifuged in a Beckman 50 TI rotor at 44,000 rpm for 65 h at 14⬚C (Achur et al. 2000). Blotting of commercial HAs onto PVDF membrane. HA samples (25 ␮l of 100 ␮g/ml solutions in 50 mM NaOAc, 150 mM NaCl, pH 6.0) were dot blotted onto PVDF membranes using a Bio-Rad dot blot apparatus. The blotted membranes were washed with distilled water and the spots were cut out and used for hexosamine analysis. Carbohydrate composition analysis. For hexosamine analysis, the HAs, PVDF membrane blots, or CSPGs were hydrolyzed with 4 M HCl at 100⬚C for 6 h. The hydrolysates were dried in a Speed-Vac and analyzed on a CarboPac PA1 high-pH anion-exchange column (4 ⫻ 250 mm) using a Dionex BioLC HPLC system coupled to a pulsed amperometric detector (Hardy 1989). The elution was with 20 mM sodium hydroxide and the response factors for the monosaccharides were determined using standard sugar solutions. Disaccharide composition analysis of GAG chains. The HA or PG samples (100 ␮g) were digested with chondroitinase ABC, treated with 4 vol of cold methanol, and centrifuged, and the supernatant was dried in a Speed-Vac. The disaccharides were analyzed on a 4.6 ⫻ 250 mm amine-bonded silica PA03 column (YMC, Inc., Milford, MA, U.S.A.), using 600E HPLC system (Waters, Milford, MA, U.S.A.). A linear gradient of 16 to 530 mM NaH2PO4 over 70 min at room temperature at a flow rate of 1 ml/min was used for elution (Achur et al. 2000). Determination of sulfate content. The sulfate contents of PGs were determined as reported previously (Achur et al. 2000). Gel electrophoresis. SDS–PAGE was performed using Bio-Rad 4–20% gradient polyacrylamide minigels (Laemmli 1970). The gels were stained sequentially with Coomassie blue and Alcian blue (Krueger and Schwartz 1987). Other analytical procedures. Protein content was estimated by using Micro BCA protein assay reagent kit (Pierce).

RESULTS To assess the nature of IRBC binding to commercially available bvhHA and hucHA, we studied samples listed in

TABLE I Protein and Hexosamine Composition of Commercial HA Preparations and Their IRBC Adherence Hexosamineb (mol %) Before blotting

After blotting

IRBCs bound (per mm2) to HA-coated plastic platesc, coating concentration (␮g/ml)

Source of HA

Proteina (weight %)

GalN

GlcN

GalN

GlcN

25

50

Human umbilical cord (Sigma Cat. No. H1504) Human umbilical cord (Sigma Cat. No. H1876) Bovine vitreous humor (Sigma Cat. No. H7630) Human umbilical cord (Sigma Cat. No. H1751) Rooster combd (Anika Therapeutics, Inc.) Streptococcus sp.d (Calbiochem Cat. No. 385908)

5 5 3 2 0 0

19 8 4 1 0.2 0

81 92 96 99 99.8 100

52 50 60 — — —

48 50 40 — — —

1800 1750 525 400 — —

1820 1800 650 420 — —

a

Estimated by the BCA method. Estimated by Dionex BioLC HPLC using CarboPac PA1 column. c Measured by cytoadherence assay as described under Materials and Methods (SD ⫾ 5%). d IRBCs were not bound due to the absence of CSPG in HA samples. b

60 Table I by an in vitro cytoadherence assay using the 3D7 parasite clone that was reported to bind both C4S and HA in equal capacity (Beeson et al. 2000). As previously reported (Beeson et al. 2000), IRBCs adhered to plastic plates coated with hucHA (Figs. 1A and 2B; see also Table I). However, in the case of bvhHA, IRBCs with ring, young, and midtrophozoite stage parasites as well as uninfected RBCs were bound and the proportion of the bound IRBCs to RBCs was similar to the proportion in the cell suspension (Figs. 1A and 2C). The adherence of RBCs to bvhHA appears to be due to nonspecific binding by a protein impurity in bvhHA. While IRBC adherence to hucHA was dose-dependent, adherence to bvhHA was not (Fig. 1A). Since pure HA do not efficiently bind plastic (Yamagata et al. 1989; Sugiura et al. 1993; Underhill and Zhang 2000; Valiyaveettil et al. unpublished results), we assessed IRBCbinding by adhesion inhibition using various HA samples as well as CSPGs purified from these HA samples (see below). The adherence of IRBCs to plastic plates coated

FIG. 1. Adhesion of Plasmodium falciparum IRBCs to commercial HA samples, CSPGs purified from HAs, and human placental CSPG. The hucHA and bvhHA from Sigma (Cat. No. H1504 and H7630, respectively) and CSPGs purified from the HAs (see Figs. 3 and 4) and human placental CSPG (Achur et al. 2000) were coated on plastic petri dishes. The spots were blocked with 2% BSA and overlaid with IRBC (25–50% parasitemia) suspension in PBS, pH 7.2. For inhibition studies (B), IRBCs were preincubated with indicated compounds (50 ␮g/ml) for 30 min and then overlaid onto the HA- or CSPG-coated spots. After 40 min, the unbound cells were washed, and the bound cells were fixed with 2% glutaraldehyde, stained with Giemsa dye. The bound IRBCs were counted under the microscope, and the average of three different assays carried out each in duplicate was plotted. (A) (䢇) CSPG purified from intervillous spaces of human placenta (Achur et al. 2000); (䡬) hucHA from Sigma (Cat. No. H1504); (䡵) CSPG purified from hucHA from Sigma (Cat. No. H1504); (䡺) bvhHA from Sigma (Cat. No. H7630); (䉱) CSPG purified from bvhHA from Sigma (Cat. No. H7630). (B) Inhibition of IRBC binding to plastic plates coated with hucHA and bvhHA (50 ␮g/ml) by HA or CS. bvhHA, the bars from left to right represent control, Streptococcus HA, bvhHA, CSPG purified from bvhHA, and bovine trachea C4S, respectively; hucHA, the bars from left to right represent control, Streptococcus HA, hucHA, CSPG purified form hucHA, and bovine trachea C4S, respectively.

VALIYAVEETTIL ET AL.

with hucHA and bvhHA was effectively inhibited by respective HAs, the CSPGs purified from the HA samples, or bovine trachea C4S (Fig. 1B). However, Streptococcus and rooster comb HA were ineffective at similar concentrations; each marginally inhibited at ⬎50 ␮g/ml (Fig. 1B and data not shown). These data suggested that the IRBC-binding to hucHA and bvhHA was mediated by CS chains associated with the HAs but not by HA chains. The adherence of IRBCs to hucHA and bvhHA samples was not affected when the coated plates were preincubated with S. hyalurolyticus hyaluronidase, an enzyme that degrades HA but not CS (Hatae and Makita 1975). However, the adherence was totally abolished upon treatment with testicular hyaluronidase, an enzyme that can degrade both HA and CS (Mathews et al. 1951; Cowman et al. 1984; Alkhalil et al. 2000; Valiyaveettil et al. unpublished results). These data indicated that the IRBC-binding to commercial hucHA and bvhHA was mediated by CSs present in the HA samples. To examine the levels of CS in commercial HA samples, we initially determined the hexosamine composition (Table I). Significant amounts of GalN were present in HA samples that supported IRBC adhesion and this sugar was absent in HAs that were unable to support the adherence, suggesting the presence of CS chains in IRBC-binding HA samples. Again, these results suggested that IRBCs adhere to CS but not to HA. Since commercial GAGs including HA are usually prepared by extraction of tissues under conditions that favor proteolysis, it is likely that CS chains in HA samples are linked to degraded core proteins. The CS chains linked to polypeptides or proteins can be selectively enriched on nylon membranes by blotting. To examine whether this is the case, various HAs were dot blotted onto PVDF membranes, and the hexosamine composition of the blotted material was determined (Table I). Materials blotted from HAs that supported cytoadherence contained markedly high contents of GalN, suggesting the presence of CSPGs in these HAs. GalN was either ⬍0.2% or not detectable in HAs, which are unable to support IRBC adherence. To characterize the CSPGs present in Sigma hucHA and bvhHA (Table II), which supported IRBC adherence, the samples were digested with S. hyalurolyticus hyaluronidase to completely remove HA chains, and the digests were chromatographed on Sepharose CL-6B columns. This procedure separated the undigested uronic-acid-containing materials (CSPGs) from the degraded HA; in each case, the CSPG was eluted as a single symmetrical peak (Fig. 3 and not shown). The uronic-acid-containing fractions were purified by cesium bromide density-gradient centrifugation (Fig. 4).

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FIG. 2. Adhesion of Plasmodium falciparum IRBCs to commercial HAs and CSPG purified from human placenta. The plastic petri dishes were coated with human placental CSPG (200 ng/ml; Achur et al. 2000), Sigma hucHA (Cat. No. H1504; 50 ␮g/ml), or Sigma bvhHA (Cat. No. H7630; 50 ␮g/ml) solutions, blocked with BSA, and IRBC adhesion was performed as outlined in Fig. 1. (A–C ) Photograph of IRBCs bound to CSPG of human placental intervillous spaces, hucHA, and bvhHA, respectively.

Compositional analysis revealed that the S. hyalurolyticus hyaluronidase-resistant polymer(s) present in hucHA and bvhHA contained high amounts of uronic acid and GalN (GlcN was a minor component) and significant levels of protein and sulfate (Table II), confirming the presence of CS chains in these HA samples. The data also showed that the CS chains exist as CSPGs. The CSPGs purified from hucHA and bvhHA samples were digested with chondroitinase ABC, and the released disaccharides were analyzed by HPLC. The CS chains of

CSPG purified from the hucHA contained 20, 54, and 26% 4-, 6-, and nonsulfated disaccharide repeats, respectively, whereas the CS chains of CSPG from bvhHA had 23, 14, and 63% of 4-, 6-, and nonsulfated disaccharides, respectively (Table II). The CSPGs purified from Sigma hucHA and bvhHA preparations supported adhesion of IRBCs (see Fig. 1A). Although the binding was dose-dependent with both CSPGs, the coating concentrations for maximum IRBC adherence were significantly higher when compared with that required for the adherence of IRBCs to CSPG of the placental intervillous spaces (Achur et al. 2000).

TABLE II Compositional Analysis of CSPG Fraction from hucHA and bvhHA CSPG composition Components Protein (weight%)a Sulfate (weight%)b Uronic acid (weight%)c Hexosamine (weight%)d Hexosamine composition (mol %) GalN GlcN Disaccharide composition (mol %)e ⌬di-0S ⌬di-4S ⌬di-6S a

hucHA (H1504)

bvhHA (H7630)

20 10 33 37

21 5 35 39

96 4

96 4

26 20 54

63 23 14

Estimated by the BCA method. Estimated as reported previously (Achur et al. 2000). c Estimated by the carbazole method (Dische 1947). d Estimated by Dionex BioLC HPLC using CarboPac PA1 column. e CSPGs were hydrolyzed with chondroitinase ABC and the released disaccharides were analyzed by HPLC (Achur et al. 2000). b

FIG. 3. Isolation of CSPGs present in commercial HAs by Sepharose CL-6B chromatography. The Sigma hucHA (20 mg; Cat. No. H1504) and Sigma bvhHA (20 mg; Cat. No. H7630) were digested with S. hyalurolyticus hyaluronidase and the digests were separately chromatographed on a Sepharose CL-6B column (2 ⫻ 65 cm) using 0.2 M NaCl. Fractions were monitored for protein (OD280) and uronic acid content (OD530). Shown is the isolation of CSPG from hucHA. The elution positions of blue dextran (BD), BSA, and glucose (Glc) are indicated.

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The CSPGs purified from hucHA and bvhHA preparations from Sigma were also analyzed by SDS–PAGE before and after treatment with chondroitinase ABC in the presence of protease inhibitors. The untreated CSPG samples purified from hucHA and bvhHA electrophoresed as broad bands corresponding to molecular weights of 20,000–115,000 and 15,000–60,000 Da, respectively, and these could be stained with Alcian blue but not with Coomassie blue (Fig. 5). These bands completely disappeared in the enzyme-treated samples; the polypeptides released by the enzyme electrophoresed as low-molecular-weight bands (⬍8000 Da; leached out of the gels during destaining).

DISCUSSION The results of this study indicate that the previously reported binding of P. falciparum IRBCs to commercial hucHA and bvhHA (Beeson et al. 2000; Chai et al. 2001) is due to the CSPGs present in the samples. Several lines of evidence support this conclusion: (1) the abrogation of IRBC binding to hucHA- and bvhHA-coated plates by pretreatment with ovine testicular hyaluronidase or chondroitinase ABC but not with S. hyalurolyticus hyaluronidase; (2) the inhibition of IRBC binding to hucHA- and bvhHAcoated plates by the respective HA that contained significant amounts of CS but not by the pharmaceutical grade rooster comb HA and Streptococcus HA, which are free of CS; (3) the binding of both RBCs and IRBCs to bvhHA that was neither dose-dependent nor saturable; (4) the specific and high-density binding of IRBCs to the CSPGs purified from hucHA and bvhHA preparations.

FIG. 5. SDS–PAGE analysis of CSPGs purified from hucHA and bvhHA. The CSPGs purified from Sigma hucHA (Cat. No. H1504) and bvhHA (Cat. No. H7630) as outlined in Fig. 4 were electrophoresed on a 4–20% gradient SDS–polyacrylamide gel under reducing conditions. The gels were stained with Coomassie blue and then with Alcian blue. Lanes 1 and 2, CSPG from bvhHA before and after chondroitinase ABC treatment, respectively. Lanes 3 and 4, CSPG from hucHA before and after chondroitinase ABC treatment, respectively. The molecular masses of the standard proteins are indicated to the right.

The commercial hucHA and bvhHA preparations contain significant levels of CSPGs. This is evident from (1) the presence of significant amounts of GalN and protein in HAs, (2) the polydispersity and molecular weights of the uronicacid-containing, S. hyalurolyticus hyaluronidase-resistant polymers present in HA samples, and (3) the composition of the enzyme-resistant polymers (high levels of GalN, protein, and sulfate and low levels of GlcN) isolated from HAs. The CSPGs purified from HA preparations appear to be proteolytically degraded as indicated by their low molecular

FIG. 4. Purification of CSPGs isolated from commercial HAs by CsBr density gradient centrifugation. The CSPGs isolated from Sigma hucHA (Cat. No. H1504) and Sigma bvhHA (Cat. No. H7630) as shown in Fig. 3 were purified by CsBr density gradient centrifugation in 4 M GdnHCl and 42% CsBr. Fractions were collected from the bottom of the centrifuge tubes and monitored for protein (OD280) and uronic acid content (OD530). Shown is the isolation of CSPG from hucHA. The CSPG-containing fractions were pooled as indicated by the horizontal bar.

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weights compared with most mammalian proteoglycans and the presence of low-molecular-weight core proteins. We have previously shown that unusually low sulfated CSPGs of the human placental intervillous spaces efficiently bind IRBCs (Achur et al. 2000). Further studies showed that a population of the placental intervillous CSPGs containing CS with 18–20% 4-sulfated disaccharide repeats bind IRBCs with relatively higher affinity compared with those containing CS chains with 8–10% 4-sulfated disaccharide repeats (Achur et al. unpublished results). Although the CS chains of the CSPGs purified from hucHA contain high levels of 6-sulfate, these sulfate groups do not appear to be involved in IRBC binding (Alkhalil et al. 2000). Interestingly, the CS chains of the CSPG purified from bvhHA sample contain unusually high levels of nonsulfated disaccharide residues, as in the case of CS chains of the CSPGs of placental intervillous spaces (Achur et al. 2000). The low sulfate content of the CS chains of CSPGs from bvhHA samples also agrees with the previously reported levels of CS content for the CSPGs of bovine cornea (Davidson and Meyer 1954). Although C4S from various sources have homogeneous polysaccharide chains consisting of glucuronic acid–GalN disaccharide repeats, they differ significantly with respect to the position and distribution of sulfate groups. Many C4Ss contain variable amounts of 6-sulfate groups, which are differentially distributed along the polysaccharide chains. A recent study reported enhanced binding by C4S in which 6sulfate groups were removed (Fried et al. 2000). However, studies in our laboratory show that the removal of 6-sulfate groups in bovine trachea C4S and whale cartilage C4S, which contain 39 and 27% 6-sulfated groups, respectively, have no significant effect on the adherence property of IRBCs (Alkhalil et al. 2000). Therefore, the observed increase in IRBC binding after 6-desulfation (Fried et al. 2000) could be due to a significant loss of 4-sulfate groups that accompanied the removal of 6-sulfate groups; C4S with 30–40% 4-sulfate groups exhibits increased IRBC adherence (Alkhalil et al. 2000). C4Ss with varying levels of 4- and 6-sulfate groups have been reported to differentially bind IRBCs treated with trypsin (Fried et al. 2000). IRBCs treated with 100 ␮g/ml trypsin at room temperature showed a 97% decrease in binding to C4S containing 6-sulfate groups, while binding to C4S lacking 6-sulfate groups decreased only by 16.7%. This was attributed to the 4-sulfate- and 6-sulfate-dependent polyvalency of the receptor, with the sulfated groups contributing differentially to adherence capacity (Fried et al. 2000). However, results of our recent study indicate that the polyvalency of C4S dodecasaccharide motif that supports optimal IRBC

binding is due to nonsulfated and 4-sulfated disaccharide repeats (Alkhalil et al. 2000). Moreover, the CS chains of placental intervillous spaces do not contain 6-sulfate groups (Achur et al. 2000). Therefore, it appears that the relative distribution of the 4-sulfated and nonsulfated disaccharide repeats within the C4S dodecasaccharide structural domain influences IRBC adhesion, and a specific distribution pattern of these structures seems to support optimal interactions (Alkhalil et al. 2000). However, since the cell-associated CSPGs (presumably those on syncytiotrophoblast surface) contain significant levels of 6-sulfate groups (Achur et al. 2000), our data do not exclude the possibility of a population of placental IRBCs exhibiting polyvalency with regard to 4- and 6-sulfate groups. If this is the case, then the relative extents of IRBC adherence in the intervillous spaces and syncytiotrophoblasts of human placenta need to be established. In conclusion, the results presented in this paper demonstrate that some commercially available hucHA and bvhHA contain significant levels of CSPGs and that the CS chains of these CSPGs contain structural features that can support IRBC adherence. These results and the finding that human placenta contains negligible amounts of HA (Achur et al. 2000) support the idea that the previously reported adherence of IRBCs to HA (Beeson et al. 2000) could be due to the CSPGs present in the HA preparations used (Fried et al. 2000). However, our data do not completely exclude the possibility of a subset of placental IRBCs binding to HA. If HA is indeed an additional receptor, it may not be the major receptor, because the level of HA in the placenta is ⬍1–2% compared with CSPGs of the intervillous spaces. In any event, further detailed studies using Streptococcus HA conjugated to a protein or a lipid and various placental IRBC isolates should resolve the issue.

ACKNOWLEDGMENTS

The study was supported by grants from the Burroughs Wellcome Fund (New Initiatives in Malaria Research Award) and AI45086 from the National Institute of Allergy and Infectious Diseases, NIH.

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