The Low Sulfated Chondroitin Sulfate Proteoglycans of Human Placenta Have Sulfate Group-clustered Domains That Can Efficiently Bind Plasmodium falciparum-infected Erythrocytes
2003; Elsevier BV; Volume: 278; Issue: 13 Linguagem: Inglês
10.1074/jbc.m211015200
ISSN1083-351X
AutoresRajeshwara N. Achur, Manojkumar Valiyaveettil, D. Channe Gowda,
Tópico(s)Research on Leishmaniasis Studies
ResumoPlasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with ∼30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only ∼8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2–3% and 9–14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6–14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20–28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation. Plasmodium falciparum infection in pregnant women results in the chondroitin 4-sulfate-mediated adherence of the parasite-infected red blood cells (IRBCs) in the placenta, adversely affecting the health of the fetus and mother. We have previously shown that unusually low sulfated chondroitin sulfate proteoglycans (CSPGs) in the intervillous spaces of the placenta are the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate motif consisting of six disaccharide moieties with ∼30% 4-sulfated residues. However, it was puzzling how the placental CSPGs, which have only ∼8% of the disaccharide 4-sulfated, could efficiently bind IRBCs. Thus, we undertook to determine the precise structural features of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which are similar by all criteria except differing in their sulfate contents; 2–3% and 9–14% of the disaccharide units of the CS chains are 4-sulfated, and the remainder are nonsulfated. The majority of the sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6–14 repeating disaccharide units. While the sulfate-rich regions of the CS chains contain 20–28% 4-sulfated disaccharides, the other regions have little or no sulfate. Further, we find that the placental CSPGs are able to efficiently bind IRBCs due to the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of placental CSPGs and that these sulfate-clustered domains have the necessary structural elements for the efficient adhesion of IRBCs, although the CS chains have an overall low degree of sulfation. infected red blood cells chondroitin sulfate chondroitin sulfate proteoglycan chondroitin 4-sulfate chondroitin 6-sulfate bovine serum albumin cesium bromide phosphate-buffered saline 2-mer, 3-mer, 4-mer, 5-mer, and 6-mer, size of C4S oligosaccharides with 1, 2, 3, 4, 5, and 6 disaccharide repeating units, respectively erythrocyte membrane protein 1 high pressure liquid chromatography A distinctive feature of Plasmodium falciparum compared with the other three human malaria parasites is its ability to express adherent protein(s) on the surfaces of the infected red blood cells (IRBCs)1 and thereby sequester in the microvascular capillaries of various organs by adhering to endothelial cell surfaces (1Pasloske B.L. 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However, over a period of time, the host develops antibodies against the exposed P. falciparum EMP1 that are able to inhibit adhesion of IRBC adhesion and aid clearance of infection (29Fried M. Nosten F. Brockman A. Brabin B.J. Duffy P.E. Nature. 1998; 395: 851-852Google Scholar, 30Maubert B. Fievet N. Tami G. Cot M. Boudin C. Deloron P. Infect. Immun. 1999; 67: 5367-5371Google Scholar, 31Gysin J. Pouvelle B. Fievet N. Scherf A. Lepolard C. Infect. Immun. 1999; 67: 6596-6602Google Scholar, 32Staalsoe T. Megnekou R. Fievet N. Ricke C.H. Zornig H.D. Leke R. Taylor D.W. Deloron P. Hviid L. J. Infect. Dis. 2001; 184: 618-626Google Scholar, 33Ricke C.H. Staalsoe T. Koram K. Akanmori B.D. Riley E.M. Theander T.G. Hviid L. J. Immunol. 2000; 165: 3309-3316Google Scholar, 34O'Neil-Dunne I. Achur R.N. Agbor-Enoh S.T. Valiyaveettil M. Naik R.S. Ockenhouse C.F. Zhou A. Megnekou R. Leke R. Taylor D.W. Gowda D.C. Infect. Immun. 2001; 69: 7487-7492Google Scholar). To overcome this defensive mechanism, the parasite constantly switches, at low frequency, to various adherent phenotypes by expressing P. falciparum EMP1s with different receptor specificity (35Taylor H.M. Kyes S.A. Newbold C.I. Mol. Biochem. Parasitol. 2000; 110: 391-397Google Scholar, 36Nielsen M.A. Staalsoe T. Kurtzhals J.A. Goka B.Q. Dodoo D. Alifrangis M. Theander T.G. Akanmori B.D. Hviid L. J. Immunol. 2002; 168: 3444-3450Google Scholar). This ability of the parasite to express P. falciparum EMP1, for which the host has not yet developed adhesion-inhibitory antibodies, enables it to selectively adhere through a different receptor. In this manner, when one adherent phenotype of parasite is eliminated by the host, another phenotype continues to thrive. In endemic areas, people by adulthood acquire a broad spectrum protective immunity against P. falciparum, including antibodies to P. falciparumEMP1s (37Baird J.K. Parasitol. Today. 1995; 11: 105-111Google Scholar, 38Riley E.M. Hviid L. Theander T.G. Kierszenbaum F. Malaria. Academic Press, Inc., New York1994: 119-143Google Scholar). Therefore, in immune-protected people, the IRBCs cannot adhere in the vascular capillaries, limiting the parasite growth. In pregnant women, however, the placenta provides a new opportunity for IRBC adhesion, because women lack immunity against placenta-adherent parasites prior to pregnancy (39Fried M. Duffy P.E. Science. 1996; 272: 1502-1504Google Scholar). Extensive adherence of IRBCs in the placenta and infiltration of mononuclear cells in response to the infection results in impaired placental function, leading to poor fetal outcome and maternal morbidity and mortality (40McGregor I.A. Wilson M.E. Billewicz W.Z. Trans. R. Soc. Trop. Med. Hyg. 1983; 77: 232-244Google Scholar, 41Snow R.W. Craig M. Diechmann U. Marsh K. Bull. WHO. 1999; 77: 624-640Google Scholar, 42Steketee R.W. Wirima J.J. Slutsker L. Heymann D.L. Breman J.G. Am. J. Trop. Med. Hyg. 1996; 55: 2-7Google Scholar, 43Steketee R.W. Nahlen B.L. Parise M.E. Menendez C. Am. J. Trop. Med. Hyg. 2001; 64: 28-35Google Scholar, 44Menendez C. Parasitol. Today. 1995; 11: 178-183Google Scholar, 45Brabin B.J. Bull. WHO. 1983; 61: 1005-1016Google Scholar, 46Marsh K. Parasitology. 1992; 104: S53-S69Google Scholar). However, women acquire placental malaria-specific immunity, including adhesion-inhibitory antibody response, during the first and second pregnancies (29Fried M. Nosten F. Brockman A. Brabin B.J. Duffy P.E. Nature. 1998; 395: 851-852Google Scholar, 30Maubert B. Fievet N. Tami G. Cot M. Boudin C. Deloron P. Infect. Immun. 1999; 67: 5367-5371Google Scholar, 31Gysin J. Pouvelle B. Fievet N. Scherf A. Lepolard C. Infect. Immun. 1999; 67: 6596-6602Google Scholar, 32Staalsoe T. Megnekou R. Fievet N. Ricke C.H. Zornig H.D. Leke R. Taylor D.W. Deloron P. Hviid L. J. Infect. Dis. 2001; 184: 618-626Google Scholar, 33Ricke C.H. Staalsoe T. Koram K. Akanmori B.D. Riley E.M. Theander T.G. Hviid L. J. Immunol. 2000; 165: 3309-3316Google Scholar, 34O'Neil-Dunne I. Achur R.N. Agbor-Enoh S.T. Valiyaveettil M. Naik R.S. Ockenhouse C.F. Zhou A. Megnekou R. Leke R. Taylor D.W. Gowda D.C. Infect. Immun. 2001; 69: 7487-7492Google Scholar). Therefore, primigravidas are at highest risk of placental malaria, and the susceptibility diminishes with increasing gravidity (44Menendez C. Parasitol. Today. 1995; 11: 178-183Google Scholar, 45Brabin B.J. Bull. WHO. 1983; 61: 1005-1016Google Scholar). C4S mediates the adhesion of IRBCs in the human placenta (39Fried M. Duffy P.E. Science. 1996; 272: 1502-1504Google Scholar, 47Pouvelle B. Meyer P. Robert C. Bardel L. Gysin J. Mol. Med. 1997; 3: 508-518Google Scholar, 48Gysin J. Pouvelle B. Tonqueze M.L. Edelman L. Boffa M.C. Mol. Biochem. Parasitol. 1997; 88: 267-271Google Scholar, 49Rogerson S.J. Brown G.V. Parasitol. Today. 1997; 134: 70-75Google Scholar). Previously, we have shown that the CSPGs localized in the intervillous spaces of the placenta are the receptors for the adherence of IRBCs in the placenta (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar). These CSPGs were found to be unusually low sulfated; on an average, only ∼8% of the disaccharide repeating units of the CS chains of placental CSPGs are 4-sulfated, and the remainder are nonsulfated (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar). In previous studies, we have also shown that IRBC adhesion involves the participation of both nonsulfated and 4-sulfated disaccharide repeating units and the optimal binding requires ∼30% 4-sulfated and ∼70% nonsulfated disaccharide repeats (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar). Further, we established that a C4S motif having six disaccharide repeating units (6-mer) with two 4-sulfated and four nonsulfated disaccharide units is the minimum structural motif required for optimal binding of IRBCs (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar). A recent study confirmed most of our findings (52Chai W. Beeson J.G. Lawson A.M. J. Biol. Chem. 2002; 277: 22438-22446Google Scholar), except that four or five rather than two of the disaccharide repeating units of the binding motif containing six-disaccharide repeating units needs to be 4-sulfated for effective IRBC binding. However, it should be noted that these investigators measured C4S-IRBC interactions by immobilizing the commercially available bovine trachea C4S/C6S copolymer (52Chai W. Beeson J.G. Lawson A.M. J. Biol. Chem. 2002; 277: 22438-22446Google Scholar), a nonrelevant glycosaminoglycan, rather than the placental CSPGs, the natural receptor used in our study (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar). Regardless of this discrepancy, it remained a puzzle how IRBCs are able to efficiently bind the unusually low sulfated CS chains of the placental intervillous space CSPGs. A full understanding of the structural requirements for IRBC binding to placenta is important for developing therapeutics or vaccine for placental malaria (53Staalsoe T. Jensen A. Theander T. Hviid L. Immunol. Lett. 2002; 84: 133-136Google Scholar). Therefore, in this study, we investigated in detail the structure of the CS chains of placental CSPGs, particularly the pattern of sulfate group distribution and its correlation to IRBC binding. The placental CSPGs were fractionated into two differentially sulfated proteoglycan populations and their CS chains isolated. The polysaccharide chains were degraded with an endoenzyme that specifically cleaves the nonsulfated regions of the CS into disaccharides, and the oligosaccharide products thus obtained were purified and examined for their ability to inhibit IRBC binding to intact CSPGs. The data demonstrate that, although the overall sulfate content of the CS chains of placental CSPGs are markedly lower than that required for optimal binding, the sulfate groups in the CS chains are clustered in uniquely size-defined domains. These sulfate-rich CS domains have the requisite structural features for the efficient binding of IRBCs. Proteus vulgarischondroitinase ABC (120 units/mg), Streptococcus dysgalactiae hyaluronidase (0.5 units/vial) andStreptomyces hyalurolyticus hyaluronidase (2000 turbidity-reducing units/mg), Flavobacterium heparinum heparitinase (113 units/mg), chondroitin, and C4S (sturgeon notochord) were purchased from Seikagaku America (Falmouth, MA); ovine testicular hyaluronidase (2160 units/mg) was from ICN Biomedicals; C4S (bovine trachea) was from Sigma; Sepharose CL-6B, Sepharose CL-4B, DEAE-Sephacel, DEAE-Sepharose, and blue dextran were from Amersham Biosciences; Bio-Gel P-6 was from Bio-Rad; HPLC grade 6m HCl, trifluoroacetic acid, and micro-BCA protein assay kit were from Pierce; polystyrene Petri dishes (Falcon 1058) were from Becton Dickinson Labware. C4S with 36% 4-sulfate was prepared by the regioselective 6-O-desulfation of bovine trachea C4S/C6S copolymer as described previously (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar) and fractionation of the product by DEAE-Sephacel chromatography. 2R. N. Achur and D. C. Gowda, unpublished results. C4S 6-mers with 36% 4-sulfate was prepared by the digestion of C4S containing 36% 4-sulfate group with testicular hyaluronidase and fractionation of the oligosaccharides by gel filtration on Bio-Gel P-6.2C4Ss with 3 and 11% 4-sulfate groups were prepared by the solvolytic desulfation of a fully 4-sulfated C4S from sturgeon notochord as described previously (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar). The low sulfated CSPGs of the placental intervillous spaces were isolated as described previously with minor modification (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar). Briefly, the placentas were cut into small pieces and extracted with PBS, pH 7.2, containing protease inhibitors, and the extract was applied onto DEAE-Sephacel columns (2.5 × 22 cm). The columns were washed with 25 mm Tris-HCl, 150 mm NaCl, 10 mmEDTA, pH 8.0, and then equilibrated with 50 mm NaOAc, 100 mm NaCl, pH 5.5. The bound material was eluted with a linear gradient of 0.1–0.9 m NaCl in 50 mmNaOAc, pH 5.5. 10-ml fractions were collected, and aliquots were analyzed for uronic acid content (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). Uronic acid-containing fractions corresponding to BCSPG-2, the major CSPG of the placental intervillous spaces (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar), were pooled and dialyzed against water. The dialysates were adjusted to 50 mm NaOAc, 100 mm NaCl, pH 5.5, and applied onto DEAE-Sepharose columns (2.5 × 17 cm). The columns were washed with 50 mm NaOAc, 0.15 mNaCl, pH 5.5, and eluted with a linear gradient of 0.15–0.55m NaCl in 50 mm NaOAc, pH 5.5. Fractions of 10-ml were collected, absorption at 260 and 280 nm was measured, and aliquots were analyzed for uronic acid content (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). The crude CSPG fractions obtained by DEAE-Sepharose chromatography were dissolved (1 mg/ml) in 25 mm sodium phosphate, pH 7.2, containing 50 mm NaCl, 0.02% NaN3, 4m guanidine hydrochloride, and 42% (w/w) CsBr. The solutions were centrifuged in a Beckman 50 TI rotor at 44,000 rpm for 65 h (55Yphantis D.A. Biochemistry. 1964; 3: 297-317Google Scholar). Gradients were collected from the bottom of the centrifuge tubes into 15 equal fractions and analyzed for uronic acid content (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar) and for proteins by measuring the absorption at 260 and 280 nm. The CSPG fractions obtained from the CsBr density gradient centrifugation step were further purified by chromatography on columns of Sepharose CL-6B (1 × 49 cm) and/or Sepharose CL-4B (1 × 48 cm) in 20 mm Tris-HCl, 150 mm NaCl, pH 7.6, containing 4m guanidine hydrochloride. Fractions were collected and monitored for absorption at 260 and 280 nm and for uronic acid content (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). The purified CSPGs (0.3 mg) were treated with S. hyalurolyticus hyaluronidase and heparitinase as described previously (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar). The enzyme-incubation mixtures were chromatographed on Bio-Gel P-6 (1 × 47 cm) in 0.1m acetic acid, 0.1 m pyridine. Fractions (0.67 ml) were collected and analyzed for uronic acid (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). The purified CSPGs were treated with 0.1 m NaOH, 1 m NaBH4for 18–20 h under nitrogen atmosphere at 45 °C (56Carlson D.M. J. Biol. Chem. 1968; 243: 616-626Google Scholar). The solutions were cooled in ice bath, neutralized with 1 m cold acetic acid, and then dried in a rotary evaporator. Boric acid was removed by repeated evaporation in a rotary evaporator by the addition of methanol, 0.1% acetic acid. The residue was applied onto a DEAE-Sepharose column (1 × 10 cm) in 20 mm Tris-HCl, pH 7.8, washed with 20 mm Tris-HCl, 0.15 mNaCl, pH 7.8, and then eluted with a linear gradient of 0.15–0.6m NaCl in the same buffer. Fractions (2.5 ml) were collected, and aliquots were assayed for uronic acid content (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). Uronic acid-positive fractions were pooled, dialyzed against distilled water, and lyophilized. The placental CS chains and chondroitin (1.4–1.8 mg each) were treated with S. dysgalactiae hyaluronidase (300 milliunits) in 250 μl of 100 mm sodium phosphate buffer, pH 6.2, containing 0.02% BSA at 37 °C for 24 h (57Hamai A. Morikawa K. Horie K. Tokuyasu K. Seikagaku. 1986; 58: 783Google Scholar). The enzyme digests were heated at 100 °C for 5 min, centrifuged, and then chromatographed on Bio-Gel P-6 columns (1 × 47 cm) in 0.1m pyridine, 0.1 m acetic acid. Fractions (0.67 ml) were collected, and aliquots were analyzed for uronic acid (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). The CS oligosaccharides were dissolved in 100 mm Tris base, 100 mm boric acid, 2 mm EDTA, pH 8.3, containing 5% glycerol (58Wall R.S. Gyi T.J. Anal. Biochem. 1988; 175: 298-299Google Scholar). The solutions were electrophoresed on 10% polyacrylamide gels (15 × 16 cm) in 100 mm Tris base, 100 mm boric acid, 2 mm EDTA, pH 8.3. The gels were stained with 0.03% Alcian Blue in 25% ethanol, 10% aqueous acetic acid for 4 h and destained with 25% ethanol, 10% aqueous acetic acid. For silver staining, the gels were treated with 10% aqueous glutaraldehyde for 30 min and washed with water three times for 30 min each. The gels were then treated with freshly prepared ammoniacal silver for 15 min, washed with water two times for 30 s each, developed with 0.005% citric acid and 0.019% formaldehyde in water, and then washed with water (59Krueger Jr., R.C. Schwartz N.B. Anal. Biochem. 1987; 167: 295-300Google Scholar). The oligosaccharides (fractions 20–35, 50 μg) obtained by the Bio-Gel P-6 chromatography of the S. dysgalactiae hyaluronidase digest of chondroitin were dissolved in 20 mm NaOAc, 50 mm NaCl, pH 5.0, and applied onto a DEAE-Sepharose microcolumn (0.1-ml bed volume). After washing with 0.5 ml of the above buffer, the bound oligosaccharides were eluted stepwise with buffer containing 0.1, 0.2, 0.4, and 0.6 m NaCl (0.5 ml each). Fractions (0.1 ml) were collected, and aliquots were analyzed for uronic acid (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). The oligosaccharide-containing fractions were pooled and digested with chondroitinase ABC, and the disaccharides formed were analyzed by HPLC. The C4Ss or C4S oligosaccharides (10–15 μg) were digested with chondroitinase ABC (10–20 milliunits) in 50 μl of 0.1 m Tris-HCl, pH 8.0, containing 30 mm NaOAc and 0.01% BSA at 37 °C for 12–15 h (60Oike Y. Kimata K. Shinomura T. Nakazawa K. Suzuki S. Biochem. J. 1980; 191: 193-207Google Scholar). The released, unsaturated disaccharides were analyzed on an amine-bond Microsorb-MV column (4.6 × 250 mm; Varian) using Waters 600E HPLC system (Milford, MA) (61Sugahara K. Shigeno K. Masuda M. Fujii N. Kurosaka A. Takeda K. Carbohydr. Res. 1994; 255: 145-163Google Scholar). The enzyme digests were injected, and the column was eluted with a linear gradient of 16–530 mm NaH2PO4 over 70 min at room temperature at a flow rate of 1 ml/min. The elution of disaccharides was monitored by measuring the absorption at 232 nm using a Waters 484 variable wavelength UV detector. The data were processed with the Millennium 2010 chromatography manager using NEC PowerMate 433 data processing system. The CSPGs or CS chains (5–10 μg) 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 HPLC column (4 × 250 mm; Dionex) (62Hardy M.R. Methods Enzymol. 1989; 179: 76-82Google Scholar). The column was eluted with 20 mm sodium hydroxide, elution of sugars was monitored by pulsed amperometric detection, and the response factors for sugars were determined using standard sugar solutions. The uronic acid contents in various column chromatography fractions were determined by the carbazole-sulfuric acid method (54Dische Z. J. Biol. Chem. 1947; 167: 189-198Google Scholar). Protein contents were measured using the Micro BCA Protein Assay Reagent kit from Pierce (63Redinbaugh M.G. Turley R.B. Anal. Biochem. 1986; 153: 267-271Google Scholar). The C4S adherent P. falciparum, selected by panning of 3D7 laboratory parasite clones on placental CSPG-coated plastic plates, were used in this study. The parasites were cultured using type O-positive human red blood cells at 3% hematocrit in RPMI 1640 medium supplemented with 25 mmHEPES, 29 mm sodium bicarbonate, 0.005% hypoxanthine,p-aminobenzoic acid (2 mg/liter), gentamycin sulfate (50 mg/liter), and 10% O-positive human serum. The cultures were incubated at 37 °C in an atmosphere of 90% nitrogen, 5% oxygen, and 5% carbon dioxide (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar). The adherence of IRBCs was performed by coating solutions (10–15 μl) of purified CSPGs as circular spots on 150 × 15-mm plastic Petri dishes as described previously (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar). The specificity of IRBC binding to CSPGs was ascertained by incubating the CSPG-coated plates with chondroitinase ABC (50 milliunits/ml) as well as by competitive inhibition with various C4Ss. For adhesion-inhibition assays, IRBCs were incubated with various C4Ss or C4S oligosaccharides at the indicated concentrations in PBS, pH 7.2, in 96-well microtiter plates at room temperature for 30 min with intermittent mixing (51Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Google Scholar). The IRBC suspension was then layered on CSPG-coated spots on Petri dishes. After 40 min at room temperature, the unbound cells were washed, and the bound cells were fixed with 2% glutaraldehyde, stained with Giemsa, and counted using a light microscope. The major low sulfated CSPG fraction (previously designated as BCSPG-2) (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar) was isolated by one-step DEAE-Sephacel chromatography of the isotonic buffer extract of placentas. This CSPG fraction represents about 93–94% of the total low sulfated CSPGs in the intervillous spaces (50Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Google Scholar). When subjected to DEAE-Sepharose chromatography, using a 0.15–0.55 m NaCl gradient, the CSPG was partially resolved into two fractions (designated BCSPG-2a and BCSPG-2b) that are distinct in their sulfation levels (Fig. 1). The proportions of BCSPG-2a and BCSPG-2b varied considerably from one placenta to another, in the range 40–65% and 35–60%, respectively. These CSPG fractions were further purified and characterized, and their ability to bind IRBCs was studied. On CsBr density
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