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• W1988221816 abstract "We have previously demonstrated that the human placenta contains a uniquely low sulfated extracellular aggrecan family chondroitin sulfate proteoglycan (CSPG). This CSPG is a major receptor for the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in placentas, causing pregnancy-specific malaria. However, it is not known whether such low sulfated CSPGs occur in placentas of other animals and, if so, whether IRBCs bind to those CSPGs. In this study, we show that rat placenta contains a uniquely low sulfated extracellular CSPG bearing chondroitin sulfate (CS) chains, which comprise only ∼2% 4-sulfated and the remainder nonsulfated disaccharides. Surprisingly, the core protein of the rat placental CSPG, unlike that of the human placental CSPG, is a spongiotrophoblast-specific protein (SSP), which is expressed in a pregnancy stage-dependent manner. The majority of rat placental SSP is present in the CSPG form, and only ∼10% occurs without CS chain substitution. Of the total SSP-CSPG in rat placenta, ∼57% is modified with a single CS chain, and ∼43% carries two CS chains. These data together with the previous finding on human placental CSPG suggest that the expression of low sulfated CSPG is a common feature of animal placentas. Our data also show that the unique species-specific difference in the biology of the rat and human placentas is reflected in the occurrence of completely different CSPG core protein types. Furthermore, the rat SSP-CSPG binds P. falciparum IRBCs in a CS chain-dependent manner. Since IRBCs have been reported to accumulate in the placentas of malaria parasite-infected rodents, our results have important implications for exploiting pregnant rats as a model for studying chondroitin 4-sulfate-based therapeutics for human placental malaria. We have previously demonstrated that the human placenta contains a uniquely low sulfated extracellular aggrecan family chondroitin sulfate proteoglycan (CSPG). This CSPG is a major receptor for the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in placentas, causing pregnancy-specific malaria. However, it is not known whether such low sulfated CSPGs occur in placentas of other animals and, if so, whether IRBCs bind to those CSPGs. In this study, we show that rat placenta contains a uniquely low sulfated extracellular CSPG bearing chondroitin sulfate (CS) chains, which comprise only ∼2% 4-sulfated and the remainder nonsulfated disaccharides. Surprisingly, the core protein of the rat placental CSPG, unlike that of the human placental CSPG, is a spongiotrophoblast-specific protein (SSP), which is expressed in a pregnancy stage-dependent manner. The majority of rat placental SSP is present in the CSPG form, and only ∼10% occurs without CS chain substitution. Of the total SSP-CSPG in rat placenta, ∼57% is modified with a single CS chain, and ∼43% carries two CS chains. These data together with the previous finding on human placental CSPG suggest that the expression of low sulfated CSPG is a common feature of animal placentas. Our data also show that the unique species-specific difference in the biology of the rat and human placentas is reflected in the occurrence of completely different CSPG core protein types. Furthermore, the rat SSP-CSPG binds P. falciparum IRBCs in a CS chain-dependent manner. Since IRBCs have been reported to accumulate in the placentas of malaria parasite-infected rodents, our results have important implications for exploiting pregnant rats as a model for studying chondroitin 4-sulfate-based therapeutics for human placental malaria. Chondroitin sulfate proteoglycans (CSPGs) 2The abbreviations used are: CSPG, chondroitin sulfate proteoglycan; IRBC, infected red blood cell; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; CS, chondroitin sulfate; GAG, glycosaminoglycan; GdnHCl, guanidine hydrochloride; PBS, phosphate-buffered saline; PVDF, polyvinyldene difluoride; SSP, spongiotrophoblast-specific protein; HPLC, high pressure liquid chromatography; BSA, bovine serum albumin; TLCK, N-α-tosyl-l-lysine chloromethyl ketone; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone. are abundantly present in the connective tissues, such as cartilage, skin, and tendon, and in cornea, bone, umbilical cord, and blood vessels (1Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 2Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (552) Google Scholar, 3Hassell J.R. Kimura J.H. Hascall V.C. Annu. Rev. Biochem. 1986; 55: 539-567Crossref PubMed Scopus (245) Google Scholar). CSPGs are also ubiquitous in tissues and cell types of mammalian and other vertebrate animals as components of extracellular matrices as well as cell surface molecules. The CSPGs of cartilage and those from a wide variety of tissues and cell types of different animals have been extensively characterized with respect to their core proteins and the structural characteristics of the constituent CS chains (1Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 2Ruoslahti E. Annu. Rev. Cell Biol. 1988; 4: 229-255Crossref PubMed Scopus (552) Google Scholar, 3Hassell J.R. Kimura J.H. Hascall V.C. Annu. Rev. Biochem. 1986; 55: 539-567Crossref PubMed Scopus (245) Google Scholar, 4Hascall V.C. Hascall G.K. Hay E. D Cell Biology of Extra-cellular Matrix. Plenum Publishing Corp., New York1981: 39-63Google Scholar, 5Poole A.R. Biochem. J. 1986; 236: 1-14Crossref PubMed Scopus (378) Google Scholar, 6Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar). However, very little is known about the CSPGs in the maternal blood space of the placentas of various animals. Previously, we have shown that human placenta contains a major extracellular, unusually low sulfated aggrecan family CSPG, localized predominantly in the intervillous space (7Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 8Muthusamy A. Achur R.N. Bhavanandan V.P. Fouda G.G. Taylor D.W. Gowda D.C. Am. J. Pathol. 2004; 164: 2013-2025Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The CS chains of the placental CSPG consist of, on average, ∼8% 4-sulfated and ∼92% nonsulfated disaccharide residues (7Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The human placental CSPG is a mixture of two major distinctively sulfated aggrecan species, one with 2–3% and the other with 9–14% sulfated disaccharide moieties (9Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Although the biological function of the low sulfated aggrecan CSPG in human placenta is not known, it is possible that the placental low sulfated CS chains are involved in mobilizing nutrients, cytokines, hormones, and growth factors required for placental function and for the development of the fetus. The low sulfated CS chains are ideally suited for this function, since they are likely to reversibly and readily release the bound factors for their function. We have previously shown that the low sulfated CSPG is the receptor for the adherence of Plasmodium falciparum IRBCs in the placentas of pregnant women and that the sulfate groupclustered regions of the C4S chains are the IRBC-binding sites (7Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 8Muthusamy A. Achur R.N. Bhavanandan V.P. Fouda G.G. Taylor D.W. Gowda D.C. Am. J. Pathol. 2004; 164: 2013-2025Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 9Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Previous studies have also shown that P. falciparum IRBC binding to C4S involves the participation of both sulfated and nonsulfated disaccharide residues, and a dodecasaccharide motif with two 4-sulfated disaccharides is the minimal structural motif for optimal binding of IRBCs (9Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Fried M. Lauder R.M. Duffy P.E. Exp. Parasitol. 2000; 95: 75-78Crossref PubMed Scopus (53) Google Scholar, 11Chai W. Beeson J.G. Lawson A.M. J. Biol. Chem. 2002; 277: 22438-22446Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Recently, we have defined the role of the key functional groups, including the acetamido and carboxyl groups of C4S for IRBC binding. 3A. S. Prakasha Gowda, S. V. Madhunapantula, R. N. Achur, M. Valiyaveettil, V. P. Bhavanandan, and D. C. Gowda, unpublished results. Although the data from these studies offer strategies for developing C4S oligosaccharide-based therapy for placental malaria, animal models for studying the therapeutic potentials of such compounds have not been defined. In pregnant rats infected with P. berghei, as in the case of human placental malaria, IRBCs and monocytes accumulate in high density in the placenta, causing severe placental pathology (12Tegoshi T. Desowitz R.S. Pirl K.G. Maeno Y. Aikawa M. Am. J. Top. Med. Hyg. 1992; 47: 643-651Crossref PubMed Scopus (22) Google Scholar, 13Oduola A.M.J. Phillips J.H. Spicer S.S. Galbraith R.M. Exp. Parasitol. 1986; 62: 181-193Crossref PubMed Scopus (16) Google Scholar, 14Desowitz R.S. Duffy P.E. Fried M. E Malaria in Pregnancy. Taylor and Francis, New York2001: 127-157Crossref Google Scholar). However, the molecular interactions involved in the observed IRBC sequestration in the rat placenta are not known. As a part of our effort to determine whether pregnant rats can be useful as a model for studying pregnancy-specific malaria, we studied, in detail, the extracellular CSPG of the rat placenta. Interestingly, we found that although the rat placenta synthesizes very low sulfated CSPG, this proteoglycan is totally unrelated to aggrecan, and the sulfate content of the CS chains of rat placental CSPG is much more lower than that of the low sulfated human placental CSPG. The core protein of the rat placental CSPG is SSP, which is expressed in a pregnancy stage-specific manner (15Iwatsuki K. Shinozaki M. Sun W. Yagi S. Tanaka S. Shiota K. Biol. Reprod. 2000; 62: 1352-1359Crossref PubMed Scopus (25) Google Scholar). The expression of similarly low sulfated CS chains both in humans and rats, although the core proteins of CSPGs from these species are entirely different, points to important biological functions for the unusually low sulfated CS chains of placentas. Here, we report the structural characterization and IRBC-binding properties of the rat placental SSP-CSPG. Materials—Proteus vulgaris chondroitinase ABC, protease-free P. vulgaris chondroitinase ABC (120 units/mg), Arthrobacter aurescens chondroitinase AC II (87 units/mg), and super special grade C6S (shark cartilage) were purchased from Seikagaku America (Falmouth, MA). Bovine tracheal chondroitin sulfate A and Streptococcus species hyaluronic acid were from Calbiochem. Phenylmethylsulfonyl fluoride, N-ethylmaleimide, and benzamidine were from Sigma. TLCK and TPCK were from Roche Applied Science. Sepharose CL-6B, DEAE-Sephacel, and blue dextran were from Amersham Biosciences. Bio-Gel P-6, 4–15% gradient and 15% Tris-HCl polyacrylamide minigels, and protein molecular weight standards for SDS-PAGE were from Bio-Rad. HPLC grade 6 m HCl, trifluoroacetic acid, and micro-BCA protein assay kit were from Pierce. Polystyrene Petri dishes (Falcon 1058) were from BD Biosciences. The rabbit antiserum against SSP was generously provided by Dr. Kunio Shiota (Laboratory of Cellular Biochemistry, University of Tokyo, Tokyo, Japan) (15Iwatsuki K. Shinozaki M. Sun W. Yagi S. Tanaka S. Shiota K. Biol. Reprod. 2000; 62: 1352-1359Crossref PubMed Scopus (25) Google Scholar). Goat anti-rabbit IgG antibodies were from KPL (Gaithersburg, MD). Isolation of Extracellular Proteoglycans from Rat Placenta—The procedures used were similar to those previously described for the isolation of CSPGs from the human term placentas (7Achur R.N. Valiyaveettil M. Alkhalil A. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40344-40356Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Achur R.N. Valiyaveettil M. Gowda D.C. J. Biol. Chem. 2003; 278: 11705-11713Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). All procedures were performed on ice or in a cold room at 4 °C. Placentas (each weighing 0.6–0.7 g) from 20- or 21-day pregnant Wistar rats were collected by surgical dissection after anesthetizing the animals. Placentas from several rats were cut into small pieces, and the combined tissue (28 g) was extracted with 50 ml of PBS, pH 7.2, containing 10 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm TLCK, 0.25 mm TPCK, 1 mm benzamidine, and 0.1 mm N-ethylmaleimide by stirring with a magnetic stirrer for 4 h. The suspension was centrifuged at 10,000 rpm for 30 min in a RC2-B Sorvall centrifuge using SS-34 rotor. The clear supernatant was collected, and the tissue pellet was extracted two more times with 40 ml each time. The combined extract (∼120 ml) was passed through a DEAE-Sephacel column (1.5 × 25 cm) equilibrated with PBS, pH 7.2, and the column was washed successively with 25 mm Tris-HCl, 150 mm NaCl, 10 mm EDTA, pH 8.0, and 20 mm NaOAc, 150 mm NaCl, pH 5.5, until absorption of the effluent at 280 nm was at background level. The bound proteoglycan was eluted with a linear gradient of 0.15–0.75 m NaCl in 50 mm NaOAc, 4 m urea, pH 5.5. Fractions (2 ml) were collected, absorption at 260 and 280 nm was measured, and aliquots analyzed for uronic acid content (16Dische Z. J. Biol. Chem. 1947; 167: 189-198Abstract Full Text PDF PubMed Google Scholar). The uronic acid-containing fractions were combined, dialyzed, and lyophilized. The yield of crude proteoglycan was 16 mg. Purification of the CSPG by Cesium Bromide Density Gradient Centrifugation—The proteoglycan preparation from the DEAE-Sephacel chromatography of the placental extract was dissolved (2 mg/ml) in 25 mm sodium phosphate, pH 7.2, containing 50 mm NaCl, 0.02% NaN3,4 m GdnHCl, and 42% (w/w) CsBr. The solutions were centrifuged (8 ml/tube) in a Beckman 50 TI rotor at 44,000 rpm for 65 h at 14 °C (17Yphantis D.A. Biochemistry. 1964; 3: 297-317Crossref PubMed Scopus (2019) Google Scholar). Gradients were fractionated from the bottom of the tubes into 15 equal portions, absorption at 260 and 280 nm was measured, and uronic acid contents were determined (16Dische Z. J. Biol. Chem. 1947; 167: 189-198Abstract Full Text PDF PubMed Google Scholar). The uronic acid-positive fractions were pooled, dialyzed, and lyophilized; yield was 7.5 mg. Purification of the CSPG by Size Exclusion Chromatography—The proteoglycan partially purified by CsBr density centrifugation was chromatographed on a column of Sepharose CL-6B (2 × 65 cm) in 50 mm NaOAc, 150 mm NaCl, pH 6.0, containing 4 m GdnHCl. Fractions (2 ml) were collected and monitored for proteins by measuring absorption at 280 nm, and aliquots were analyzed for uronic acid contents (16Dische Z. J. Biol. Chem. 1947; 167: 189-198Abstract Full Text PDF PubMed Google Scholar); yield was 4.9 mg. Isolation of the CS Chains of Rat Placental CSPG—The purified CSPG (0.6 mg) was dissolved in 0.5 ml of 0.1 m NaOH, 1 m NaBH4 and incubated at 45 °C for 24 h under nitrogen (18Carlson D.M. J. Biol. Chem. 1968; 243: 616-626Abstract Full Text PDF PubMed Google Scholar). The solution was cooled in an ice bath, neutralized with 1 m HOAc, and dried in a rotary evaporator. Boric acid was removed by repeated evaporation with 0.1% acetic acid in methanol. The residue was dissolved in 0.2 m NaCl and chromatographed on a Sepharose CL-6B column (1 × 49 cm) in 0.2 m NaCl. Fractions (0.67 ml) were collected, and aliquots were assayed for uronic acid content (16Dische Z. J. Biol. Chem. 1947; 167: 189-198Abstract Full Text PDF PubMed Google Scholar). Fractions containing the CS chains were combined, dialyzed (molecular weight cut-off 3,500) against distilled water, and lyophilized. Carbohydrate Composition Analysis—The purified proteoglycan or the CS chains (5–10 μg each) released by alkaline β-elimination of the CSPG were hydrolyzed with 400 μl of 4 m HCl at 100 °C for 4 h, and the hydrolysates were dried in a SpeedVac and analyzed for hexosamines. For analysis of neutral sugar in the core proteins, the purified CSPG was treated with protease-free chondroitinase ABC (see below) and analyzed by SDS-PAGE using 15% gels, and the protein bands in the gels were transferred onto PVDF membranes, stained for 30 s with 0.1% Coomassie Blue in 40% MeOH and 1% AcOH, and destained with 50% MeOH. The membrane portions corresponding to the protein bands were cut into pieces, suspended in 400 μl of 2.5 m trifluoroacetic acid and hydrolyzed at 100 °C for 5 h, and the hydrolysates were dried as above. The hydrolysates from both procedures were analyzed on a CarboPac PA1 high pH anion exchange column (4 × 250 mm) using a Dionex BioLC HPLC system with a pulsed amperometric detector (19Hardy M.R. Townsend R.R. Methods Enzymol. 1994; 230: 208-225Crossref PubMed Scopus (157) Google Scholar). The elution was performed with 20 mm sodium hydroxide, and the response factors for the monosaccharides were determined using standard sugar solutions. Treatment with Chondroitinases—For GAG chain analysis, the purified CSPG or GAG chains obtained by alkaline β-elimination (100 μg) were treated with chondroitinase ABC (20 milliunits) in 50 μl of 100 mm Tris-HCl, pH 8.0, containing 30 mm NaOAc and 0.01% BSA at 37 °C for 5 h (20Oike Y. Kimata K. Shinomura T. Nakazawa K. Suzuki S. Biochem. J. 1980; 191: 193-207Crossref PubMed Scopus (201) Google Scholar). The CSPG (50 μg) was also treated with chondroitinase AC II (200 milliunits) in 50 μl of 100 mm NaOAc, pH 6.0, containing 0.01% BSA at 37 °C for 30 min (21Saito H. Yamagata T. Suzuki S. J. Biol. Chem. 1968; 243: 1536-1542Abstract Full Text PDF PubMed Google Scholar). Disaccharide Compositional Analysis of GAGs—The GAGs (10–15 μg), obtained by the treatment of the purified proteoglycan with 0.1 m NaOH, 1 m NaBH4, followed by Sepharose CL-6B chromatography, were digested with chondroitinase ABC (5 milliunits) as above. The released unsaturated disaccharides were analyzed on a 4.6 × 250-mm amine-bonded silica PA03 column (YMC Inc., Milford, MA), using a Waters 600E HPLC system (Milford, MA), and eluted with a linear gradient of 16–530 mm NaH2PO4 over 70 min at room temperature at a flow rate of 1 ml/min (22Sugahara K. Shigeno K. Masuda M. Fujii N. Kurosaka A. Takeda K. Carbohydr. Res. 1994; 255: 145-163Crossref PubMed Scopus (82) Google Scholar). The elution of disaccharides was measured by recording the absorption at 232 nm with a Waters 484 variable wavelength UV detector. The data were processed with the Millennium 2010 chromatography manager using NEC PowerMate 433 data processing system. Analysis of the CSPG Core Protein by SDS-PAGE and Western Blotting—The purified CSPG (100 μg) was treated with protease-free chondroitinase ABC (20 milliunits; protease inhibitors were also added to the incubation mixture as a precaution) in 100 μl of 100 mm Tris-HCl, 30 mm NaOAc, pH 8.0, at 37 °C for 6 h (20Oike Y. Kimata K. Shinomura T. Nakazawa K. Suzuki S. Biochem. J. 1980; 191: 193-207Crossref PubMed Scopus (201) Google Scholar). The enzyme digest corresponding to 20 μg of CSPG and 30 μg of untreated CSPG was electrophoresed on 4–15% polyacrylamide gradient gels under reducing conditions using 2-mercaptoethanol. The gels were stained sequentially with Coomassie Blue followed by Alcian Blue. For Western blot analysis, the protein bands (corresponding to 6 μg of CSPG) on gels were transferred onto PVDF membranes, and the membranes blocked with 1% BSA in 50 mm Tris-HCl, 150 mm NaCl, pH 8.0, containing 0.1% Tween 20. The membranes were incubated with 1:1000 diluted rabbit anti-SSP antiserum, and the bound antibodies were detected using alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibodies and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color-developing reagent. NH2-terminal Sequencing of the CSPG Core Proteins—The core proteins released from the purified CSPG (100 μg) by protease-free chondroitinase ABC (20 milliunits) were electrophoresed (20 μg CSPG/well) on 15% gels under reducing conditions. The protein bands on gels were transferred onto PVDF membranes using 10 mm 3-(cyclohexylamino)propane sulfonic acid buffer, pH 11.0, containing 10% methanol. The membranes were stained with Ponceau S, and the protein bands were cut out and sequenced by the Edman degradation method at the protein sequencing facility of the Penn State University College of Medicine using Applied Biosystems Procise 491 peptide sequencer. Analysis of Nonglycosylated and CSPG Form of SSP in Rat Placenta—The isotonic buffer extracts of rat placentas were analyzed on 15% polyacrylamide minigels before and after treatment with protease-free chondroitinase ABC, and the protein bands in the gels were transferred onto PVDF membranes. The membranes were treated with 1:1000-diluted rabbit anti-SSP antiserum followed by 1:2000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG. The bound secondary antibody was detected using chemiluminescence substrate. Other Analytical Procedures—The uronic acid contents were determined by the Dische method (16Dische Z. J. Biol. Chem. 1947; 167: 189-198Abstract Full Text PDF PubMed Google Scholar). Protein contents were estimated by using the micro-BCA protein estimation kit from Pierce (23Redinbaugh M.G. Turley R.B. Anal. Biochem. 1986; 153: 267-271Crossref PubMed Scopus (402) Google Scholar). P. falciparum Culture—The C4S adherent P. falciparum were panselected from a 3D7 parasite clone as described earlier (24Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Parasites were cultured in RPMI 1640 medium supplemented with 25 mm HEPES, 29 mm sodium bicarbonate, 0.005% hypoxanthine, p-aminobenzoic acid (2 mg/liter), gentamycin sulfate (50 mg/liter), and 10% O+ human serum using type O+ human red blood cells at 3% hematocrit (24Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Ockenhouse C.F. Ho M. Tandon N.N. Van Seventor G.A. Shaw S. White N.J. Jamieson G.A. Chulay J.D. Webster H.K. J. Infect. Dis. 1991; 164: 163-169Crossref PubMed Scopus (187) Google Scholar). The cultures were incubated at 37 °C in an atmosphere of 90% nitrogen, 5% oxygen, and 5% carbon dioxide. Parasites with 20–30% parasitemia at the early trophozoite stage were used for the IRBC binding and inhibition assays. IRBC Adherence Assay—The assay was performed as described previously (24Alkhalil A. Achur R.N. Valiyaveettil M. Ockenhouse C.F. Gowda D.C. J. Biol. Chem. 2000; 275: 40357-40364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly, the purified rat placental CSPGs at various concentrations were coated onto plastic Petri dishes as 0.4-cm circular spots, blocked with 2% BSA in PBS, pH 7.2, and then overlaid with a 2% suspension of parasite culture in PBS, pH 7.2. The unbound IRBCs and RBCs were washed with PBS, and the bound IRBCs were fixed with 2% glutaraldehyde. The bound IRBCs were stained with Giemsa and examined under the light microscope. IRBC Adherence Inhibition Assay—Solutions (1 μg/ml) of the purified rat placental CSPG were coated onto plastic plates as 4-mm circular spots, and the spots were blocked with 2% BSA. The IRBC suspensions in PBS, pH 7.2, were preincubated with the indicated amounts of partially sulfated C4S (40% 4-sulfate, 59% nonsulfated, and 1% 6-sulfate), C6S, chondroitin, or hyaluronic acid at room temperature for 30 min with occasional mixing. The cell suspensions were overlaid onto the CSPG-coated spots and allowed to stand at room temperature for 30 min. After washing the unbound cells, the bound cells were fixed, stained, and counted by light microscopy. Isolation and Purification of the Extracellular Proteoglycan of Rat Placenta—DEAE-Sephacel chromatography of the isotonic buffer extracts of the placentas from 20- or 21-day-old pregnant rats using a 0.15–0.75 m NaCl gradient eluted the uronic acid-containing material (proteoglycan) as a single homogeneous peak at 0.35 m NaCl (Fig. 1). Final elution of the column with 1.2 m NaCl did not elute any uronic acid-containing material (not shown). Upon CsBr gradient centrifugation, the proteoglycan was fractionated as a broad peak to the middle of the density gradient, separating from most of the associated proteins, which remained at the top of the gradient (Fig. 2). Upon further purification by size exclusion chromatography on Sepharose CL-6B column, the proteoglycan was eluted as a single but somewhat asymmetrical peak (Kav = 0.67) with an estimated average molecular weight of ∼120,000 (Fig. 3). The yield and composition of the proteoglycan is given in Table 1. The presence of protein, uronic acid, and hexosamine in high amounts is consistent with the proteoglycan nature of the purified material from rat placentas.FIGURE 2Purification of rat placental CSPG by CsBr density gradient centrifugation. The rat placental CSPG, isolated by DEAE-Sephacel chromatography (Fig. 1), was dissolved in 25 mm sodium phosphate, pH 7.2, containing 4 m GdnHCl, 0.02% sodium azide, and 42% CsBr. The solutions were centrifuged in a Beckman 50 TI rotor at 44,000 rpm for 65 h at 14 °C. The gradients were collected into 15 equal fractions from the bottom of the tube, and the aliquots were monitored for uronic acid (•) and protein (○). The CSPG-containing fractions were pooled as indicated by the horizontal bar.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Purification of rat placental CSPG by size exclusion chromatography. The rat placental CSPG purified by CsBr gradient centrifugation was chromatographed on a Sepharose CL-6B column (2 × 65 cm) in 50 mm NaOAc, 150 mm NaCl, pH 6.0, containing 4 m GdnHCl. The eluted fractions (2 ml) were monitored for uronic acid (•) and for protein (○). The CSPGs were recovered by pooling the fractions, as indicated by the horizontal bar. The elution positions of blue dextran (BD) and glucose (Glc) are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Yield and composition of rat placental CSPGYield/10 g of pooled rat placentaaThe average weight of one rat placenta is about 0.65 g.CompositionHexNDisaccharidebBy chondroitinase ABC digestion followed by the HPLC analysis.ProteincBy the BCA method (23).Uronic aciddBy the carbazole method (16).HexNeBy high pH anion exchange HPLC of 4 m HCl hydrolysates of the CSPG.GalNGlcNΔdi-4SΔdi-0Sweight %mol ratio %1.8 mg193942>99<1298a The average weight of one rat placenta is about 0.65 g.b By chondroitinase ABC digestion followed by the HPLC analysis.c By the BCA method (23Redinbaugh M.G. Turley R.B. Anal. Biochem. 1986; 153: 267-271Crossref PubMed Scopus (402) Google Scholar).d By the carbazole method (16Dische Z. J. Biol. Chem. 1947; 167: 189-198Abstract Full Text PDF PubMed Google Scholar).e By high pH anion exchange HPLC of 4 m HCl hydrolysates of the CSPG. Open table in a new tab Characterization of the GAG Chains of Rat Placental Blood Space Proteoglycan—The carbohydrate compositional analysis revealed that the purified proteoglycan contains >99% GalN and a trace amount of GlcN (Table 1). Upon treatment with chondroitinase ABC or chondroitinase AC II, the GAG chains were quantitatively converted into disaccharides (not shown). These results indicated that the proteoglycan is a CSPG. The CSPG was subjected to alkaline β-elimination using NaOH/NaBH4, and the released CS chains were purified by gel filtration on a Sepharose CL-6B column calibrated with chondroitin sulfates of known molecular weight (Fig. 4). The CS chains of the CSPG were eluted as a single symmetrical peak with an estimated molecular weight of ∼40,000. Digestion of the CSPG with chondroitinase ABC and HPLC analysis of the disaccharides formed showed that the CS chains consist of 2% 4-sulfated and 98% nonsulfated disaccharides, demonstrating that the CS chains of the CSPG are extremely low sulfated, resembling nonsulfated chondroitin chains (Table 1). Characterization of Core Proteins of the Rat Placental CSPG—Upon SDS-PAGE analysis, the purified CSPG was electrophoresed as two partially separated broad bands with mobility corresponding to molecular weights of 60,000–80,000 (average ∼70,000) and 80,000–180,000 (∼130,000) (Fig. 5A). The core proteins released by the treatment of the purified CSPG with chondroitinase ABC were electrophoresed as a doublet corresponding to molecular mass of ∼17 and ∼18 kDa (Fig. 5A). The relative abundance of the 17- and 18-kDa core protein bands in gels, as assessed by the densitometric scan, was ∼57 and ∼43%, respectively (Fig. 5B). The NH" @default.
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• W1988221816 date "2006-10-01" @default.
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