Chondroitin sulfate proteoglycans of the endothelia of human umbilical vein and arteries and assessment for the adherence of Plasmodium falciparum-infected erythrocytes

Molecular & Biochemical Parasitology 134 (2004) 115–126

Chondroitin sulfate proteoglycans of the endothelia of human umbilical vein and arteries and assessment for the adherence of Plasmodium falciparum-infected erythrocytes Manojkumar Valiyaveettil1 , Rajeshwara N. Achur, Arivalagan Muthusamy, D. Channe Gowda∗ Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA Received 31 July 2003; received in revised form 13 November 2003; accepted 13 November 2003

Abstract Infection with Plasmodium falciparum during pregnancy leads to chondroitin 4-sulfate-mediated adhesion of the infected red blood cells (IRBCs) in the placenta, causing severe health complications to fetus and the mother. The IRBCs are also frequently found in low density in the umbilical cord of infected placentas. In this study, the CSPGs of umbilical vein and arteries were purified, characterized, and their localization and IRBC-binding abilities were studied. While a versican type CSPG was found both in the vein and arteries, a serglycin type CSPG was present exclusively in the vein. The CSPGs were present at significant level on the endothelial surface of the umbilical vein but not on that of arteries. Although the purified versican and serglycin type CSPGs could bind IRBCs, their binding abilities were significantly less compared to the low sulfated CSPGs of the placenta because of the predominance of 6-sulfated disaccharide moieties in the CS chains. Therefore, IRBCs were unable to bind efficiently onto the umbilical cord endothelial surface. Unexpectedly, however, the IRBCs adhered densely in the blood vessels of fetal villi in the placental tissue sections and sparingly in the blood spaces of the umbilical cord vein, presumably because the CSPG that can efficiently bind IRBCs is present at high levels in the fetal blood vessels and at very low levels in the umbilical cord blood vessels. Since the C4S-adherent IRBCs that enter the fetal blood vessels cannot adhere to the cord endothelial surface and parasites cannot efficiently grow due to fetal hemoglobin toxicity and protection by maternal antibodies, transplacental infection may be quickly cleared without clinical episodes. © 2003 Elsevier B.V. All rights reserved. Keywords: Plasmodium falciparum; Umbilical cord; Chondroitin sulfate proteoglycans; Infected erythrocytes; Adherence

1. Introduction

Abbreviations: IRBCs, infected red blood cells; HUVECs, human umbilical vein endothelial cells; GAG, glycosaminoglycan; CS, chondroitin sulfate; PG, proteoglycan; CSPG, chondroitin sulfate proteoglycan; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; DS, dermatan sulfate; DSPG, dermatan sulfate proteoglycan; HA, hyaluronic acid; HS, heparan sulfate; BSA, bovine serum albumin; BD, blue dextran; Glc, glucose; HexN, hexosamine; GalN, galactosamine; GlcN, glucosamine; CsBr, cesium bromide; HRP, horseradish peroxidase; DEAE, diethylaminoethyl; EDTA, ethylenediamine tetraacetic acid; GdnHCl, guanidine hydrochloride; NEM, N-ethylmaleimide; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; TLCK, N-␣-tosyl-l-lysine chloromethyl ketone; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PVDF, polyvinylidene difluoride ∗ Corresponding author. Tel.: +1-717-531-0992; fax: +1-717-531-7072. E-mail address: [email protected] (D.C. Gowda). 1 Present address: Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA. 0166-6851/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2003.11.009

Sequestration of Plasmodium falciparum infected red blood cells (IRBCs) in the vascular capillaries of various organs by the adhesion to endothelial cell lining is a central event during falciparum malaria pathogenesis [1]. Accumulated evidence suggests that the IRBC adhesion is mediated by the interaction of the host endothelial cell surface adhesion molecules with an antigenic variant parasite protein, P. falciparum erythrocyte membrane protein 1, expressed on the surfaces of infected erythrocytes [2,3]. Thus, extensive accumulation of IRBCs leads to capillary obstruction and deprivation of essential nutrients for vital metabolic function [4]. These functional impairments together with the production of high level of proinflammatory cytokines at IRBC adhesion sites are likely to cause endothelial damage, organ dysfunction, resulting in severe illness [4,5]. In malaria endemic areas, regardless of gender, adults have natural immunity against developing severe malaria;

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thus, they rarely develop clinical complications although they may harbor P. falciparum infection [6,7]. However, in the case of women, this immunological balance changes when they become pregnant. It has long been known that, during pregnancy, women are susceptible to malaria despite the acquisition of immunity against clinical malaria by the time they become pregnant [8,9]. This is because of an immunologically distinct P. falciparum is selected opportunistically by binding to the chondroitin sulfate proteoglycans (CSPGs) in the placenta through the expression of chondroitin 4-sulfate (C4S)-binding protein(s) on the surfaces of infected erythrocytes [10–15]. Since the host lacks immunity against this adherent phenotype, the parasite can thrive efficiently until a specific immunity is developed, which occurs during the first and second pregnancies [16–19]. Thus, primigravidas are highly susceptible to placental malaria and the risk decreases with increased gravidity [16]. Several studies have shown the presence of infected erythrocytes in the umbilical cord as well as in the newborn infants [20–24], although clinical cases of congenital malaria are rare [25,26]. The observed infection is due to the existence of bilateral trafficking of erythrocytes and other circulatory cells between the mother and the fetus [27–30]. The transplacental passage of erythrocytes and mononuclear cells from the mother to the fetus has been demonstrated and this phenomenon has been proposed to be involved in the vertical transmission of infectious agents to the fetus [27]. Consistent with these observations, by measuring parasitemia in the placenta as well as cord blood, it has been recognized that the transfer of IRBCs across the placenta is a common event [22,23]. Placental malaria infection may further facilitate infiltration of IRBCs to the fetus through breaks in the syncytiotrophoblast cell lining that could occur due to the necrosis and damage to microvilli. A recent study has shown that P. falciparum is commonly found in the umbilical cord [20]. The in vitro cultured human umbilical vein endothelial cells (HUVECs), human lung epithelial cells, and Saimiri monkeys brain microvascular endothelial cells have been shown to support the C4S-mediated adhesion of IRBCs [31–33]. To determine whether HUVECs in vivo produce the CSPGs on their surface and, if so, whether the CSPGs can support the adherence of IRBCs, we purified and characterized the CSPGs of the umbilical cord and studied their IRBC binding characteristics. The results of these studies and those of the immunohistochemical analysis show that a versican and possibly a serglycin CSPGs are present as surface adsorbed molecules on the endothelium of the umbilical vein but not arteries. Our data also demonstrate that the CS chains of CSPGs cannot efficiently support the IRBC adherence due to the predominance of 6-sulfate groups. Thus, the IRBCs that entered the fetal blood from the infected placentas, presumably in low numbers, may eventually be cleared without causing clinical symptoms since C4S-adherent parasites are unable to adhere to the vascular endothelial surfaces.

2. Materials and methods 2.1. Materials Proteus vulgaris chondroitinase ABC, protease-free P. vulgaris chondroitinase ABC, Arthrobacter aurescens chondroitinase AC II, Flavobacterium heparinum chondroitinase B, F. heparinum heparitinase, Streptomyces hyalurolyticus hyaluronidase, antiproteoglycan
di-6S (IgM) and
di-4S (IgG) monoclonal antibodies, Sturgeon notochord C4S, and shark cartilage C6S were purchased from Seikagaku America (Falmouth, MA). Anti-C4/6S monoclonal antibody (clone CS-56, IgM), C4S (bovine trachea), bovine pancreas RNase, and protein molecular weight standards for gel filtration were from Sigma. DNase I (grade II) was from Roche Molecular Biochemicals. Human blood and serum were obtained from the Hershey Medical Center, Hershey. Polyclonal rabbit antiserum (LF-99) against the synthetic peptide that corresponds to LHKVKVGKSPPVRC sequence of human versican core protein was a generous gift from Dr. Larry Fisher, Craniofacial and Skeletal Disease Branch, NIDR, NIH, Bethesda, MD. 2.2. Isolation of CSPGs from the human umbilical cord vein and arteries The vein and arteries (40 g each), dissected from the human normal umbilical cords of term placentas, were cut into pieces and separately extracted by stirring three times with 250 ml of PBS, pH 7.2, containing 10 mM EDTA, 0.1 mM PMSF, 0.1 mM TLCK, 0.25 mM TPCK, 1 mM benzamidine, and 0.1 mM NEM. The extracts were centrifuged at 10 000 rpm for 30 min in a Sorvall centrifuge using SS-34 rotor, and the clear supernatants collected. The pellets of the tissues were then extracted with 3 × 250 ml of PBS, 10 mM EDTA, pH 7.2, containing 0.5% Triton X-100, and protease inhibitors. Finally, the pellets were extracted with 3 × 250 ml of PBS, pH 7.2, containing 10 mM EDTA, 0.5% Triton X-100, 6 M urea, and protease inhibitors, by homogenizing for 30 min using a Polytron homogenizer. The extracts were chromatographed on DEAE–sephacel columns (2.5 cm × 15 cm) and the columns were washed with 25 mM Tris–HCl, 10 mM EDTA, 150 mM NaCl, pH 8.0, followed by 20 mM NaOAc, 150 mM NaCl, pH 5.5, and finally with 20 mM NaOAc, 250 mM NaCl, pH 5.5. In each case, 0.25 M NaCl-containing buffer eluted significant amounts of hyaluronic acid, which was not studied further. The columns were then equilibrated with 50 mM NaOAc, 0.1 M NaCl, pH 5.5, containing 4 M urea, and eluted with a linear gradient of 0.1–1.0 M NaCl in 50 mM NaOAc, 4 M urea, pH 5.5. Fractions (2.3 ml) were monitored for absorption at 260 and 280 nm. The aliquots were analyzed for uronic acid and the positive fractions were combined, dialyzed and lyophilized as described [12].

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2.3. Purification of CSPGs The nucleic acid contaminants in the proteoglycans (PGs) were removed by incubation with a mixture of DNase I and bovine pancreas RNase, as reported previously [12]. The PGs (20–25 mg) were incubated with S. hyalurolyticus hyaluronidase (50 turbidity reducing units) at 60 ◦ C for 2 h, dialyzed against water, lyophilized, dissolved in 0.5 ml of 50 mM Tris–HCl, pH 8.0, and incubated with heparitinase (25 milliunits) at 43 ◦ C for 18 h. The solutions were chromatographed on Sepharose CL-4B column (1.5 cm × 83 cm) in 20 mM Tris–HCl, 150 mM NaCl, 4 M GdnHCl, pH 7.6. Fractions (2 ml) were collected, absorption at 280 nm was measured, and aliquots of fractions were analyzed for uronic acid content. The uronic acid-containing fractions were pooled, dialyzed and lyophilized. The CSPGs were further purified by CsBr density gradient centrifugation as described previously for placental CSPGs [12]. 2.4. Identification of the core proteins of the CSPGs The purified CSPGs (10–15 ␮g) were treated with chondroitinase ABC in 100 mM Tris–HCl, pH 8.0, containing 30 mM NaOAc at 37 ◦ C for 5 h [12]. The released core proteins and untreated CSPGs were electrophoresed on 4–15% gradient polyacrylamide mini gels under reducing condition, and visualized by sequential staining with Coomassie Blue, Alcian Blue, and then with ammoniacal silver [34]. For amino acid analysis, the core proteins after SDS– PAGE were electroblotted onto PVDF membranes using 10 mM CAPS, pH 11.0, containing 10% methanol. The membranes were stained with Ponceau S, and the protein bands were hydrolyzed under vacuum with 100 ␮l of 6 M HCl, 0.2% phenol for 16 h at 115 ◦ C. The hydrolysates were analyzed at the Biotechnology Resource Laboratory (Yale University, New Haven, CT). 2.5. Western blot analysis The chondroitinase ABC-treated VPG-IIa was electrophoresed on 4–15% gradient gel and electrotransferred onto PVDF membranes, blocked with 1% BSA, and then incubated with 1:1000 diluted anti-versican peptide antisera. The bound antibodies were detected with alkaline phosphatase-conjugated goat anti-rabbit IgG and nitro blue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate color development substrates. 2.6. Isolation and characterization of glycosaminoglycan (GAG) chains The purified CSPGs (200–400 ␮g) were treated with 0.5 ml of 0.1 M NaOH, 1 M NaBH4 at 45 ◦ C for 18 h under nitrogen [12], and the released GAG chains were isolated by the chromatography on a Sepharose CL-6B column (1 cm × 49 cm) in 0.2 M NaCl. Fractions (0.67 ml) were

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collected and aliquots assayed for uronic acid content. The uronic acid-containing fractions were combined, dialyzed, and lyophilized. The CSPGs or GAGs (25–50 ␮g) were digested with chondroitinase ABC, chondroitinase AC II or chondroitinase B as described previously and the released unsaturated disaccharides were analyzed by HPLC [12]. The CSPGs (10–20 ␮g) were hydrolyzed with 4 M HCl at 100 ◦ C for 6 h. The hydrolysates were dried in a Speed-Vac and analyzed for the carbohydrate composition by Dionex BioLC HPLC as described earlier [12]. 2.7. Immunohistochemical localization of CSPGs One-cm portions of the umbilical cord were cut out from fresh term placentas, suspended in 50 ml of PBS, pH 7.2, containing 2% formalin, 0.5% glutaraldehyde, and heated to 45 ◦ C in a microwave oven [35]. The paraffin-embedded tissues were sectioned (5 ␮m thickness) onto glass slides, deparaffinized, and rehydrated. The sections were treated with 0.3% H2 O2 to inactivate any endogenous peroxidase, and then blocked with 1:60 diluted normal goat serum in PBS, pH 7.2, for 20 min according to manufacturer’s instructions. The sections were incubated with the purified monoclonal antibody (1:100 diluted as specified by the supplier) or with 1:100 diluted rabbit antisera against PG peptide motifs, in PBS, pH 7.2. After 1 h, sections were washed, incubated with 1:200 diluted biotinylated goat anti-mouse IgM in PBS, pH 7.2, containing 1:60 diluted normal goat serum for 30 min. For staining with rabbit antisera, sections were incubated with 1:200 biotinylated goat anti-rabbit IgG as above. The sections were washed, incubated with HRP-conjugated avidin for 30 min, washed, and treated with 3,3 -diaminobenzidine tetrahydrochloride color developing reagent for 2–3 min. Sections treated with chondroitinase ABC prior to incubation with the monoclonal antibody or rabbit antiserum were similarly processed [36]. All incubations were at room temperature in a humidified chamber, and washings were with PBS, pH 7.2. The tissue sections were counterstained with methyl green, mounted under glass cover slips using permount, examined under microscope, and photographed. 2.8. Culturing of parasites C4S adherent P. falciparum, selected by panning of the 3D7 laboratory parasite strains on plastic plates coated with placental CSPG [12] were used for the assays. The parasites were cultured in RPMI 1640 medium using 10% O-positive human serum using type O-positive human red blood cells at 3% hematocrit [37]. 2.9. IRBC adhesion to the purified CSPGs and adhesion–inhibition assays The adherence of IRBCs was assessed by coating various concentrations (4 ng to 10 ␮g/ml) of the purified CSPGs

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as 0.4 cm circular spots on 150 mm × 15 mm plastic Petri dishes as described previously [37]. The specificity of IRBC binding to CSPGs was ascertained by treating the CSPG-coated plates for 2 h with chondroitinase ABC (50 milliunits/ml) at 37 ◦ C, heparitinase (20 milliunits/ml) at 43 ◦ C or S. hyalurolyticus hyaluronidase (40 turbidity reducing units/ml) at 60 ◦ C prior to IRBC overlaying. For adhesion–inhibition assays, parasite cultures were pre-incubated with various concentrations of inhibitors for 30 min, and then layered on CSPG-coated spots on Petri dishes [37]. After 40 min at room temperature, the unbound cells were washed. The bound cells were fixed with 2% glutaraldehyde, stained with Giemsa, and counted under light microscope. 2.10. IRBC adhesion to umbilical cord and placental tissue sections The placental tissues (1 cm × 1 cm) were fixed and sectioned as described above for the umbilical cord. The slides were washed twice with PBS for 5 min each and blocked with 2% BSA in PBS for 30 min. The parasites (25–30% parasitemia) were stained with 1:10 000 diluted SYBR Green (as recommended by the supplier) in PBS by incubating at room temperature for 5 min, and washed. A 2% suspension of cells in PBS was overlaid onto the tissue sections and incubated at room temperature for 5 min. The unbound cells were washed three times with PBS, mounted using 15 ␮l of 0.5% 1-propyl gallate and 50% glycerol in PBS, pH 7.2, and viewed under fluorescence and light microscope at 40× magnification. Parasite cultures pre-incubated with 40 ␮g/ml bovine C4S were used to confirm the specificity of adherence. Tissue sections treated with chondroitinase ABC were also tested to confirm the specificity of IRBC adherence.

3. Results 3.1. Isolation and purification of CSPGs of the human umbilical cord vein and arteries The umbilical vein and arteries were extracted with PBS alone to solubilize the CSPGs adsorbed on cell surfaces and those unbound and/or very weakly bound in the extracellular matrices. The tissue pellets were then extracted with PBS containing 0.5% Triton X-100 to solubilize the cell-associated PGs (intracellular or integral plasma membrane). The tissues were finally extracted with PBS containing 0.5% Triton X-100 and 6 M urea to solubilize PGs that are tightly bound in the extracellular matrices. Structural analysis of the PGs extracted by PBS/detergent and PBS/detergent/urea indicated that, in these extracts, predominantly decorin and biglycan family chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) of tissue matrix and to a lesser extent heparan sulfate proteoglycans were present (Valiyaveettil et al., unpublished results).

Fig. 1. DEAE–sephacel chromatography of the extracts of human umbilical vein (A) and arteries (B). The PGs were eluted using NaCl gradient and the fractions were monitored for proteins at 280 nm and for uronic acid at 530 nm. Horizontal bars denote pooled fractions. NaCl gradient in panel B is similar to that in panel A.

CSPGs other than these were not present in significant amounts in these two extracts. These results suggest that the surface of the vascular endothelia either lack or contain only very low level of the integral membrane CSPGs. Immunohistochemical analysis using antibodies specific to core proteins of decorin and biglycans suggested that these PGs are present predominantly in the blood vessel walls and in the cord tissue, but are absent on the endothelial surfaces of the umbilical vein and arteries (Valiyaveettil et al., unpublished results). The characterization of these tissue matrix decorins and biglycans will be published elsewhere. The PGs in the PBS extracts of the umbilical vein and arteries were fractionated by chromatography on DEAE–sephacel (Fig. 1). In the case of vein, the PGs were separated into two unresolved peaks, a minor peak (VPG-I) and a major peak (VPG-II), eluting at 0.54 and 0.67 M NaCl, respectively (Fig. 1A). Hexosamine analysis of VPG-I revealed the presence of ∼30% galactosamine (GalN) and ∼70% glucosamine (GlcN), suggesting that this PG fraction is associated with substantial amounts of hyaluronic acid (HA) and/or heparan sulfate (HS). Therefore, VPG-I was treated with S. hyalurolyticus hyaluronidase and heparitinase to remove HA and HS; 70% GAGs were degraded into oligosaccharides (Fig. 2A). The enzyme-resistant material in VPG-I had ∼96% GalN and ∼4% GlcN, suggesting that this material is a CSPG. The fraction VPG-II from the umbilical vein had ∼80% GalN and ∼20% GlcN. Treatment of VPG-II with S. hyalurolyticus hyaluronidase

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In the case of umbilical arteries, DEAE–sephacel chromatography of the PBS extract yielded a single PG peak (APG-II) at 0.72 M NaCl (Fig. 1B); PGs similar to VPG-I of the umbilical vein were absent (compare Fig. 1A with Fig. 1B). APG-II had significant amounts of GalN and GlcN, and treatment with S. hyalurolyticus hyaluronidase and heparitinase degraded ∼32% of GAGs. Sepharose CL-4B chromatography yielded predominantly GalN-containing PG fractions APG-IIa and APG-IIb (Fig. 2C). VPG-IIb and APG-IIb were found to be, in each case, a mixture of tissue matrix decorin and biglycans, and therefore, these were not described here. VPG-I, VPG-IIa, and APG-IIa, obtained as above, were further purified by CsBr density gradient centrifugation. In all the three fractions, the PGs sedimented to the high-density regions (ρ = 1.43–1.55 g/ml) separated from the associated protein contaminants, which remained in the low-density fractions (ρ = 1.30–1.35 g/ml) at the top of the CsBr gradients (data not shown). The yield and compositions of the purified PGs are summarized in Table 1. Consistent with their PG nature, all fractions contained high levels of proteins, sulfate, hexosamine, and uronic acid. The presence of 92–96% GalN and only 4–8% GlcN in various purified fractions indicates that the PGs have CS, DS, or CS/DS chains. Fig. 2. Sepharose CL-4B elution profile of PGs of umbilical vein and arteries. The PGs from DEAE–sephacel chromatography (see Fig. 1) were digested with S. hyalurolyticus hyaluronidase and heparitinase and chromatographed on Sepharose CL-4B columns. The fractions were monitored for proteins at 280 nm and for uronic acid at 530 nm. Horizontal bars denote pooled fractions. (A) VPG-I, (B) VPG-II, and (C) APG-II. The arrows from the left to right indicate the elution positions of BD, BSA, and Glc, respectively.

and heparitinase degraded ∼15% of the GAG chains into oligosaccharides. On Sepharose CL-4B chromatography, the undigested VPG-II eluted as two unresolved peaks, a minor peak eluting as a tail at the leading edge of the major peak (Fig. 2B). The enzyme-resistant, PGs were pooled into two fractions, VPG-IIa and VPG-IIb (Fig. 2B), which contained predominantly GalN.

3.2. Structural characterization of the GAG chains of the CSPGs The GAG chains of VPG-I, VPG-IIa, and APG-IIa, released by ␤-elimination, were eluted on Sepharose CL-6B column as single peaks indistinguishable from one another with an estimated Mr of ∼70 000 (data not shown). In all cases, the GAG chains were completely degraded into unsaturated disaccharides by both chondroitinase ABC and chondroitinase AC II, suggesting that they are exclusively chondroitin sulfates. Consistent with these results, the GAG chains were completely resistant to chondroitinase B. The composition of the GAG chains is given in Table 1.

Table 1 Yield and composition of CSPGs purified from human umbilical vein and arteries Location in the cord/PGs

Yielda (mg/100 g tissue)

Composition (wt.%) Proteinc

Uronic acidd

HexNe

Sulfatef

GalNe

GlcNe


di-0S


di-4S


di-6S

Vein VPG-I VPG-IIa

1.2 2.9

13 21

37 33

41 36

9 10

96 94

4 6

19 15

25 16

56 69

Arteries APG-IIa

3.4

20

34

36

10

94

6

14

14

72

a

Disaccharidesb (mol% ratio)

HexN (mol% ratio)

Yields of CSPGs after purification by gel filtration on Sepharose CL-4B column (see Fig. 2) and CsBr density gradient centrifugation. b Calculated from the areas of the peaks by assuming that the non-sulfated and sulfated disaccharides have similar molar extinction coefficients. c By the modified Lowry protein assay. d By the carbazole method. e By the high pH anion-exchange HPLC of the 4 M HCl hydrolysates of the purified CSPGs. f Approximate values estimated based on the proportions of sulfated and non-sulfated disaccharides formed by chondroitinase ABC digestion.

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Fig. 3. SDS–PAGE and Western blot analysis of CSPGs and core proteins of the umbilical vein. (A) The intact CSPGs and chondroitinase ABC-released core proteins were electrophoresed on 4–15% gradient polyacrylamide mini gels under reducing condition, and stained successively with Coomassie Blue, Alcian Blue, and then with ammoniacal silver. Lane 1, VPG-I; lane 2, core protein of VPG-I; lane 3, VPG-IIa; lane 4, core protein of VPG-IIa. The positions of the protein molecular weight markers are indicated to the left. (B) Chondroitinase ABC-treated VPG-IIa was electrophoresed as above, transferred onto PVDF membrane and probed with anti-rabbit versican peptide antisera. The arrow on the right indicates the position of chondroitinase ABC.

3.3. Characterization of the core proteins of the CSPGs The purified CSPGs of umbilical vein were treated with chondroitinase ABC in the presence of protease inhibitors, and the released core proteins analyzed by SDS–PAGE and Western blotting (Fig. 3). On SDS–PAGE, a significant amount of VPG-I was either barely entered or unable to enter the gel and the remainder was electrophoresed over a broad range in the molecular mass range of 35–230 kDa (Fig. 3A, lane 1). The core protein of VPG-I electrophoresed as a diffused band with a mobility corresponding to ∼10 kDa (Fig. 3A, lane 2). Since the intact VPG-I, upon chromatography on Sepharose CL-4B, eluted as a high molecular size species corresponding to an estimated average Mr of 200 000 (see Fig. 2A) and considering the average Mr of the GAG chains (70 000), it appears that the majority of VPG-I contain 1-3 GAG chains. Treatment of VPG-IIa with chondroitinase ABC released mainly a >200 kDa, polydispersed protein and minor amounts of 43–48 kDa core proteins (Fig. 3A, lane 4). The 43–48 kDa protein bands were immunoreactive to antisera

against decorin and biglycan core proteins (Valiyaveettil et al, unpublished data); these were presumably due to the overlapping of VPG-IIb with VPG-IIa during chromatography on Sepharose CL-4B (see Fig. 2B). The molecular weight of VPG-IIa, estimated based on the elution volume on a calibrated Sepharose CL-4B column (Fig. 2B), is between 0.5 × 106 and 0.8 × 106 . Based on this and the GAG chain size of ∼70 kDa, VPG-IIa must contain several CS chains linked to its core protein. SDS–PAGE analysis showed that the core proteins of APG-IIa were similar to those of VPG-IIa (not shown). Based on these results and the similarities in GAG chain sizes and types (see above), VPG-IIa and APG-IIa are similar CSPGs and thus only VPG-IIa was used for further characterization and binding studies. The amino acid compositions of the core proteins of the purified umbilical vein CSPGs are shown in Table 2. The core protein of VPG-I contains high proportions of Ser, Gly and acidic amino acids and has low proportions of basic amino acids. The characteristic ∼10 kDa molecular mass of the core protein, and high proportions of Ser and Gly suggest that VPG-I belongs to the serglycin family of CSPGs [38]. The core protein of VPG-IIa also has high proportions of Ser, Gly, and acidic amino acids (Table 2). In this regard, and with respect to the overall molecular size and core protein Table 2 Amino acid composition of the core proteins of umbilical vein CSPGs and the core proteins of the previously reported, serglycins and versicans (residues/1000 amino acidsa ) Amino acid

VPG-I Serglycinb Serglycinc VPG-IIa Versicand PG-Me

Asx Thr Ser Glx Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Trp Cys

37 60 196 160 47 217 73 38 8 69 31 12 11 11 15 15 NDf ND

a

104 20 270 93 41 280 10 10 20 42 20 30 30 0 0 20 10 0

138 31 131 131 62 92 23 15 23 23 116 31 54 15 23 69 8 15

88 74 97 141 73 95 59 62 13 55 69 23 33 28 58 32 ND ND

89 107 105 147 65 56 61 66 15 46 57 26 39 23 41 33 10 14

79 89 105 147 34 132 60 53 14 41 73 31 32 21 44 39 ND 6

The average value from two separate hydrolyses. The amino acid composition of the rat L2 yolk sac tumor cell serglycin sequence deduced from the cloned cDNA [47]. c The amino acid composition of the human platelet serglycin sequence deduced from the cloned cDNA [48]. d The amino acid composition of the human fetal fibroblast cell versican sequence deduced from the cloned cDNA [39]. e The amino acid composition of the large versican-like CSPG of the chick embryo limb buds [44]. f ND indicates not determined. b

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Fig. 4. Immunohistochemical localization of CSPGs in the umbilical cord. The umbilical cord tissue sections were incubated with either anti-versican antisera or CS-56 monoclonal antibody, and the bound antibodies were stained using biotin-labeled secondary antibodies and HRP-conjugated avidin. Panels A and B: Untreated (A) and chondroitinase ABC-treated (B) tissue sections stained with anti-versican antisera. Shown are the portions of the tissue sections corresponding to the umbilical vein (A-1, A-2, B-1 and B-2), arteries (A-3, A-4, B-3 and B-4), and tissue around the blood vessels (A-5 and B-5). Panels C and D: Untreated (C) and chondroitinase ABC-treated (D) tissue sections stained with CS-56 monoclonal antibody. Shown are the portions of the tissue sections corresponding to the umbilical vein (C-1, C-2, D-1 and D-2), arteries (C-3, C-4, D-3 and D-4), and tissue around the blood vessels (C-5 and D-5). Tissue sections treated with pre-immune sera prepared from the corresponding animals showed background levels of staining (not shown). Panels A-1, B-1, C-1, D-1, A-3, B-3, C-3, and D-3 are at 4× magnification; panels, A-2, B-2, C-2, D-2, A-4, B-4, C-4, D-4, A-5, B-5, C-5, and D-5 are at 20× magnification. E, endothelial cell layers; L, lumen of the vein or the arteries.

size and composition, VPG-IIa resembles versican family CSPGs [39–41]. Upon Western blot analysis of VPG-IIa core protein, the antiserum against the versican peptide reacted with the diffused >200 kDa band (Fig. 3B). The size of the immunoreactive core protein was similar to the Coomassie-stained diffused >200 kDa band observed on SDS–PAGE of the chondrotinase ABC-treated VPG-IIa (see Fig. 3A, lane 4). 3.4. Localization of the CSPGs in the umbilical vein and arteries The localization of CSPGs in the umbilical cord was studied by immunohistochemical analysis of tissue sections using antisera against human versican core protein, and a monoclonal antibody (CS-56) specific to C4S/C6S chains. The anti-versican antisera strongly stained the endothelial surface of the umbilical vein, the tissue surrounding the blood vessels, and the connective tissue of the Wharton’s jelly (Fig. 4A-1–A-5). In the arteries, only the smooth muscle cells were stained, but not the endothelial bed and endothelial surface (Fig. 4A-4). The antibody staining was considerably increased upon pre-incubation of the tissue

sections with chondroitinase ABC (Fig. 4B-1–B-5). This is presumably due to the exposure of peptide epitopes that were masked by the GAG chains. The CS-56 monoclonal antibody strongly stained the endothelial bed and endothelial surface of the umbilical vein (Fig. 4C-1 and C-2). Interestingly, however, the antibody staining was absent on the endothelial surface of the umbilical arteries (Fig. 4C-3 and C-4). This is consistent with the localization of versican containing C4S/C6S chains on the endothelial surface of the umbilical vein but not on that of the arteries as inferred by the results of anti-versican antibody staining (see Fig. 4A-2 and 4B-2). The monoclonal antibody also intensely stained the blood vessel walls (Fig. 4C-1 and 4C-3). Staining with anti-C4S/C6S monoclonal antibody was abolished upon pre-incubation of tissue sections with chondroitinase ABC (compare Fig. 4C-1–C-5 with Fig. 4D-1–D-5), suggesting that the staining was specific to the GAG chains. 3.5. Adhesion of P. falciparum IRBCs to the umbilical vein CSPGs The IRBC-binding to VPG-I and VPG-IIa was assessed by an in vitro cytoadherence assay [37]. The parasites used

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space BCSPG-2 [12] is due to poor coating of the umbilical vein CSPGs onto plastic surfaces. Consequently, the measurement of the number of IRBCs bound on the plates might not accurately reflect their relative binding strength. Therefore, the binding strengths of the umbilical vein CSPGs were measured by the abilities of their GAG chains to inhibit IRBC binding to the placental CSPG (Fig. 5B). In agreement with the results of binding assay (see Fig. 5A), at all concentrations tested, the inhibitory capacity of the GAGs from VPG-I was significantly higher than the GAG chains of VPG-IIa, but the activity was markedly lower compared with that of bovine trachea C4S (Fig. 5B). Thus, the density of IRBCs bound to the CSPGs of umbilical cord vein coated on plastic plates directly relates to the IRBC binding strengths of the respective CSPGs (compare Fig. 5A with B). Based on the sites of their localization, it is obvious that VPG-IIa and, most likely VPG-I, are accessible for the adherence of IRBCs in the umbilical vein. 3.6. Adhesion of P. falciparum IRBCs to the umbilical cord and placental tissue sections

Fig. 5. Adhesion of IRBCs to the umbilical vein CSPGs. (A), The purified umbilical vein CSPGs were coated at the indicated concentrations on plastic Petri dishes, blocked, and the IRBC binding was assessed as described in the text. Assays were carried out two times each in duplicate, and average values plotted. The spots not coated with CSPGs but blocked with BSA were used as negative controls. (䊉), placental intervillous space BCSPG-2 [12]; (䊊), VPG-I; and (䉱), VPG-IIa. (B), Plastic Petri dishes were coated with 0.2 ␮g/ml solution of placental BCSPG-2 [12], blocked and the adhesion–inhibition assay was performed as described in the text. (䊉), Bovine trachea C4S; (䊊), GAG chains of VPG-I; and (䉱), GAG chains of VPG-IIa; (䊐), HA; (䊏), heparin.

were selected for binding to the placental low-sulfated CSPG, the major natural receptor for IRBC binding to placenta [12] and therefore these parasites should resemble the placental isolates in C4S-binding characteristics. Both CSPGs supported the adhesion of IRBCs, when coated on plastic plates (Fig. 5A). The IRBC adhesion to the umbilical vein CSPGs was abolished by pre-treatment of the CSPG-coated plates with chondroitinase ABC, but not with heparitinase or S. hyalurolyticus hyaluronidase (data not shown). Further, the IRBC adhesion could be competitively inhibited by the bovine trachea C4S; heparin, HS, and HA had no inhibitory effect (Fig. 5B, and data not shown). These results suggest that GAG chains mediate the adhesion of IRBCs to the CSPGs. Of the two CSPGs, VPG-I binds IRBCs at significantly higher level than VPG-IIa (Fig. 5A), but the level of IRBC binding was significantly lower compared with that of the placental BCSPG-2 [12]. It is possible that the lower IRBC binding capacity of VPG-I and VPG-IIa compared to the placental intervillous

While working with placental tissue sections, we found that intervillous space CSPGs are preserved in tissues fixed with formaldehyde/glutaraldehyde and microwave heating rather than in the snap-frozen tissues (Muthusamy et al., unpublished results). Therefore, this procedure was used to examine IRBC binding to the umbilical cord tissue. Parasites were not adhered onto the umbilical cord tissue and endothelial surface, indicating that versican and serglycin CSPGs on the umbilical cord are unable to support the IRBC adhesion (not shown). This is presumably due to the low binding capacity of the CS chains of the umbilical cord CSPGs as noted above. However, IRBCs adhered in low density in the umbilical vein blood space (about 5–8 IRBCs per 40× field were observed), likely due to the presence of a CSPG that can support IRBC binding (Fig. 6A and B). Interestingly, we observed that IRBCs bound densely in the blood vessels of the fetal villi (Fig. 6E and F). In both cases, prior treatment of the tissue sections with chondroitinase ABC or pre-incubation of the cells with 40 ␮g/ml bovine trachea C4S completely abolished IRBC binding in the blood vessels of the fetal villi (Fig. 6C, D, G, H, and data not shown). These results suggest that blood vessels of the fetal villi contain high levels of CSPG that can efficiently bind IRBCs. Because it is difficult to obtain the fetal villi free of other tissue components, it was not possible to determine the nature of this CSPG. The adherence of IRBCs seen in the vein of the umbilical cord tissue sections was not due to nonspecific random adherence of cells during the assay because only a few IRBCs were bound through C4S in the umbilical vein although culture with 25–30% parasitemia was used for the assay. The non-stained RBCs in the tissue sections represent RBCs of the fetal blood preserved in the microwave-fixed tissues (compare Fig. 6 with Fig. 4C-2 and D-2).

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Fig. 6. Adhesion of IRBCs to the umbilical vein and to the fetal blood vessels of placenta sections. The umbilical cord and placental tissue sections were assayed using SYBR Green stained IRBCs and photographed. (A and B) Untreated and (C and D) chondroitinase ABC treated umbilical cord tissue sections. (E and F) Untreated and (G and H) chondroitinase ABC treated placental tissue sections. IRBCs are densely bound in the fetal blood vessels. The unstained cells in the umbilical vein and fetal blood vessels represent RBCs that were present in the tissue sections but not RBCs of the parasite culture. Panels A, C, E, and G are under fluorescence and B, D, F, and H are under light microscope. FBV, fetal blood vessel; SYN, syncytiotrophoblast lining; IVS, intervillous space.

4. Discussion In this study, we demonstrate that the umbilical vein contains a serglycin and a versican CSPGs, whereas the umbilical arteries have only versican but not serglycin. In addition to these PGs, the vein and arteries contain tissue matrix decorin and biglycan CS/DSPGs (Valiyaveettil et al., unpublished data). Although versican is distributed throughout the umbilical cord, it is also present at significant levels on the endothelial surfaces of the umbilical vein but not on that of arteries. Considering that serglycin, a secretory CSPG, can present as adsorbed molecules on cell surfaces, it is likely that this CSPG is also present in significant levels on the umbilical vein endothelial surface. Although IRBCs could bind in moderate levels to the purified versican and serglycin CSPGs coated on plastic plates, the binding strength is weak. Therefore, the IRBCs were bound sparingly to the umbilical cord endothelial surface. However, interestingly, IRBCs were bound densely in the blood vessels of the fetal villi in a C4S-dependent manner. Because IRBCs cannot adhere to the endothelial surface and the fetal hemoglobin is toxic to P. falciparum [42,43], the C4S-adherent parasites that enter the umbilical vein may not efficiently multiply and sequester at high density in the umbilical cord. This may partly contribute to the low incidences of congenital clinical malaria. VPG-IIa is a member of versican family of CSPGs [39–41]. The core protein of VPG-IIa is fairly rich in Ser and Gly, but the proportion of these amino acids is markedly less compared with that in the cartilage aggrecan core protein, suggesting the absence of extensive Ser–Gly repeats in the former. However, considering the levels of Ser and Gly

and the presence of several CS chains, it is likely that the core protein of VPG-IIa has either short Ser–Gly repeats and/or a number of discontinuous Ser–Gly dipeptides. The amino acid composition of the core protein of VPG-IIa is similar to that of the core protein of versican, the high molecular weight CSPG of the human fetal fibroblast and the core proteins of chick embryonic limb bud mesenchyme CSPG, a versican-like CSPG ([39,44]; see Table 2). The results of immunohistochemical studies indicate that the versican and possibly serglycin are present on the endothelial surfaces of the umbilical vein but not on that of arteries. Although information on the precise location of the serglycin CSPG of the umbilical vein could not be obtained in this study, it is likely that significant levels of VPG-I is present as adsorbed molecules on the endothelial surface. In vitro cultured HUVECs have been reported to synthesize serglycin as a component of secretory granules and are secreted into the culture medium [38]. A notable feature of the serglycin family CSPGs, in general, is the association of these macromolecules with the secretory granules, extracellular secretion upon cell activation, and adsorption onto cell surfaces. These features together with the exclusive presence in the umbilical vein and extraction readily by isotonic buffer is consistent with VPG-I being present as an adsorbed molecule on the umbilical vein endothelial surface. The presence of versican, and serglycin CSPGs on the umbilical vein endothelial surface is also supported by staining of this site by anti-C4S/C6S chains and anti-versican peptide antisera, but not that of the umbilical arteries. The CS chains of the umbilical cord serglycin and versican are predominantly 6-sulfated, 56 and 69%, respectively (see

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Table 1). These features, when compared to the structural requirements for IRBC adhesion, and that the high levels of 6-sulfated disaccharide moieties in the CS chains adversely affect the adherence [37], suggest the weak binding of IRBCs to these CSPGs. Consistent with this prediction, IRBCs were unable to bind onto the endothelial surface in the umbilical cord tissue sections. However, it is possible that IRBCs with different binding characteristics may bind to endothelial surface. Interestingly, IRBCs were bound in the blood vessels of the fetal villi in a C4S-dependent manner when placental tissue sections were analyzed for binding. Thus, fetal blood vessels are likely to contain a different type of CSPG(s) with CS chains which can efficiently bind IRBCs. The nature of this IRBC-binding CSPG of the fetal blood vessels could not be studied because of the practical difficulty in selectively isolating this PG. Since IRBCs could bind only at low densities in the umbilical vein blood space, it is likely that the CSPG is present at a relatively low concentration in the umbilical cord blood and appears to accumulate in the blood vessels of the fetal villi. Previous studies have shown that in vitro cultured HUVECs express CSPGs on their surface and support IRBC adhesion [11]. Other studies have shown that in vitro cultured endothelial cells of Saimiri monkey brain can also support IRBC adhesion in a C4S-dependent manner [31,32]. However, caution should be exercised in extrapolating these observations to in vivo conditions and in concluding that IRBCs can also bind to the human vascular endothelia in a C4S-dependent manner. Whereas, in vitro cultured endothelial cells can abundantly express both secreted and cell surface CSPGs, the CSPGs are expressed at low levels on vascular endothelia. Because of the entirely different environment of cells under in vitro and in vivo conditions, structural features of the CS chains could be markedly different. Moreover, the structural features of CS chains are species specific. As indicated by our data, the CSPGs that are present at significant levels as adsorbed molecules on the human umbilical cord endothelial surface have low capacity to bind IRBCs and thus unable to support IRBC adhesion to the tissue sections. Our results also rule out the presence of other minor CSPGs that could support the adherence of IRBCs onto the umbilical cord endothelial surface. Based on the results of IRBC binding to the CSPG form of a purified recombinant thrombomodulin (TM), it has been argued that TM expressed on vascular endothelial surface is involved in IRBC binding [15,45]. However, TM is a part time CSPG, (only 10–20% of the protein is known to bear C4S chain) and moreover, it is expressed at very low levels on the vascular endothelia. This is consistent with our observation that the CSPG form of TM was not present in detectable levels in the detergent extract of the umbilical vein. Further, although the recombinant TM expressed in human kidney 293 cells [46] that has 87% 4-sulfated, 2% 6-sulfated and 11% non-sulfated disaccharides [Valiyaveettil and Gowda, unpublished] can bind IRBCs, the TM

expressed on the vascular endothelia need not necessarily support IRBC binding. Since the structures of CS chains are cell- and tissue-type dependent, the CS chains of the recombinant and in vivo produced TM are likely to be different. Because the CS structures are cell-type specific, it is more likely that the CS chains of the TM expressed by the umbilical cord vein endothelia are similar to those of the serglycin, which is known to be synthesized by the HUVECs [38]. This conclusion is in agreement with the inability of the C4S-adherent IRBCs to bind to the umbilical cord tissue sections. Several studies have reported transplacental infection of P. falciparum in significant number of newborns (3–30%) [20–24]. However, although clinical conditions are rare in newborns, development of fever in some babies has been reported, and the parasite density is usually low [25,26]. Although C4S-adherent IRBCs can bind in the blood vessels of fetal villi, they are unable to bind at significant level in the cord vascular endothelia because versican and serglycin, and any other minor CSPGs on the endothelial surface cannot support high IRBC binding. Moreover, the parasite growth is limited in newborns because of the growth inhibition by fetal hemoglobin and protection by the maternal antibodies [42,43]. Therefore, the IRBCs that enter the fetal blood are likely to be cleared from the circulation without causing clinical conditions. In conclusion, although CSPGs are present at significant levels on the endothelial surface of the vein of the umbilical cord, they are unable to bind IRBCs because their CS chains cannot efficiently support the adherence of IRBCs. Therefore, the C4S-adherent IRBCs that are vertically transferred from the P. falciparum-infected placenta do not accumulate in the umbilical cord although these IRBC type can adhere in the blood vessels within the fetal villi. Furthermore, those IRBCs that enter the fetal vein may not efficiently survive because of fetal hemoglobin toxicity and protection by the maternal antibodies. In any case, it would be interesting to examine the nature of CSPG receptors present in the blood vessels of the fetal villi.

Acknowledgements This study was supported by the grant AI45086 from the National Institute of Allergy and Infectious Diseases, NIH. Part of the work reported here was done when the authors were at the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20007. We thank Dr. Christian F. Ockenhouse, Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, MD, for providing C4S-binding 3D7 P. falciparum parasites, and Dr. Larry Fisher, Craniofacial and Skeletal Disease Branch, NIDR, NIH, for anti-versican antisera.

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