Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum–infected erythrocytes

Arivalagan Muthusamy, Rajeshwara N. Achur, Manojkumar Valiyaveettil, Subbarao V. Madhunapantula, Ikuko Kakizaki, Veer P. Bhavanandan and Channe D. Gowda1

Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033

Received on January 21, 2004; accepted on March 10, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Sequestration of Plasmodium falciparum–infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S–IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure–activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that ~80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4–12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%–92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.

Key words: bovine tracheal chondroitin sulfate proteoglycan / chondroitin sulfate chains / infected erythrocytes adherence / Plasmodium falciparum / structure–adherence relationship


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The protozoan parasite Plasmodium falciparum causes severe malaria in human, resulting in millions of death predominantly in children younger than 5 years (Gilles and Warrel, 1993Go; Snow et al., 1999Go). The ability of P. falciparum to adhere in the vascular capillaries of vital organs is believed to be an important factor in the high morbidity and mortality of the disease (Greenwood and Mutabingwa, 2002Go; Miller et al., 2002Go). However, people in endemic areas acquire immunity against malaria pathogenesis as a result of continuous exposure to the parasite during childhood (Baird, 1995Go). Thus most adults are resistant to clinical malaria. In spite of this, women become highly susceptible to malaria during pregnancy despite the presence of the acquired immunity. This is because P. falciparum with a different adherence property than those that sequester in the endothelial capillaries is specifically selected for adherence of parasite-infected red blood cells (IRBCs) in the placenta (Nielsen et al., 2002Go; Taylor et al., 2000Go). The adhered parasite multiplies to a high density within the placenta, affecting pregnancy outcomes and causing severe malaria and even death in the mother (Menendez et al., 2000Go).

Chondroitin 4-sulfate (C4S) has been shown to mediate the adherence of IRBCs in the placenta (Achur et al., 2000Go; Chai et al., 2002Go; Fried et al., 2000Go; Rogerson and Brown, 1997Go). A number of studies have addressed various aspects of the adherence phenomenon, including the presence of adhesion specific antibodies in pregnant women, nature of the placental receptors, C4S structural requirements for adhesion, and the characteristics of the parasite adhesive protein (Achur et al., 2000Go; Alkhalil et al., 2000Go; Baruch, 1999Go; Fried et al., 1998Go; Gamain et al., 2001Go; O'Neil-Dunne et al., 2001Go; Pouvelle et al., 1998Go; Smith et al., 2000Go; Staalsoe et al., 2001Go).

A cytoadherence assay is commonly used to study the interactions of IRBCs with C4S and to analyze for IRBC–C4S adhesion inhibitory antibodies in sera of pregnant women in endemic areas (Baruch, 1999Go; Chai et al., 2002Go; Fried and Duffy, 1996Go; Fried et al., 1998Go; Staalsoe et al., 2001Go). This assay is based on the adhesion of IRBCs to C4S immobilized onto plastic. The most widely used chondroitin sulfate (CS) for these studies is a commercially available bovine trachea cartilage preparation. However, this commercial chondroitin sulfate A (cCSA; the term CSA rather than C4S is used for the bovine trachea CS because it is a copolymer of C4S and C6S) is deficient in core protein because it is isolated by proteolytic treatment of the cartilage tissue. Therefore cCSA is expected to adsorb poorly onto the plastic surface.

The chondroitin sulfate proteoglycan (CSPG) of bovine trachea has been studied with regard to its purification, polydispersity, variation in the total number of attached CS chains, core protein structure, and ability to bind hyaluronic acid to form large aggregates and structures of the linkage regions of the CS chains (de Beer et al., 1996Go; Hascall and Heinegard, 1974Go; Morgelin et al., 1994Go; Wight et al., 1991Go). However, relatively little is known about the structure of the CS chains. According to the manufacturer's specification, cCSA is a mixture of ~70% C4S and 30% C6S (Sigma, St Louis, MO, catalog number C8529). An earlier study determined that the CS chains of this PG are copolymers containing variable amounts of 4- and 6-sulfated disaccharide moieties (Seno et al., 1975Go). However the available information on the distribution of 4- and 6-sulfate groups in the bovine tracheal CS chains is contradictory. For example, an earlier study reported that the distribution is nonrandom (Cheng et al., 1992Go), but a more recent study reported that the distribution is random (Desaire et al., 2001Go).

Previously, we have shown that (1) CSPGs bearing unusually low-sulfated CS chains (consist of 8%–10% 4-sulfated and 90%–92% nonsulfated disaccharides) mediate the P. falciparum IRBC adherence to human placenta (Achur et al., 2000Go), (2) a C4S dodecasaccharide motif is the minimum chain length required for IRBC binding, and (3) the dodecasaccharides with two to four sulfated disaccharide units optimally bind IRBCs, whereas those with one, five, or six sulfated disaccharide units groups bind poorly (Alkhalil et al., 2000Go). We have also shown that clusters of 4-sulfated domains in the CS chains of placental CSPGs provide the sites for IRBC binding in the placenta (Achur et al., 2003Go). Furthermore, based on the efficient C4S–IRBC adhesion inhibitory activity of bovine tracheal CSA, we reported that 6-sulfated disaccharides in this glycosaminoglycan minimally interfere with the IRBC binding (Alkhalil et al., 2000Go). However, others found that 6-sulfated groups in the bovine CSA significantly affect the inhibitory activity of the CSA (Chai et al., 2002Go; Fried et al., 2000Go). The structural basis for the efficient C4S–IRBC adhesion inhibitory activity of bovine tracheal CSA, despite the presence of a relatively high proportion of 6-sulfated disaccharides, remained unclear. Therefore, the aim of this study was (1) to purify the intact CSPG from the bovine trachea (pCSPG) and investigate its ability to bind IRBCs compared to cCSA, and (2) to determine the distribution of 4- and 6-sulfate in the CS chains with the view of understanding the structural features that support IRBC adhesion. Our work shows that (1) the pCSPG bind IRBCs at 1.5-fold higher density compared to the cCSA and (2) the efficient binding of IRBCs to bovine tracheal CS chains is due to the presence of the majority of the 4-sulfated disaccharides as either single and in blocks of two or three separated by one to three 6-sulfated disaccharide moieties.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Purification of bovine tracheal cartilage CSPG and characterization of the purified CSPG and commercial bovine tracheal CSA
The CSPG from bovine trachea was extracted with buffer containing 4 M guanidine HCl (GdnHCl) and subjected to CsCl density gradient centrifugation as described previously (Antonopoulos et al., 1974Go). The majority of the CSPG fractionated to the bottom one-third of the gradient and separated from most of the proteins. The CSPG was further purified by gel filtration followed by a second density gradient centrifugation. On Sepharose CL-4B chromatography, under dissociative conditions, the majority of the CSPG (I) eluted at a slightly included volume (Figure 1A, Kav = ~0.05; Mr = >1 x 106). A minor relatively low-molecular-weight CSPG fraction (II), representing ~18% of the total CSPG, eluted as a shoulder at the tailing edge of the major peak (Figure 1A). Hexosamine analyses revealed predominantly galactosamine in both the major (I) and minor (II) PG fractions, suggesting that these are CSPGs. On density gradient centrifugation using CsBr, both CSPG species sediment to the bottom of the gradient, separating from any remaining protein contaminants (data not shown). The yield and composition of the purified CSPGs are summarized in Table I. The fraction I contained predominantly N-acetylgalactosamine with minor amounts of N-acetylglucosamine, galactose, and mannose in addition to uronic acid, sulfate, and protein (Table I). Although the disaccharide composition of the fraction II was similar to that of the fraction I (designated as pCSPG), the former contained a significantly higher level of N-acetylglucosamine, galactose, and mannose (Table I). Therefore, only fraction I was studied further.



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Fig. 1. Size-exclusion chromatography of the bovine tracheal CSPG, cCSA, and their CS chains. The CSPG isolated by CsCl density gradient centrifugation of the bovine tracheal extract (A) and cCSA (B) were chromatographed on Sepharose CL-4B columns (1.5 x 83 cm) in 50 mM Tris-HCl, 0.2 M NaCl, pH 7.5, containing 6 M GdnHCl. The elution was monitored for uronic acid at 530 nm (closed circles) and for protein at 280 nm (open circles). The CSPG and cCSA fractions were recovered by pooling the fractions as indicated by the horizontal bars. (C) The CS chains of the pCSPG (closed circles) and cCSA (open triangles), released by alkaline ß-elimination, were subjected to chromatography on Sepharose CL-6B column (1 x 49 cm) in 0.2 M NaCl. The CS chains were recovered by pooling fractions as shown by horizontal bars. The elution positions of blue dextran (BD) and glucose (Glc) are indicated.

 

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Table I. Compositions of the CSPG purified from the bovine tracheal cartilage and cCSA

 
The carbohydrate and sulfate compositions of CSPG fraction I was very similar to that of the cCSA (Table I). However, the protein content of cCSA was significantly lower (4%) than that of CSPG fraction I (14%) (Table I). The CS chains of cCSA, bovine tracheal CSPG fractions I and II and cCSA fractions I and II were digested with chondroitinase ABC and the released disaccharides analyzed by high-performance liquid chromatography (HPLC) (Table II). The disaccharide compositions of tracheal CSPG fraction I, CSPG fraction II and cCSA were similar.


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Table II. Disaccharide composition of the CS chains of CSPG purified from bovine tracheal cartilage and cCSA

 
The elution profile of cCSA on Sepharose CL-4B, as expected, was distinct compared to that of the pCSPG (Figure 1B versus 1A). The CSA eluted as an included peak (Kav = 0.74) with a shoulder at the leading edge. The material eluting in the symmetrical and shoulder portions of the peak were pooled separately and recovered as cCSA fraction I and cCSA fraction II (Figure 1B). Fractions I and II differed considerably in their protein contents, 6.4% and 3.5%, respectively, and in sulfation pattern, 59% and 48% 4-sulfate and 33% and 42% 6-sulfated disaccharides (Table II).

The purity of the pCSPG was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The pCSPG and even the core protein released by treatment with chondroitinase ABC did not enter a 5% polyacrylamide gel (data not shown). Even on 4% polyacrylamide and 0.5% agarose copolymer gels, the pCSPG and core protein barely entered the gel (Figure 2A). However, these electrophoretic analyses indicated that the pCSPG is completely free of the contaminating lower-molecular-weight proteins. To investigate the size of the residual core protein (or peptide moiety) in total cCSA and in the cCSA fractions I and II, the samples were analyzed by SDS–PAGE using 15% gel before and after treatment with chondroitinase ABC (Figure 2, and data not shown). The majority of the cCSA electrophoresed as a broad diffused band in the size range of 5–45 kDa, with a very minor portion of the sample not entering the gel (Figure 2B). After chondroitinase ABC treatment, a minor level of high-molecular-weight polypeptides (some barely entering the gels) electrophoresed as a broad diffused band, suggesting that a small portion of the cCSA preparation has relatively large core protein polypeptides. A small amount of peptides in the size range of 2000–5000 Da were also present. These results confirm that the major portion of the bovine tracheal CSPG core protein is removed during the cCSA preparation, and the CS chains are attached to small peptides. The results are also consistent with the gel filtration profile on Sepharose CL-4B (see Figure 1B), showing a very minor amount of relatively high-molecular-weight species (CSA fraction I) with relatively high proportion (6.4%) of protein. These low-abundance molecules containing significant portion of the core protein in the cCSA preparation are those that are likely adsorbed preferentially onto the plastic surface and bind IRBCs when cCSA is used for cytoadherence assay.



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Fig. 2. SDS–PAGE analysis of bovine tracheal CSPG and cCSA. (A) Bovine tracheal pCSPG electrophoresed on 4%-acrylamide gel containing 0.5% agarose under reducing conditions, and the gels were stained with Coomassie blue followed by Alcian blue. Lane 1, untreated pCSPG (30 µg); lane 2, chondroitinase ABC–treated pCSPG (30 µg). (B) cCSA was electrophoresed on 15% polyacrylamide gel under reducing conditions, and the gel was stained with Coomassie blue followed by Alcian blue and finally with ammoniacal silver. Lane 1, untreated cCSA (10 µg); lane 2, chondroitinase ABC–treated cCSA (25 µg). In both panels, the positions of the molecular mass (kDa) of marker proteins are indicated to the right.

 
The CS chains of the pCSPG and cCSA were released by alkaline beta-elimination in the presence of NaBH4 and analyzed by Sepharose CL-6B chromatography (see Figure 1C). In each case, the released CS chains eluted as a single peak at the elution volume corresponding to a molecular weight of ~25,000 Da. The elution patterns for the CS chains of the pCSPG and cCSA were similar. Furthermore, in the case of cCSA, there was no significant change in the peak position of the material eluted from the column whether or not it was subjected to alkaline beta-elimination (not illustrated), supporting the conclusion that the majority of commercial CSA molecules lack the major portion of core protein.

Structural analysis of the CS chains of bovine tracheal CSPG
The structure–activity relationship studies were performed for both cCSA and the CS chains of CSPG purified from bovine trachea (pCSPG). Because the results for these two samples were similar (see Figures 3, 4, and 6, and Tables III and IV), only the data for cCSA is described. The bovine tracheal CS chains were analyzed by DEAE-Sephacel chromatography to examine the homogeneity of the chains with respect to their overall charge density. A sharp symmetrical peak was observed revealing that the CS chains are sulfated at similar levels (Figure 3A). In contrast, anion-exchange chromatography of the regioselectively 6-O-desulfated CS chains eluted as a broad unresolved peak (Figure 3B), suggesting that the CS chains vary widely with respect to the ratio of 4- and 6-sulfated disaccharides. This is in agreement with a previous report that the bovine tracheal CS chains are mixture of several discrete species differing in the ratios of 4- and 6-sulfate groups (Inerot and Heinegard, 1983Go). The eluted material was pooled into different fractions as indicated (Figure 3B), and the disaccharide composition determined (Table III). All the fractions contained significant proportions of nonsulfated disaccharide residues but differed substantially in the 4-sulfate content that increased from fractions I to VI (see Figure 3B and Table III). Because the untreated (non-6-O-desulfated) bovine tracheal CS chains contain only ~10% nonsulfated disaccharide moieties (Table II), the majority of the nonsulfated disaccharides in these 6-O-desulfated CS chains must be derived from 6-sulfated disaccharides (Table III).



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Fig. 3. Fractionation of CSA and 6-O-desulfated CSA by DEAE-Sephacel chromatography. Bovine tracheal CSA before (A) and after 6-O-desulfation (B) was chromatographed on DEAE-Sephacel columns (1.5 x 10 cm) using NaCl gradient and elution was monitored by the uronic acid assay (closed circles). Chondroitin was similarly chromatographed and monitored as a reference sample (open circles). The 6-O-desulfated CSA fractions (B) were pooled as indicated and the disaccharide composition determined (see Table III). The NaCl gradient in B (data not shown) was similar to that of A. Note: The elution profile of the regioselectively 6-O-desulfated CS chains isolated from the purified pCSPG was also identical to that shown for cCSA in panel B (not illustrated).

 


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Fig. 4. Analysis of the chondroitinase C digestion products of bovine tracheal CSA. The cCSA, digested with chondroitinase C, was chromatographed on a Bio-Gel P-6 column (1.5 x 70 cm), and the elution was followed by uronic acid. The oligosaccharide fractions were pooled as indicated by the horizontal bars and analyzed for disaccharide composition (see Table IV). The elution positions of blue dextran (BD) and glucose (Glc) are indicated. Note: The elution profile of the CS chains of pCSPG was indistinguishable from that illustrated for cCSA (data not shown).

 


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Fig. 6. Inhibition of IRBC binding to CSPG by variously 6-O-desulfated CSA. The plastic petri dishes were coated as circular spots with the placental low sulfated CSPG (Ref. 1) at 0.2 µg/ml concentrations. The assays were performed as described under Materials and methods using CSAs with different 6-sulfate content. The assays were carried out three times each in duplicate and the average values plotted. The 4- and 6-sulfate contents (%) of the partially 6-O-desulfated CSA (CSA1 to CSA5), used for inhibition, are given in parentheses; the remainder is nonsulfated disaccharides. X's, CSA1 (48, 42); open circles, CSA2 (52, 39); closed circles, CSA3 (52, 30); open squares, CSA4 (60, 17); closed squares, CSA5 (52, 1); closed triangles, cCSA fraction 8 from Table IV (88, 10); open triangles, cCSA fraction 9 from Table IV (92, 8). Data are expressed as the mean ± SEM of three independent experiments. The CS chains of pCSPG were studied similarly and the results (not shown) were comparable to those of cCSA.

 

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Table III. Disaccharide composition of the partially 4-sulfated CS obtained by the DEAE-Sephacel chromatography of the regioselectively 6-O-desulfated bovine tracheal CSA

 

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Table IV. Compositions and structural features of oligosaccharide fractions formed by the chondroitinase C digestion of bovine tracheal CSA

 
To determine the distribution pattern of 4- and 6-sulfated moieties, the bovine tracheal CS chains were digested with chondroitinase C, which specifically cleaves the ß(1-4)-glycosidic bonds between N-acetylgalactosamine-6-sulfate and glucuronic acid. Bio-Gel P-6 chromatography of the enzymatic digestion products showed predominantly di-, tetra-, hexa-, and octasaccharides (Figure 4; fractions 1, 2 + 3, 4, and 5, respectively). Based on the yield and compositions of these oligosaccharides (Table IV), it can be inferred that the majority of the 4-sulfated disaccharides are present in the CS chains as single units and also as blocks of two or three consecutive 4-sulfated disaccharides interspersed by one to three 6-sulfated disaccharides (Table IV, fractions 2–5). The remainder of the 4-sulfated disaccharides is present in blocks of four or more disaccharides separated by one or more 6-sulfated disaccharides (Table IV, fractions 6–9).

IRBC adherence to pCSPG and the cCSA
The IRBC binding abilities of pCSPG, cCSA, and cCSA fractions I and II (Figure 1B) were determined by adherence and adherence inhibition assays. All samples were able to bind IRBCs in a dose-dependent manner, but their binding capacity was significantly low compared to human placental CSPGs (Figure 5A), which are the receptors for IRBC adherence in the intervillous spaces of the placenta (Achur et al., 2000Go). Of the trachea samples, pCSPG with intact core protein and cCSA fraction I with 6.4% protein bound, respectively, 1.5-fold and ~2-fold more IRBCs per unit area compared to cCSA (Figure 5A). Because the structural features of the CS in these three preparations are marginally different, the noted difference in IRBC binding was thought to be mainly due to the limited ability of cCSA to adsorb onto the plastic surface. Enzyme-linked immunosorbent assay was performed to directly evaluate the coating efficiency of various CSA preparations onto the petri dishes. It was found that the coating of the three samples was saturated at 5–10 µg/ml, but they differed in their coating capacity. The pCSPG and cCSA fraction I adsorbed onto the plastic up to eightfold more than cCSA (data not shown). This is consistent with the higher proportion of protein in pCSPG and cCSA fraction I compared to total cCSA, in which cCSA fraction II with low protein content predominates. However, the eightfold-higher coating capacity of samples with high level of protein is surprising, considering that they could only bind 1.5- to 2-fold more IRBCs than cCSA (Figure 5A). One possibility for this observation could be that the ability of the glycosaminoglycan (GAG) chains of coated CSPGs to bind IRBCs is saturating at the levels lower than those the plastic plate can adsorb.




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Fig. 5. Adherence of P. falciparum IRBCs to the bovine tracheal pCSPG and CSA fractions. (A) The pCSPG, cCSA, and cCSA fractions I and II were coated at 7 ng to 8 µg/ml concentrations (based on uronic acid content) as circular spots on plastic petri dishes. The assays were performed four times, each in duplicate, and average values plotted. Adherence to human placental low-sulfated CSPG (Ref. 1) was also assayed as a reference. Closed circles, placental low-sulfated CSPG; open circles, cCSA fraction I; closed triangles, pCSPG from bovine trachea; open triangles, cCSA; closed squares, cCSA fraction II; open squares, the CS chains released from cCSA by alkaline ß-elimination. (B) Inhibition of IRBC binding to the placental low-sulfated CSPG (black), bovine trachea pCSPG (dark hatched), cCSA fraction I (gray), cCSA (light hatched), and cCSA fraction II (open) by C4S with 36% 4-sulfated and 64% nonsulfated disaccharides. Data are expressed as the mean ± SEM of three independent experiments.

 
Because the IRBC binding assay that measures the number of cells bound to the immobilized CSPGs does not reflect the relative binding strengths, we assessed the inhibition of IRBC binding to pCSPG and cCSA using a C4S containing 36% 4-sulfate, obtained by the regioselective 6-O-desulfation of cCSA, as the inhibitor. At various concentration of the inhibitor tested, no significant difference in the binding capacity between the pCSPG and cCSA or its fractions (I and II) was evident (Figure 5B). Thus IRBCs can bind with similar strengths to the CS chains of pCSPG and cCSA. However, the IRBC-binding strengths of pCSPG and cCSA were significantly lower than that of the human placental low-sulfated CSPGs (Figure 5B).

Role of 4- and 6-sulfated residues in the binding of IRBCs to bovine tracheal CSA
As noted, the 4- and 6-sulfated disaccharides in the GAG chains of bovine tracheal CSPG are distributed in segregated blocks of varying chain lengths (Figure 4 and Table IV). To determine which of these structural features can more avidly bind IRBCs, the oligosaccharides higher than dodecamers formed by the chondroitinase C digestion of the bovine tracheal CS chains (see Figure 4) were assessed for their ability to inhibit IRBC binding. At the range of concentrations tested, oligosaccharide fractions 8 and 9 containing longer 4-sulfated blocks with only 8%–10% of 6-sulfated moieties were significantly less inhibitory compared to the intact cCSA (see Table IV and Figure 6). These results indicate, albeit indirectly, that the blocks with fewer 4-sulfated disaccharides interspersed by one or more 6-sulfated disaccharides as in fractions 1–7 (Figure 4 and see Table IV) can bind IRBCs more efficiently than the blocks with continuous 4-sulfate. Previous studies have shown that IRBC binding involves the participation of both 4-sulfated and 4-nonsulfated disaccharides of the minimal dodecasaccharide motif and that 6-sulfated residues can act as 4-nonsulfated moieties (Alkhalil et al., 2000Go), although the 6-sulfate groups considerably interfere with the inhibitory activity of C4S (Chai et al., 2002Go; Fried et al., 2000Go). To assess the extent to which 6-sulfated moieties affect the inhibitory activity of CSA, 6-sulfate groups were removed to varying degrees and then tested for inhibition of IRBC adhesion to placental CSPG. The binding capacity of the partially 6-O-desulfated CSA increased gradually with decrease in 6-sulfate content to 17%, and further decrease in 6-sulfate did not show appreciable effect (Figure 6). These results are consistent with the avid binding of IRBCs to regions consisting of shorter blocks of 4-sulfated residues alternating with blocks of two to three residues of 6-sulfate. Based on the previous findings (Alkhalil et al., 2000Go), it is likely that such regions consists of one to three 4-sulfated disaccharides with or without one nonsulfated moiety interspersed with two to three 6-sulfated disaccharides.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We purified and characterized pCSPG, which represents >75% of the total CSPG in the tracheal cartilage tissue (Antonopoulos et al., 1974Go). The composition of the pCSPG closely resembles that of the previously characterized high-molecular-mass CSPG in the AI-DI aggrecan fraction obtained by the CsCl density gradient centrifugation (Heinegard, 1972Go). The molecular weight of the PG (>1 x 106) suggests the presence of large number of CS chains (~25,000) attached to the core protein of the pCSPG. In addition, the significant amount of neutral sugars and N-acetylglucosamine in the molecule suggests that the core protein is also substituted with N- and O-linked oligosaccharides and possibly keratan sulfate chains (Antonopoulos et al., 1974Go).

We found that both the pCSPG and cCSA bound P. falciparum IRBCs in a dose-dependent manner. A comparison of the IRBC binding capacities revealed that when coated at similar uronic acid concentrations, the pCSPG and cCSA fraction I could bind ~1.5- and 2-fold more IRBCs per unit area, respectively, compared with the cCSA. However direct evaluation of the efficiency of these samples to adsorb to plastic petri dishes indicated that pCSPG and cCSA fraction I were up to eightfold better compared to cCSA. Therefore the more efficient coating of pCSPG and cCSA fraction I appear to have little bearing on their only 1.5- to 2-fold better IRBC binding than cCSA. An explanation for this disparity could be that beyond a certain level of CSPG coating the spatial disposition of the GAG chain may not be favorable for continued increase in IRBC binding. That is, at some critical concentration, the IRBC binding is saturated and the GAG chains cannot accommodate more IRBCs. This is consistent with the observation that, in the case of the low-sulfated CSPGs, the IRBC binding saturates at a coating concentration of 100–200 ng/ml, even though the level of CSPGs coated on the dishes continue to increase markedly with increase in coating concentration (data not shown).

It was interesting that despite the high content of 6-sulfate groups, the CS chains of pCSPG can support high-density binding of IRBCs, and cCSA is an effective inhibitor of IRBC binding (Alkhalil et al., 2000Go). This is surprising because high levels of 6-sulfated disaccharides in the C4S chains have been reported to markedly interfere with IRBC binding (Chai et al., 2002Go; Fried et al., 2000Go). Therefore we performed structure–IRBC adherence relationship studies to determine the structural features of the bovine tracheal CSA that efficiently binds IRBCs. Structural analysis of the bovine tracheal CSA by specific degradation using chondroitinase C indicates that both 4- and 6-sulfated disaccharides are distributed predominantly as single units or blocks of two to four consecutive residues. In the case of the 4-sulfated disaccharide residues, ~80% are distributed as single and also as blocks of 2 or 3 units, and the remainder is present in blocks of 4 to 12 disaccharide units. Based on the total content of the 6-sulfate in the CSA chains and the amount of 6-sulfated disaccharide unit released by chondroitinase C, it can be estimated that ~75% of the 6-sulfated disaccharide moiety is present as single residues and the remainder as blocks of two, three, or possibly four units separated by 4-sulfated disaccharides. Interestingly, the majority of the nonsulfated disaccharide moieties, although present in significantly lower proportion compared to the sulfated moieties, are present as either single unit or adjacent to a 4-sulfated unit that are separated by either a single or blocks of 6-sulfated moieties. Thus our data indicate that the 4- and 6-sulfated disaccharides of the bovine tracheal CS chains are segregated such that the majority of the 4-sulfated disaccharides are present as a single unit and in blocks of two or three units interrupted by blocks comprised of one to three 6-sulfated disaccharides. This is likely to be related to the distribution pattern of 4- and 6-sulfotransferases in the Golgi and their ability to transfer sulfate groups to either single or several consecutive disaccharide moieties of the chondroitin chains.

Based on our results, it was not possible to infer the relative distribution of various 4- and 6-sulfated blocks in the CSA chains of the pCSPG. However, our data demonstrated that the nonsulfated and 4-sulfated di- and tetrasaccharide moieties are in preponderance. This strongly suggests the relatively high probability that such di- and tetrasaccharide moieties are situated next to each other or separated by motifs consisting of three or four 4-sulfated disaccharide moieties. Considering that IRBCs bind more strongly to dodecasaccharide motifs containing two to four 4-sulfate groups and poorly to those containing five or six 4-sulfate groups (Alkhalil et al., 2000Go), it appears likely that the regions that contain shorter blocks of 4- and nonsulfated disaccharide moieties separated by one or two 6-sulfated moieties, such as -4S-0S-4S-4S-0S-6S-6S-, -0S-4S-6S-4S-4S-0S-6S-, -4S-6S-0S-4S-4S-6S-6S-, and -4S-4S-6S-4S-6S-0S-6S-, provide sites for binding of IRBCs. In these motifs, the overall 6-sulfate content will account for 17% to 33% per dodecasaccharide unit, which may not drastically affect the IRBC binding but can moderately lower the binding strengths. These conclusions are consistent with our observation that the larger fragments of CSA, obtained by chondroitinase C digestion, that consist of 88%–92% 4-sulfated and 8%–10% 6-sulfated disaccharides were significantly less inhibitory compared to the whole cCSA chains. Thus our data indicate that shorter blocks of 4-sulfated residues interspersed by two to three residues of 6-sulfated disaccharides can efficiently bind IRBCs. Because IRBC binding requires the participation of 4-sulfated and 4- nonsulfated residues, it appears likely that the 6-sulfated disaccharides satisfies the crucial requirement of the availability of free 4-OH on N-acetylgalactosamine. Nonetheless, the 6-sulfate groups also significantly lower the IRBC-CSA interactions (see later discussion), which otherwise would be much more efficient.

The data presented here clearly show the inhibitory effect of 6-sulfate on IRBC binding by bovine tracheal CSA. Removal of 6-sulfate groups from bovine CSA to varying degree by regioselective 6-O-desulfation increased the ability of CSA to bind IRBCs. Although reduction of 6-sulfate content from 39% to 17% gradually and significantly increased the IRBC binding, further decrease of 6-sulfate showed either only marginal or no increase in IRBC binding. Thus our data demonstrate that although moderate to high levels of 6-sulfate can lower the ability of chondroitin 4-sulfate to bind IRBCs as reported previously (Chai et al., 2002Go; Fried et al., 2000Go), low levels of 6-sulfate have no significant effect.

The IRBC binding strength of the bovine tracheal CS chains is lower by an order of magnitude compared to those of the placental low-sulfated CSPGs. This is despite the presence of only a few clusters of 4-sulfated domains, which are sites for IRBC adherence in the placental CS chains. Thus it appears likely that although 4-sulfated disaccharides are distributed through the CSA chains of bovine tracheal CSPG, because of the relative distribution of 4- and 6-sulfated residues, only a few sites are able to bind IRBCs with sufficient strength. Regardless of this, the bovine tracheal CSA chains support IRBC adherence better than the CS chains from other sources, such as the whale cartilage CSA (69% 4-sulfate and 27% 6-sulfate), sturgeon notochord C4S (fully 4-sulfated), and shark cartilage C2,6diS (28% 4-sulfate, 46% 6-sulfate and 23% 2,6-disulfate) (Alkhalil et al., 2000Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Trachea were collected from a local slaughterhouse immediately after the cow was sacrificed and stored frozen at –80°C until used. Protease-free chondroitinase ABC (Proteus vulgaris), anti-{Delta}di-4S mouse monoclonal IgG, and anti-{Delta}di-6S mouse monoclonal IgM were purchased from Seikagaku America (Falmouth, MA). Bovine trachea CSA (catalog number C8529), chondroitinase C, phenylmethylsulfonyl fluoride (PMSF), N-ethylmaleimide (NEM), benzamidine, and protein molecular weight standards for gel filtration were from Sigma (St. Louis, MO). N-{alpha}-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were from Roche Molecular Biochemicals (Indianapolis, IN). Sepharose CL-4B, Sepharose CL-6B, DEAE-Sephacel, and blue dextran were from Amersham Pharmacia Biotech (Piscataway, NJ). HPLC-grade 6 N HCl, trifluoroacetic acid, and micro BCA protein assay kit were from Pierce (Rockford, IL). Polystyrene petri dishes (Falcon 1058) were from Becton-Dickinson Labware.

Isolation of CSPG from bovine trachea
All procedures were performed at 4°C. Bovine tracheal cartilage tissue (14 g) was cut into pieces; suspended in 60 ml 4 M GdnHCl in 50 mM NaOAc buffer, pH 5.8, containing 10 mM ethylenediamine tetra-acetic acid (EDTA), 0.1 mM PMSF, 0.1 mM TLCK, 0.25 mM TPCK, 1 mM benzamidine, and 0.1 mM NEM for 6 h; and homogenized with a Polytron homogenizer (Brinkmann, Switzerland), and the homogenate was stirred overnight. The homogenate was centrifuged at 10,000 rpm, and the clear supernatant was used for further purification. Solid CsCl was added to a final concentration of 42% and the solution centrifuged at 44,000 rpm in a Beckman 70 Ti rotor at 14°C for 48 h (Achur et al., 2000Go). Gradients were collected from the bottom of the tubes into 13 equal fractions. Aliquots of the fractions were assayed for uronic acid contents (Achur et al., 2000Go). The high-density fractions having a high uronic acid content were pooled, dialyzed, and lyophilized to recover CSPGs.

Purification of CSPG
The crude preparation of CSPG (16 mg) was dissolved in 50 mM Tris-HCl, pH 7.5, containing 6 M GdnHCl and 0.2 M NaCl, and chromatographed on a Sepharose CL-4B column (1.5 x 83 cm). Fractions (2 ml) were collected and aliquots assayed for uronic acid at 530 nm and for protein at 280 nm. The CSPG-containing fractions were combined, dialyzed, and lyophilized. The recovered CSPG (8 mg) was dissolved in 8 ml 25 mM sodium phosphate buffer, pH 7.2, containing 4 M GdnHCl, 10 mM EDTA, 0.1 mM PMSF, 0.1 mM TLCK, 0.25 mM TPCK, 1 mM benzamidine, 0.1 mM NEM, and 42% CsBr and centrifuged in a Beckman 70 Ti rotor at 44,000 rpm for 48 h at 14°C (Achur et al., 2000Go). Gradients were collected from the bottom of the tubes into 13 equal fractions, and the absorption at 260 and 280 nm was measured. An aliquot of each fraction was also assayed for uronic acid content and the fractions containing CSPG were pooled, dialyzed, and lyophilized.

Electrophoresis
The core proteins or peptides were prepared from the pCSPG and cCSA by treatment with chondroitinase ABC. The material was dissolved in 60 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, and 0.5 M 2-mercaptoethanol and electrophoresed on 4% polyacrylamide gel containing 0.5% agarose (Sachdev et al., 1978Go). The gels were stained with Coomassie blue followed by Alcian blue (Krueger and Schwartz, 1987Go). The peptides from the CSA were analyzed by SDS–PAGE using 15% gels (Laemmli, 1970Go).

Isolation of the CS chain
The pCSPG or cCSA (25–50 mg each) were treated with 5–10 ml 0.1 M NaOH containing 1 M NaBH4 at 45°C for 18 h (Achur et al., 2000Go). The solution was cooled in an ice bath, neutralized with 1 M HOAc, and dried in a Speed-Vac. Boric acid was removed by repeated evaporation with 0.1% acetic acid in methanol. The residues were dried and chromatographed on a Sepharose CL-6B column (1 x 49 cm) in 0.2 M NaCl. Fractions were collected and aliquots assayed for uronic acid. The positive fractions were combined, dialyzed against distilled water, and lyophilized.

Monosaccharide composition analysis
The CSPG samples and CSA (20 µg each) were hydrolyzed with either 4 M HCl at 100°C for 4 h (for hexosamine analysis) or 2.5 M trifluoroacetic acid at 100°C for 5 h (neutral sugar analysis). The hydrolysates were dried in a Speed-Vac and analyzed on a CarboPac PA10 high pH anion-exchange column using a Dionex BioLC HPLC (Hardy, 1989Go) using 18 mM NaOH. The response factors for the monosaccharides were determined using standard sugars.

Disaccharide composition analysis of the CS chains
The CS chains (50 µg) were digested with chondroitinase ABC (20 mU) in 50 µl 100 mM Tris-HCl, pH 8.0, containing 30 mM NaOAc and 0.01% bovine serum albumin (BSA) at 37°C for 5 h (Oike et al., 1980Go). The samples were dried in a Speed-Vac, and the digests corresponding to 12–15 µg of CS chains were analyzed by HPLC (Waters, Milford, MA) on a 4.6 x 250-mm amino bonded silica PA03 column (YMC, Milford, MA) using a linear gradient of 16–530 nm NaH2PO4 at a flow rate of 1 ml/min (Sugahara et al., 1994Go).

Regioselective 6-O-desulfation of the CS chains
CSA was regioselectively 6-O-desulfated as described previously (Alkhalil et al., 2000Go). Briefly, CSA (10–20 mg), deionized on Dowex 50W-X8 (H+), was neutralized with pyridine, lyophilized, and dissolved in dry pyridine (5 ml). N,O-Bis(trimethylsilyl)acetamide (1 ml) was added to a final concentration of 20% and heated in a screw-cap glass tube at 80°C for 4 h (Alkhalil et al., 2000Go). The reaction mixture was cooled in an ice bath, and ice-cold water (6 ml) was added to decompose excess silylating reagent; then the mixture was dialyzed against water and lyophilized.

DEAE-Sephacel chromatography of the intact and 6-O-desulfated CSA
The regioselective 6-O-desulfated CSA (5.0 mg) was applied onto a DEAE-Sephacel column (1.5 x 10 cm) preequilibrated with 50 mM sodium acetate buffer, pH 5.5, and washed with the same buffer. The bound material was eluted with a NaCl gradient (0–1.0 M) in the same buffer; fractions (3 ml) were collected and aliquots analyzed for uronic acid by the carbazole method. Similarly, the intact CSA and chondroitin were applied on identical DEAE-Sephacel columns, eluted with same NaCl gradients, and aliquots of the fractions were analyzed for uronic acid.

Digestion of CS chains with chondroitinase C and fractionation of products by gel filtration
The CS chains (5 mg) were incubated with 25 U chondroitinase C enzyme in a total volume of 1 ml in 0.1 M Tris-HCl buffer, pH 8.0, containing 30 mM NaOAc at 25°C for 40 h. The sample was fractionated on a Bio-Gel P-6 column (1.5 x 70 cm) preequilibrated with 0.1 N pyridine/0.1 M acetic acid, pH 5.2, and aliquots of fractions (2 ml) were analyzed for uronic acid.

P. falciparum cell culture
The C4S-adherent P. falciparum, selected by panning of the FCR-3 laboratory parasite strains on plastic plates coated with placental low-sulfated CSPG, were used in this study. The parasites were cultured in RPMI 1640 medium using O-positive type human blood and serum at 3% hematocrit. The cultures were incubated at 37°C in an atmosphere of 90% nitrogen, 5% oxygen, and 5% carbon dioxide (Alkhalil et al., 2000Go).

IRBC adhesion and adhesion-inhibition assays
The adherence of IRBCs was assessed by coating solutions (15 µl) of CSPGs, cCSA, and cCSA fractions at 7 ng to 8 µg/ml concentrations, based on uronic acid content, as circular spots on 150 x 15-mm plastic petri dishes as described previously (Alkhalil et al., 2000Go). Nonspecific sites were blocked by incubating with 2% BSA in phosphate buffered saline (PBS), pH 7.2, at room temperature for 2 h. Parasite cultures with 20%–30% parasitemia were used for the assay. After 30-min incubation at room temperature, the unbound cells were removed by washing and the bound cells fixed with 2% glutaraldeyde, stained with Giemsa reagent, and counted under light microscope.

For adhesion-inhibition assays, 2x IRBC suspension was preincubated with equal volume of 200 ng/ml to 100 µg/ml desulfated bovine C4S containing 36% 4-sulfate in PBS, pH 7.2, in 96-well microtiter plates at room temperature for 30 min with intermittent mixing (Alkhalil et al., 2000Go). The IRBC suspensions were layered on CSPG-coated spots on petri dishes, and the assay was carried out as described.

Assessment of CSA coating onto the petri dish by enzyme-linked immunosorbent assay
Plastic petri dishes were coated with 50 µl/spot of proteoglycans at 0.2–20 µg/ml in PBS, pH 7.2, overnight at 4°C. The spotted areas were sequentially treated as follows: (1) The nonspecific sites were blocked with 55 µl 2% BSA in PBS at room temperature for 2 h. (2) Incubated with protease free chondroitinase ABC (4 mU/spot) for 2 h at 37°C in a moisture chamber. (3) Washed three times with PBS and then incubated with 50 µl 1:200 diluted anti-{Delta}di-4S monoclonal IgG or 1:50 diluted anti-{Delta}di-6S monoclonal IgM at room temperature for 2 h. (4) The antibody solution was aspirated, washed three times with PBS containing Tween-20, followed by twice with PBS, and then incubated with 50 µl 1:2000 diluted horseradish peroxidase–conjugated goat anti-rabbit IgG or IgM for 1 h at room temperature. (5) The bound antibodies were measured using ABTS substrate.


    Acknowledgements
 
This work was supported by Public Health Service grant AI-45086 from the National Institute of Allergy and Infectious Diseases.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: gowda{at}psu.edu


    Abbreviations
 
BSA, bovine serum albumin; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; CS, chondroitin sulfate; CSA, chondroitin sulfate A; CSPG, chondroitin sulfate proteoglycan; EDTA, ethylenediamine tetra-acetic acid; GAG, glycosaminoglycan; HPLC, high-performance liquid chromatography; IRBC, infected red blood cell; NEM, N-ethylmaleimide; PBS, phosphate buffered saline; PG, proteoglycan; PMSF, phenylmethylsulfonyl fluoride; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TLCK, N-{alpha}-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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