From the Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Received for publication, October 28, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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Plasmodium falciparum infection in
pregnant women results in the chondroitin 4-sulfate-mediated
adherence of the parasite-infected red blood cells (IRBCs) in the
placenta, adversely affecting the health of the fetus and mother. We
have previously shown that unusually low sulfated chondroitin sulfate
proteoglycans (CSPGs) in the intervillous spaces of the placenta are
the receptors for IRBC adhesion, which involves a chondroitin 4-sulfate
motif consisting of six disaccharide moieties with ~30% 4-sulfated
residues. However, it was puzzling how the placental CSPGs, which have
only ~8% of the disaccharide 4-sulfated, could efficiently bind
IRBCs. Thus, we undertook to determine the precise structural features
of the CS chains of placental CSPGs that interact with IRBCs. We show that the placental CSPGs are a mixture of two major populations, which
are similar by all criteria except differing in their sulfate contents;
2-3% and 9-14% of the disaccharide units of the CS chains are
4-sulfated, and the remainder are nonsulfated. The majority of the
sulfate groups in the CSPGs are clustered in CS chain domains consisting of 6-14 repeating disaccharide units. While the
sulfate-rich regions of the CS chains contain 20-28% 4-sulfated
disaccharides, the other regions have little or no sulfate. Further, we
find that the placental CSPGs are able to efficiently bind IRBCs due to
the presence of 4-sulfated disaccharide clusters. The oligosaccharides corresponding to the sulfate-rich domains of the CS chains efficiently inhibited IRBC adhesion. Thus, our data demonstrate, for the first time, the unique distribution of sulfate groups in the CS chains of
placental CSPGs and that these sulfate-clustered domains have the
necessary structural elements for the efficient adhesion of IRBCs,
although the CS chains have an overall low degree of sulfation.
A distinctive feature of Plasmodium falciparum compared
with the other three human malaria parasites is its ability to express adherent protein(s) on the surfaces of the infected red blood cells
(IRBCs)1 and thereby
sequester in the microvascular capillaries of various organs by
adhering to endothelial cell surfaces (1-5). The extensive accumulation of IRBCs in vital organs causes capillary blockage with
deprivation of oxygen and nutrients and production of toxic levels of
proinflammatory cytokines (3, 6-10), damaging the endothelial lining
and causing organ dysfunction and severe pathological conditions. A
number of studies have shown that the adherent protein expressed on the
surfaces of IRBCs to be P. falciparum erythrocyte membrane
protein 1 (EMP1), a multidomain, antigenic var gene
family protein (11-17). P. falciparum EMP1 can bind, in a
domain-specific manner, CD36, intercellular adhesion molecule-1,
vascular cell adhesion molecule-1, E-selectin, platelet
endothelial cell adhesion molecule-1/CD31, and thrombospondin on
vascular endothelial cell surface (18-24). In addition, P. falciparum EMP1 can also bind complement receptor (25), heparan
sulfate (20), and chondroitin 4-sulfate (C4S) (22-28). Thus, the
parasite, by its ability to express divergent P. falciparum EMP1s using the var gene repertoire, can
adhere to various organs. However, over a period of time, the host
develops antibodies against the exposed P. falciparum EMP1
that are able to inhibit adhesion of IRBC adhesion and aid clearance of
infection (29-34). To overcome this defensive mechanism, the parasite
constantly switches, at low frequency, to various adherent phenotypes
by expressing P. falciparum EMP1s with different receptor
specificity (35, 36). This ability of the parasite to express P. falciparum EMP1, for which the host has not yet developed
adhesion-inhibitory antibodies, enables it to selectively adhere
through a different receptor. In this manner, when one adherent
phenotype of parasite is eliminated by the host, another phenotype continues to thrive. In endemic areas, people by adulthood acquire a broad spectrum protective immunity against P. falciparum, including antibodies to P. falciparum
EMP1s (37, 38). Therefore, in immune-protected people, the IRBCs cannot
adhere in the vascular capillaries, limiting the parasite growth.
In pregnant women, however, the placenta provides a new opportunity for
IRBC adhesion, because women lack immunity against placenta-adherent
parasites prior to pregnancy (39). Extensive adherence of IRBCs in the
placenta and infiltration of mononuclear cells in response to the
infection results in impaired placental function, leading to poor fetal
outcome and maternal morbidity and mortality (40-46). However, women
acquire placental malaria-specific immunity, including
adhesion-inhibitory antibody response, during the first and second
pregnancies (29-34). Therefore, primigravidas are at highest risk of
placental malaria, and the susceptibility diminishes with increasing
gravidity (44, 45).
C4S mediates the adhesion of IRBCs in the human placenta (39, 47-49).
Previously, we have shown that the CSPGs localized in the intervillous
spaces of the placenta are the receptors for the adherence of IRBCs in
the placenta (50). These CSPGs were found to be unusually low sulfated;
on an average, only ~8% of the disaccharide repeating units of the
CS chains of placental CSPGs are 4-sulfated, and the remainder are
nonsulfated (50). In previous studies, we have also shown that IRBC
adhesion involves the participation of both nonsulfated and 4-sulfated
disaccharide repeating units and the optimal binding requires ~30%
4-sulfated and ~70% nonsulfated disaccharide repeats (51). Further,
we established that a C4S motif having six disaccharide repeating units
(6-mer) with two 4-sulfated and four nonsulfated disaccharide units is
the minimum structural motif required for optimal binding of IRBCs
(51). A recent study confirmed most of our findings (52), except that
four or five rather than two of the disaccharide repeating units of the
binding motif containing six-disaccharide repeating units needs to be
4-sulfated for effective IRBC binding. However, it should be noted that
these investigators measured C4S-IRBC interactions by immobilizing the
commercially available bovine trachea C4S/C6S copolymer (52), a
nonrelevant glycosaminoglycan, rather than the placental CSPGs, the
natural receptor used in our study (50).
Regardless of this discrepancy, it remained a puzzle how IRBCs are able
to efficiently bind the unusually low sulfated CS chains of the
placental intervillous space CSPGs. A full understanding of the
structural requirements for IRBC binding to placenta is important for
developing therapeutics or vaccine for placental malaria (53).
Therefore, in this study, we investigated in detail the structure of
the CS chains of placental CSPGs, particularly the pattern of sulfate
group distribution and its correlation to IRBC binding. The placental
CSPGs were fractionated into two differentially sulfated proteoglycan
populations and their CS chains isolated. The polysaccharide chains
were degraded with an endoenzyme that specifically cleaves the
nonsulfated regions of the CS into disaccharides, and the
oligosaccharide products thus obtained were purified and examined for
their ability to inhibit IRBC binding to intact CSPGs. The data
demonstrate that, although the overall sulfate content of the CS chains
of placental CSPGs are markedly lower than that required for optimal
binding, the sulfate groups in the CS chains are clustered in uniquely size-defined domains. These sulfate-rich CS domains have the requisite structural features for the efficient binding of IRBCs.
Materials--
Proteus vulgaris
chondroitinase ABC (120 units/mg), Streptococcus
dysgalactiae hyaluronidase (0.5 units/vial) and
Streptomyces hyalurolyticus hyaluronidase (2000 turbidity-reducing units/mg), Flavobacterium
heparinum heparitinase (113 units/mg), chondroitin, and C4S
(sturgeon notochord) were purchased from Seikagaku America (Falmouth,
MA); ovine testicular hyaluronidase (2160 units/mg) was from ICN
Biomedicals; C4S (bovine trachea) was from Sigma; Sepharose CL-6B,
Sepharose CL-4B, DEAE-Sephacel, DEAE-Sepharose, and blue dextran were
from Amersham Biosciences; Bio-Gel P-6 was from Bio-Rad; HPLC grade 6 M HCl, trifluoroacetic acid, and micro-BCA protein assay
kit were from Pierce; polystyrene Petri dishes (Falcon 1058) were from
Becton Dickinson Labware. C4S with 36% 4-sulfate was prepared by the
regioselective 6-O-desulfation of bovine trachea C4S/C6S
copolymer as described previously (51) and fractionation of the product
by DEAE-Sephacel
chromatography.2 C4S 6-mers
with 36% 4-sulfate was prepared by the digestion of C4S containing
36% 4-sulfate group with testicular hyaluronidase and fractionation of
the oligosaccharides by gel filtration on Bio-Gel P-6.2
C4Ss with 3 and 11% 4-sulfate groups were prepared by the solvolytic desulfation of a fully 4-sulfated C4S from sturgeon notochord as
described previously (51).
Isolation of CSPGs from Human Placenta--
The low sulfated
CSPGs of the placental intervillous spaces were isolated as described
previously with minor modification (50). Briefly, the placentas were
cut into small pieces and extracted with PBS, pH 7.2, containing
protease inhibitors, and the extract was applied onto DEAE-Sephacel
columns (2.5 × 22 cm). The columns were washed with 25 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, pH 8.0, and then equilibrated with 50 mM NaOAc, 100 mM NaCl, pH 5.5. The bound material was eluted with a
linear gradient of 0.1-0.9 M NaCl in 50 mM
NaOAc, pH 5.5. 10-ml fractions were collected, and aliquots were
analyzed for uronic acid content (54). Uronic acid-containing fractions
corresponding to BCSPG-2, the major CSPG of the placental intervillous
spaces (50), were pooled and dialyzed against water. The dialysates
were adjusted to 50 mM NaOAc, 100 mM NaCl, pH
5.5, and applied onto DEAE-Sepharose columns (2.5 × 17 cm). The
columns were washed with 50 mM NaOAc, 0.15 M
NaCl, pH 5.5, and eluted with a linear gradient of 0.15-0.55 M NaCl in 50 mM NaOAc, pH 5.5. Fractions of
10-ml were collected, absorption at 260 and 280 nm was measured, and
aliquots were analyzed for uronic acid content (54).
Cesium Bromide Density Gradient Centrifugation of CSPGs--
The
crude CSPG fractions obtained by DEAE-Sepharose chromatography were
dissolved (1 mg/ml) in 25 mM sodium phosphate, pH 7.2, containing 50 mM NaCl, 0.02% NaN3, 4 M guanidine hydrochloride, and 42% (w/w) CsBr. The
solutions were centrifuged in a Beckman 50 TI rotor at 44,000 rpm for
65 h (55). Gradients were collected from the bottom of the
centrifuge tubes into 15 equal fractions and analyzed for uronic acid
content (54) and for proteins by measuring the absorption at 260 and
280 nm.
Size Exclusion Chromatography of CSPGs--
The CSPG fractions
obtained from the CsBr density gradient centrifugation step were
further purified by chromatography on columns of Sepharose CL-6B
(1 × 49 cm) and/or Sepharose CL-4B (1 × 48 cm) in 20 mM Tris-HCl, 150 mM NaCl, pH 7.6, containing 4 M guanidine hydrochloride. Fractions were collected and
monitored for absorption at 260 and 280 nm and for uronic acid content
(54).
Analysis of CSPGs for Purity--
The purified CSPGs (0.3 mg)
were treated with S. hyalurolyticus hyaluronidase and
heparitinase as described previously (50). The enzyme-incubation
mixtures were chromatographed on Bio-Gel P-6 (1 × 47 cm) in 0.1 M acetic acid, 0.1 M pyridine. Fractions (0.67 ml) were collected and analyzed for uronic acid (54).
Isolation of the CS Chains of CSPGs--
The purified CSPGs were
treated with 0.1 M NaOH, 1 M NaBH4
for 18-20 h under nitrogen atmosphere at 45 °C (56). The solutions were cooled in ice bath, neutralized with 1 M cold acetic
acid, and then dried in a rotary evaporator. Boric acid was removed by
repeated evaporation in a rotary evaporator by the addition of
methanol, 0.1% acetic acid. The residue was applied onto a DEAE-Sepharose column (1 × 10 cm) in 20 mM Tris-HCl,
pH 7.8, washed with 20 mM Tris-HCl, 0.15 M
NaCl, pH 7.8, and then eluted with a linear gradient of 0.15-0.6
M NaCl in the same buffer. Fractions (2.5 ml) were
collected, and aliquots were assayed for uronic acid content (54).
Uronic acid-positive fractions were pooled, dialyzed against distilled
water, and lyophilized.
Digestion of CS with S. dysgalactiae Hyaluronidase--
The
placental CS chains and chondroitin (1.4-1.8 mg each) were treated
with S. dysgalactiae hyaluronidase (300 milliunits) in
250 µl of 100 mM sodium phosphate buffer, pH 6.2, containing 0.02% BSA at 37 °C for 24 h (57). The enzyme
digests were heated at 100 °C for 5 min, centrifuged, and then
chromatographed on Bio-Gel P-6 columns (1 × 47 cm) in 0.1 M pyridine, 0.1 M acetic acid. Fractions (0.67 ml) were collected, and aliquots were analyzed for uronic acid
(54).
Gel Electrophoresis--
The CS oligosaccharides were dissolved
in 100 mM Tris base, 100 mM boric acid, 2 mM EDTA, pH 8.3, containing 5% glycerol (58). The
solutions were electrophoresed on 10% polyacrylamide gels (15 × 16 cm) in 100 mM Tris base, 100 mM boric acid,
2 mM EDTA, pH 8.3. The gels were stained with 0.03% Alcian
Blue in 25% ethanol, 10% aqueous acetic acid for 4 h and
destained with 25% ethanol, 10% aqueous acetic acid. For silver
staining, the gels were treated with 10% aqueous glutaraldehyde for 30 min and washed with water three times for 30 min each. The gels
were then treated with freshly prepared ammoniacal silver for 15 min,
washed with water two times for 30 s each, developed with 0.005%
citric acid and 0.019% formaldehyde in water, and then washed with
water (59).
DEAE-Sepharose Chromatography of Oligosaccharides--
The
oligosaccharides (fractions 20-35, 50 µg) obtained by the Bio-Gel
P-6 chromatography of the S. dysgalactiae hyaluronidase digest of chondroitin were dissolved in 20 mM NaOAc, 50 mM NaCl, pH 5.0, and applied onto a DEAE-Sepharose
microcolumn (0.1-ml bed volume). After washing with 0.5 ml of the above
buffer, the bound oligosaccharides were eluted stepwise with buffer
containing 0.1, 0.2, 0.4, and 0.6 M NaCl (0.5 ml each).
Fractions (0.1 ml) were collected, and aliquots were analyzed for
uronic acid (54). The oligosaccharide-containing fractions were pooled
and digested with chondroitinase ABC, and the disaccharides formed were
analyzed by HPLC.
Disaccharide Composition Analysis--
The C4Ss or C4S
oligosaccharides (10-15 µg) were digested with chondroitinase ABC
(10-20 milliunits) in 50 µl of 0.1 M Tris-HCl, pH 8.0, containing 30 mM NaOAc and 0.01% BSA at 37 °C for
12-15 h (60). The released, unsaturated disaccharides were analyzed on
an amine-bond Microsorb-MV column (4.6 × 250 mm; Varian) using Waters 600E HPLC system (Milford, MA) (61). The enzyme digests were
injected, and the column was eluted with a linear gradient of 16-530
mM NaH2PO4 over 70 min at room
temperature at a flow rate of 1 ml/min. The elution of disaccharides
was monitored by measuring the absorption at 232 nm using a Waters 484 variable wavelength UV detector. The data were processed with the
Millennium 2010 chromatography manager using NEC PowerMate 433 data
processing system.
Carbohydrate Composition Analysis--
The CSPGs or CS chains
(5-10 µg) were hydrolyzed with 4 M HCl at 100 °C for
6 h. The hydrolysates were dried in a Speed-Vac and analyzed on a
CarboPac PA1 high pH anion exchange HPLC column (4 × 250 mm;
Dionex) (62). The column was eluted with 20 mM sodium
hydroxide, elution of sugars was monitored by pulsed amperometric detection, and the response factors for sugars were determined using
standard sugar solutions.
Other Analytical Procedures--
The uronic acid contents in
various column chromatography fractions were determined by the
carbazole-sulfuric acid method (54). Protein contents were measured
using the Micro BCA Protein Assay Reagent kit from Pierce (63).
P. falciparum Cell Culture--
The C4S adherent P. falciparum, selected by panning of 3D7 laboratory parasite clones
on placental CSPG-coated plastic plates, were used in this study. The
parasites were cultured using type O-positive human red blood cells at
3% hematocrit in RPMI 1640 medium supplemented with 25 mM
HEPES, 29 mM sodium bicarbonate, 0.005% hypoxanthine,
p-aminobenzoic acid (2 mg/liter), gentamycin sulfate (50 mg/liter), and 10% O-positive human serum. The cultures were incubated
at 37 °C in an atmosphere of 90% nitrogen, 5% oxygen, and 5%
carbon dioxide (51).
IRBC Adhesion and Adhesion-Inhibition Assays--
The adherence
of IRBCs was performed by coating solutions (10-15 µl) of purified
CSPGs as circular spots on 150 × 15-mm plastic Petri dishes as
described previously (51). The specificity of IRBC binding to CSPGs was
ascertained by incubating the CSPG-coated plates with chondroitinase
ABC (50 milliunits/ml) as well as by competitive inhibition with
various C4Ss.
For adhesion-inhibition assays, IRBCs were incubated with various C4Ss
or C4S oligosaccharides at the indicated concentrations in PBS, pH 7.2, in 96-well microtiter plates at room temperature for 30 min with
intermittent mixing (51). The IRBC suspension was then layered on
CSPG-coated spots on Petri dishes. After 40 min at room temperature,
the unbound cells were washed, and the bound cells were fixed with 2%
glutaraldehyde, stained with Giemsa, and counted using a light microscope.
Fractionation and Characterization of Differentially Sulfated CSPGs
of Human Placental Intervillous Spaces--
The major low sulfated
CSPG fraction (previously designated as BCSPG-2) (50) was isolated
by one-step DEAE-Sephacel chromatography of the isotonic buffer extract
of placentas. This CSPG fraction represents about 93-94% of the total
low sulfated CSPGs in the intervillous spaces (50). When subjected to
DEAE-Sepharose chromatography, using a 0.15-0.55 M NaCl
gradient, the CSPG was partially resolved into two fractions
(designated BCSPG-2a and BCSPG-2b) that are distinct in their sulfation
levels (Fig. 1). The proportions of BCSPG-2a and BCSPG-2b varied considerably from one placenta to another,
in the range 40-65% and 35-60%, respectively. These CSPG fractions
were further purified and characterized, and their ability to bind
IRBCs was studied.
On CsBr density gradient centrifugation, BCSPG-2a and BCSPG-2b
sedimented to the middle of the gradients (average
BCSPG-2a and BCSPG-2b were further purified by Sepharose CL-6B and
Sepharose CL-4B chromatography, which removed any residual protein
contaminants. In each case, the CSPG fraction eluted as a single
nonsymmetrical peak, and the chromatograms were similar to that
previously reported for the total BCSPG-2 (not shown) (50). The yields
and compositions of the purified BCSPG-2a and BCSPG-2b are shown in
Table I. As in the case of total BCSPG-2, BCSPG-2a and BCSPG-2b each contained high and low molecular mass (~1000- and ~570-kDa, respectively) proteoglycan species (50). The
proportions of ~1000- and ~570-kDa species in BCSPG-2a and BCSPG-2b
were similar (not shown). Chondroitinase ABC degraded the
glycosaminoglycan chains of both BCSPG-2a and BCSPG-2b completely into
unsaturated disaccharides, as assessed by chromatography of the enzyme
digests on Bio-Gel P-6 column (not shown), indicating the absence of
hyaluronic acid and/or heparan sulfate in these fractions. Consistent
with these results, the CS chains of the proteoglycan fractions contain
predominantly galactosamine (Table II),
and they were completely resistant to the action of S. hyalurolyticus hyaluronidase and heparitinase (not shown). As
evident from the disaccharide compositions of the CS chains (see below;
Table II), BCSPG-2a and BCSPG-2b differ significantly in their sulfate
contents. Thus, the differences in the overall charge density of the
proteoglycan fractions, as indicated by their elution at different salt
concentrations from DEAE-Sepharose columns (Fig. 1), is mainly due to
the differences in sulfate contents of the CS chains.
SDS-PAGE analysis of the core proteins released after chondroitinase
ABC treatment revealed that BCSPG-2a and BCSPG-2b each contain two
distinct core proteins, a high (~670-kDa) and a low (~56-kDa)
molecular mass species, respectively, in ~40 and ~60% proportion
(Table I). The proteoglycan fractions closely resembled one another in
the amino acid compositions of their core proteins and the molecular
weight (~60,000) of the CS chains, which were similar to that
reported previously for BCSPG-2 (50). The results, when considered
together, indicate that BCSPG-2a and BCSPG-2b are similar to one
another with respect to proteoglycan types but differ in the amounts of
sulfate groups in their CS chains (Table II).
Adhesion of P. falciparum IRBCs to BCSPG-2a and BCSPG-2b--
The
purified BCSPG-2a and BCSPG-2b were assessed for their abilities to
adhere IRBCs by an in vitro cytoadherence assay (Fig. 2). Both CSPGs efficiently bound IRBCs in
a concentration-dependent manner. Significant levels
of IRBC binding were observed at coating concentration as low as 12 ng/ml. At 50 ng/ml, both fractions efficiently bound IRBCs, and at
100-200 ng/ml they exhibited saturated levels of binding. Thus,
despite significant difference in the sulfate contents of the CS
chains, BCSPG-2a and BCSPG-2b were indistinguishable from one another
with regard to the number of IRBCs adhering when immobilized on solid
surfaces (Fig. 2A). It is possible that similar IRBC binding
capacities of BCSPG-2a and BCSPG-2b might merely reflect the number of
IRBCs bound, even if the IRBCs adhered to these proteoglycans with
different affinities. Therefore, to determine the relative affinities
of IRBC binding by BCSPG-2a and BCSPG-2b, we performed adhesion
inhibition assays. BCSPG-2a and BCSPG-2b were coated on plastic plates
and tested in parallel for competitive inhibition of IRBC binding by a
regioselectively 6-O-desulfated bovine trachea C4S fraction
with 36% 4-sulfate and a C4S 6-mer with 36% 4-sulfate groups. Both
compounds were marginally (10-20%) more inhibitory to IRBC binding to
BCSPG-2a compared with BCSPG-2b, suggesting that BCSPG-2b binds
IRBCs with slightly higher affinity than BCSPG-2a (Fig.
2B). This agrees with the difference in the sulfate content
and the number of IRBC binding sites in the CS chains of BCSPG-2a
and BCSPG-2b. However, the difference in the levels of inhibition is
not as much as that expected based on the number of IRBC binding sites
in the CS chains of BCSPG-2b; the CS chains of BCSPG-2b has 4-5 times
more binding sites than those of BCSPG-2a (see below).
The Level and Distribution Pattern of Sulfate Groups in the CS
Chains of Placental CSPGs--
The CSPGs of the intervillous spaces
can efficiently bind IRBCs despite markedly lower total sulfate
contents of their CS chains than that required for the optimal IRBC
binding (51). This suggested that the sulfate groups may be clustered
in their CS chains and that these sulfate-rich regions could
efficiently bind IRBCs. To investigate this further, we performed a
detailed structural characterization of the CS chains of placental
CSPGs. The CS chains of BCSPG-2a and BCSPG-2b were isolated by the
alkaline
The CS chains of BCSPG-2a and BCSPG-2b, CS-2a and CS-2b, fractionated
by DEAE-Sepharose chromatography, were recovered by pooling the
fractions as shown in Fig. 3. The molecular sizes of the CS chains were
assessed by chromatography on a Sepharose CL-6B column calibrated with
glycosaminoglycans of known molecular weights (50). The CS-2a and CS-2b
were eluted as single symmetrical peaks, indistinguishable from one
another, with an estimated molecular weight of ~60,000 (not shown).
Hexosamine compositional analysis indicated that both CS-2a and CS-2b
have predominantly N-acetylgalactosamine (Table II). HPLC
analysis of the unsaturated disaccharides released by the digestion
with chondroitinase ABC revealed that CS-2a and CS-2b, obtained from
CSPGs of various placentas, consist of, respectively, 2-3% and
9-14% 4-sulfated and 97-98% and 86-91% nonsulfated disaccharide repeating units. Together, these results suggest that CS-2a and CS-2b
are similar in molecular sizes but differ in the levels of sulfate groups.
To determine the distribution of sulfate groups, the CS chains of
placental CSPG fractions were digested with S. dysgalactiae hyaluronidase, an endo-
Since the oligosaccharide Fractions I and II (Fig. 4) obtained by the
digestion of the CS chains of placental CSPGs with S. dysgalactiae hyaluronidase contained only 20-28% sulfated
disaccharides, it is possible that the oligosaccharides are a mixture
of sulfated and nonsulfated species. Because the placental CS chains
were available only in limited amounts, the specificity of the enzyme, under the conditions used for the placental CS chains, was studied using a commercially available chondroitin, which consisted of ~4%
6- and 4-sulfated disaccharide moieties and ~96% nonsulfated. Bio-Gel P-6 chromatography showed that the enzyme degraded ~90% of
chondroitin into disaccharides and ~10% (~6% in fractions 20-25 and ~4% in fractions 26-35; the pattern was similar to that of CS-2a in Fig. 4, not shown) into a mixture of oligosaccharides with >5
disaccharide repeating units. The oligosaccharides in fractions 20-25
and 26-35 had 12-15% 6-sulfated and 6-8% 4-sulfated disaccharide
moieties and the remainder nonsulfated. Ion exchange chromatography on
DEAE-Sepharose using stepwise elution with NaCl and compositional
analysis of the fractions showed the presence of predominantly sulfated
oligosaccharides. These results suggested that S. dysgalactiae hyaluronidase readily degrades the nonsulfated regions of CS and slowly acts at the sulfated domains, forming partially sulfated oligosaccharides.
To determine the exact sizes of the larger oligosaccharides formed by
the digestion of CS-2a and CS-2b with S. dysgalactiae hyaluronidase, Fractions I and II (see Fig. 4) were analyzed by polyacrylamide gel electrophoresis (Fig.
5). In both the cases (CS-2a and CS-2b),
the oligosaccharides in Fractions I and II ranged in size from 8 to 14 and from 6 to 11 disaccharide units, respectively (Fig. 5). These
results indicate that the sulfate groups in the CS chains of BCSPG-2a
and BCSPG-2b are clustered in CS chain motifs composed of 6-14
disaccharide units.
Inhibition of P. falciparum IRBC Adherence to the Placental CSPGs
by the CS Chains of Placental CSPGs and Their
Oligosaccharides--
The intact CS chains, CS-2a and CS-2b, and the
oligosaccharides obtained by the digestion of CS chains with
S. dysgalactiae hyaluronidase (see Fig. 4) were assessed for
their ability to inhibit the adhesion of IRBCs to placental CSPGs. The
CS chains as well as their oligosaccharide Fractions I and II inhibited the IRBC adhesion to BCSPG-2a and BCSPG-2b in a
dose-dependent manner (Fig.
6). Consistent with the prediction based
on the level of 4-sulfated disaccharide clustered domains (see Table
II), the inhibition of IRBC binding by CS-2b was 2-3-fold higher than
that by CS-2a. Further, oligosaccharide Fractions I and II, obtained from CS-2a and CS-2b, were significantly better inhibitors than the
corresponding intact chains (Fig. 6 and data not shown). The inhibitory
capacity of Fractions I and II of CS-2a was only marginally lower than
those of Fractions I and II from CS-2b. The inhibitory ability of the
oligosaccharide from CS-2b was comparable with that of C4S, with 36%
4-sulfated disaccharide residues prepared by the regioselective
6-O-desulfation of bovine trachea C4S/C6S copolymer
(Fig. 6). In contrast, the C4S with 3 and 11% 4-sulfate groups
prepared by solvolytic desulfation of a fully 4-sulfated sturgeon
notochord C4S were markedly less inhibitory compared with the CS chains
of BCSPG-2a, which have only 2-3% sulfated disaccharide repeating
units. These results agree with the finding described above that the
sulfate groups in the CS chains of BCSPG-2a and BCSPG-2b are clustered
in CS chain motifs consisting of 6-14-disaccharide repeating units.
The results also agree with our previous finding that optimal binding
of IRBCs requires a 6-mer motif in which two of six disaccharide
repeating units sulfated on C-4 of N-acetylgalactosamine. Since the oligosaccharides obtained by the digestion of CS-2a and CS-2b
with S. dysgalactiae hyaluronidase are larger than six disaccharide repeating units, the sulfate content in the 6-mer IRBC-binding motif is likely to be ~30% or more. This satisfies the
level of 4-sulfation required for optimal IRBC binding. These results
indicate that the sulfate groups in the CS chains of placental CSPGs
are uniquely distributed and provide the necessary structural elements
for the efficient adhesion of IRBCs.
Recently, we showed that unusually low sulfated CSPGs of the
intervillous spaces of human placenta can efficiently support the
adherence of P. falciparum IRBCs in the placenta (50). The IRBC binding involves critical interactions by both 4-sulfated and
nonsulfated repeating units of the CS chains, and the optimal binding
requires a 6-mer motif with two 4-sulfated disaccharide repeating units
(51). In this study, the placental intervillous space CSPGs and the
structural features of their CS chain motifs that bind IRBCs were
investigated. The new findings are as follows. 1) The placental CSPGs
are a mixture of two distinct populations, which are similar with
regard to proteoglycan type and sizes but different with respect to the
levels of sulfation. 2) The majority of the sulfate groups in the
glycosaminoglycan chains of both CSPGs are clustered in size-defined
domains that comprised 6-14 disaccharide repeating units. Within these
domains, about 20-28% of the disaccharide repeating units are
4-sulfated, whereas the other regions of the CS chains have essentially
no sulfate groups. 3) The oligosaccharides corresponding to
these sulfate group-rich domains can efficiently inhibit IRBC binding
to placental CSPGs. Thus, our data define, for the first time, the
distribution of sulfate groups in the CS chains of the low sulfated
placental CSPGs and establish that P. falciparum uses the
sulfate group-clustered domains of the CS chains for IRBC adhesion in
the placenta.
The data show that the low sulfated CSPGs of the intervillous spaces of
placenta consist of two major, differentially sulfated proteoglycan
populations, BCSPG-2a and BCSPG-2b. These CSPG populations resemble one
another very closely with respect to their physical properties,
including hydrodynamic size, buoyant density, and the core protein
type, proportion, and composition. The CSPG species are also very
similar with respect to the number and molecular sizes of the CS
chains, but they differ significantly in the sulfate contents of the CS
chains (2-3% and 9-14%, respectively, depending on placentas).
A CSPG population with 2% sulfate groups in the CS chains has
been previously identified as a minor CSPG in the placental intervillous spaces (50). This CSPG species, designated as BCSPG-1, accounted for only 6-7% of the total CSPGs and was eluted from the
DEAE-Sephacel column as a distinct peak, completely separated from the
remainder of the intervillous space CSPG molecules, BCSPG-2 (50). A
comparison of the data from this study with those of the previous study
indicates that BCSPG-1 is very similar to BCSPG-2a by all criteria
including overall charge density, core protein types, and the content
of sulfate groups in CS chains. Therefore, BCSPG-1 and BCSPG-2a appear
to represent similar CSPG populations. The results of the present study
clearly show that these proteoglycan species represent 40-65% of the
total CSPGs of the placental intervillous spaces. Based on the results
of the previous study, it is evident that only a portion of BCSPG-2a
aggregates with the low amount of hyaluronic acid present in the
intervillous spaces and eluted as a distinct peak during DEAE-Sephacel
chromatography (50).
The results of this study conclusively establish that the majority of
sulfate groups of the CS chains of the CSPGs of placental intervillous
spaces are clustered in size-defined motifs, consisting of 6-14
disaccharide repeating units. The sizes of the oligosaccharides formed
by the digestion of the CS chains of BCSPG-2a and BCSPG-2b with
S. dysgalactiae hyaluronidase support this conclusion. The enzyme converted >90% of the CS chains of BCSPG-2a and ~80% of the
CS chains of BCSPG-2b into predominantly nonsulfated disaccharides and
a minor amount of tetrasaccharides; only a small portion of the latter
appears to be 4-sulfated (Table III). Based on the specificity of the
enzyme (degrades chondroitin but not chondroitin sulfate), the amount
of sulfate groups in the tetrasaccharide fraction must represent the
level of 4-sulfated disaccharide units that occur as single residues
randomly distributed in relatively large nonsulfated regions of the CS
chains. This accounts for 3-5% of the total sulfate groups in the CS
chains of BCSPG-2a and BCSPG-2b. These results clearly show that the
majority of the sulfate groups in CS chains of placental CSPGs are
clustered in domains consisting of 6-14 disaccharide repeating units.
Our data demonstrate that P. falciparum IRBCs bind to the
sulfate group-clustered motifs but not to the nonsulfated regions of
the CS chains of placental intervillous space CSPGs. Consistent with
this conclusion, chondroitin, the nonsulfated glycosaminoglycan, has no
inhibitory effect on the adhesion of IRBCs to placental CSPGs. The C4Ss
with 3 and 11% 4-sulfate content, prepared by partial desulfation of
fully sulfated sturgeon notochord C4S, could only marginally inhibit
the IRBC binding (Fig. 6). In these C4Ss, the majority of the sulfate
groups are likely to be distributed as single residues separated by a
number of nonsulfated moieties. In contrast, the CS chains of BCSPG-2a
and BCSPG-2b, containing clustered sulfate residues, are severalfold
more inhibitory. The oligosaccharides corresponding to the sulfate
group-clustered domains (Fractions I and II), obtained by the S. dysgalactiae hyaluronidase digestion of the CS chains of the
placental CSPGs, are superior inhibitors compared with the
corresponding intact chains (Fig. 6). This is especially evident in the
case of the CS chains of BCSPG-2a. This is not surprising, because the
major portion of CS-2a and significant amounts of CS-2b have no sulfate groups, and thus these portions are not able to bind IRBCs. Therefore, on weight basis, the oligosaccharide Fractions I and II should be much
more active in inhibiting IRBC binding than the intact CS chains, CS-2a
and CS-2b, from which they were obtained.
We have previously shown that two of the six disaccharide residues of
the CS 6-mer motif must be 4-sulfated for optimal IRBC binding (51).
This translates to 33% 4-sulfated residues in the oligosaccharide. The
level of 4-sulfate determined in this study for the sulfate
group-clustered domains of the CS chains of placental CSPGs is
20-28%. However, it should be noted that the sizes of the predominant
oligosaccharides (Fractions I and II in Fig. 4) formed by the action of
S. dysgalactiae hyaluronidase are larger than the 6-mer, the
minimum chain length required for optimal inhibition. Therefore, it is
possible that the 6-mer binding motifs in the CS chains of placental
CSPGs have the level of 4-sulfation required for optimal IRBC adhesion.
Although the capacity of the CS chains of BCSPG-2a to inhibit the IRBC
binding to placental CSPGs is relatively low, immobilized BCSPG-2a
efficiently binds IRBCs. When coated on plastic plates, the density of
IRBC binding by BCSPG-2a is remarkably similar to that by BCSPG-2b.
Further, the CS chains of BCSPG-2b contain significantly higher
4-sulfate, and the inhibitory capacities of these chains were 4-5-fold
higher compared with those of BCSPG-2a. Based on the sulfate contents,
size of the CS chains, and requirements of two sulfate groups per 6-mer
IRBC-binding motif, it can be calculated that the CS chains of BCSPG-2a
and BCSPG-2b have about 1-2 and 4-10 binding sites, respectively.
Thus, the difference in the inhibitory capacities of the CS chains of
these proteoglycan species agree with that expected based on the number
of available IRBC binding sites in the CS chains. However, when IRBC
binding and inhibitory data considered together, it appears that the
density of IRBCs adhered to CSPG-coated plastic plates merely
represents the number of cells bound and does not reflect the binding
strength. When IRBC binding affinity to immobilized BCSPG-2a and
BCSPG-2b was measured by adhesion inhibition using C4S with 36%
4-sulfate, marginal differences were observed, only at very low
inhibitor concentrations (Fig. 6). This was also the case with C4S
6-mer having 36% 4-sulfate or oligosaccharide Fractions I and II from the CS chains of BCSPG-2b. Therefore, it appears that, when CSPGs are
immobilized on solid surface, because of clustering of molecules, the
CS chains of BCSPG-2a, despite containing fewer binding sites, can
provide a sufficient number of binding sites for IRBCs using adjacent
CS chains. Thus, they are able to bind IRBCs with capacity almost
similar to that of the CS chains of BCSPG-2b. Alternatively, it
is possible that because of the steric constraints due to the one-dimensional disposition of IRBCs with respect to the immobilized CS
chains, IRBCs might not be able to interact with all available binding
sites in the CS chains of BCSPG-2b. Therefore, the CS chains of both
proteoglycan fractions could provide a similar number of binding sites
to interact with IRBCs, and thus the CS chains of both BCSPG-2a and
BCSPG-2b bind with almost comparable capacity. In solution phase, on
the other hand, the disposition of IRBCs to binding by CS chains is
multidirectional, which might allow for effective interactions with all
of the available binding sites in the CS chains. Therefore, the CS
chains of BCSPG-2b can inhibit the IRBC adhesion more effectively than
the CS chains of BCSPG-2a. However, in the placenta, since the CSPGs
are immobilized in the intervillous spaces, it is likely that both
proteoglycans are equally efficient in binding IRBCs.
The clustering of almost all of the sulfate groups present in CS chains
of the placental CSPGs in size-defined oligosaccharide domains suggests
a well regulated mechanism of biosynthesis. This clustering of sulfate
groups in the CS chains of the CSPGs might have important roles in the
function of placenta. Clearly, P. falciparum has evolved to
exploit these sulfated group-clustered motifs of the CS chains for
adherence in the placenta. Recent studies have demonstrated that
versican, a high molecular weight CSPG expressed in a variety of cells
and tissues, including fibroblasts, arterial smooth muscle cells,
keratinocytes, kidney, brain, aorta, and skin, can bind L-selectin,
P-selectin, CD44, and a number chemokines (64-66). The last class of
proteins include secondary lymphoid chemokines, macrophage inflammatory
protein, stromal cell-derived factor 1, monocyte chemoattractant
protein, regulated on activation normal T-cell-expressed and secreted
proteins,
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Isolation and fractionation of the
intervillous space CSPGs of human placenta by DEAE-Sepharose
chromatography. The isotonic buffer extracts of placentas were
chromatographed on DEAE-Sephacel columns (2.5 × 22 cm) using a
linear gradient of 0.1-0.9 M NaCl in 50 mM
NaOAc, pH 5.5, as described previously (50). The uronic acid-containing
fractions corresponding to the major CSPG fraction (designated BCSPG-2
in Ref. 50) were pooled, dialyzed, and chromatographed on
DEAE-Sepharose (2.5 × 17 cm) in 50 mM NaOAc, 0.15 M NaCl, pH 5.5. The column was washed with the same buffer,
and the bound CSPGs were eluted with a linear gradient of 0.15-0.55
M NaCl in 50 mM NaOAc, pH 5.5. 10-ml fractions
were collected, absorptions at 280 and 260 nm were measured, and
aliquots were assayed for uronic acid (54). The CSPG peaks were pooled
as indicated by horizontal bars. Note that the
previously reported minor BCSPG-1 (50), which represents 6-7% of the
total CSPGs of the intervillous spaces of the placenta, was not studied
here.
= 1.43 g/ml) separating from protein and nucleic acid contaminants (not shown). The sedimentation patterns of BCSPG-2a and BCSPG-2b were indistinguishable from each other and very similar to that previously reported for the total BCSPG-2 (50). In both cases, fractions of
the gradient containing significant levels of CSPGs (
= 1.35-1.5 g/ml density regions) were pooled, and the material was recovered.
Yield and composition of the differentially sulfated populations of the
CSPGs of intervillous spaces of human placenta
Composition of the CS chains of differentially sulfated CSPGs of
intervillous spaces of human placenta
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Fig. 2.
Adhesion of P. falciparum
IRBCs to the differentially sulfated CSPG fractions of human
placenta and inhibition of adhesion by partially sulfated C4S. The
solutions of purified CSPG fractions in PBS, pH 7.2, were coated at the
indicated concentrations as 4-mm circular spots on plastic Petri dishes
overnight at 4 °C. The nonspecific sites were blocked with 2% BSA
for 2 h at room temperature. A, the blocked spots were
overlaid with a 2% suspension of IRBCs in PBS, pH 7.2, for 40 min at
room temperature. The unbound cells were washed, and the bound cells
were fixed with 2% glutaraldehyde, stained with Giemsa, and counted
under light microscopy. Assays were carried out three times each in
duplicate. The spots coated only with PBS and the uninfected RBCs
layered on CSPG-coated spots were used as negative controls. Shown is
the dose-dependent binding of IRBCs to CSPG-coated plates
(mean of all three assays). , total BCSPG-2;
, BCSPG-2a;
,
BCSPG-2b. B, a 2% suspension of IRBCs in PBS, pH 7.2, were
incubated with various concentrations of the indicated C4S for 30 min
at room temperature and then overlaid onto the CSPG-coated spots. After
40 min at room temperature, the unbound cells were washed, and the
bound cells were fixed, stained with Giemsa, and measured using light
microscopy. Assays were performed two times each in duplicate, and mean
values were plotted.
and
, inhibition of IRBC binding to
BCSPG-2a and BCSPG-2b, respectively, by C4S with 36% 4-sulfate
prepared by regioselective 6-O-desulfation of bovine trachea
C4S/C6S copolymer;
and
, inhibition of IRBC binding to
BCSPG-2a and BCSPG-2b, respectively, by 6-mer prepared from C4S
with 36% 4-sulfate.
-elimination of the proteoglycans followed by
chromatography on DEAE-Sepharose columns (Fig.
3). In the case of BCSPG-2a, about 68%
of CS chains (designated as CS-2a) eluted as a single peak at 0.28 M NaCl, and the remainder eluted as heterogeneous peaks at
a mean NaCl concentration of 0.36 M. In the case of
BCSPG-2b, on the other hand, only 28% of the CS chains eluted at 0.28 M NaCl, and the remainder (CS-2b) eluted as a single peak
at 0.37 M NaCl. The presence of additional CS population
(Fig. 3) in both BCSPG-2a and BCSPG-2b was probably due to the
overlapping separation of the CSPG fractions on
DEAE-Sepharose columns (see Fig. 1). Thus, these results
demonstrate that BCSPG-2a and BCSPG-2b carry differentially sulfated CS
chains. The presence of two intervillous space CSPGs with distinctively
sulfated CS chains was also evident from the elution pattern of the CS
chains released by alkaline
-elimination of the total placental
intervillous space CSPGs (BCSPG-2) on the DEAE-Sepharose column (see
inset in Fig. 3).
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Fig. 3.
DEAE-Sepharose chromatography of CS chains of
the placental CSPG fractions. The CS chains (1.5-2.0 mg) released
by NaOH/NaBH4 -elimination of the purified placental
CSPGs, BCSPG-2a and BCSPG-2b, were chromatographed on
DEAE-Sepharose columns (1 × 10 cm) equilibrated with 20 mM Tris-HCl, pH 7.8. The columns were washed with 20 mM Tris-HCl, 0.15 M NaCl, pH 7.8, and the bound
glycosaminoglycans eluted with a linear gradient of 0.15-0.6
M NaCl in 20 mM Tris-HCl, pH 7.8. Fractions
(2.5 ml) were collected, and 50-µl aliquots were analyzed for uronic
acid (530 nm). The CS chain fractions were pooled as shown by the
horizontal bars and recovered for further
studies.
, the CS chains (CS-2a) of BCSPG-2a;
, the CS chains
(CS-2b) of BCSPG-2b. The inset shows the elution pattern of
the CS chains obtained from the total BCSPG-2 (corresponding to
fractions 23-49 in Fig. 1) from another placenta.
-N-acetylhexosaminyl lyase. This
enzyme degrades hyaluronic acid and chondroitin, but not chondroitin sulfate, to produce disaccharides with nonreducing 4,5-unsaturated uronic acid residue (57). Chromatography of the enzyme digests on
Bio-Gel P-6 indicated that ~95 and 80%, respectively, of CS-2a and
CS-2b were degraded predominantly into disaccharides with minor amount
of tetrasaccharides. The remainders of the CS chains were converted
into oligosaccharides larger than decasaccharide (5-mer) (Fig.
4). The disaccharide composition of the
oligosaccharide fractions (see Fig. 4), determined by HPLC after
digestion with chondroitinase ABC, are given in Table
III. In both cases, the larger
oligosaccharides (Fractions I and II), formed by the action of S. dysgalactiae hyaluronidase, contained significant levels of
sulfate groups. Fractions I and II from CS-2a comprised 20-22% 4-sulfated and 78-80% nonsulfated disaccharide repeating units, whereas Fractions I and II from CS-2b had 25-28% 4-sulfated and 72-75% nonsulfated disaccharide repeating units. The tetrasaccharides (Fraction III) from CS-2a were ~97% nonsulfated and ~3% sulfated, whereas those from CS-2b were ~95% nonsulfated and ~5% sulfated. As expected, based on the specificity of S. dysgalactiae
hyaluronidase, the disaccharides (Fraction IV), formed by the action of
this enzyme, were exclusively nonsulfated. Interestingly, however, hexa- to decasaccharides (3- to 5-mers) were not formed in appreciable amounts from either CS-2a or CS-2b. These results indicate that, in the
CS chains of both BCSPG-2a and BCSPG-2b, the majority of sulfate groups
are clustered such that these regions have 20-28% or more of
4-sulfated disaccharide repeating units. The yields of the larger
oligosaccharides (Fractions I and II) from CS-2a and CS-2b should
correspond to the number of sulfate group-rich domains in the CS
chains.
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Fig. 4.
Size exclusion chromatography of the
oligosaccharides formed by the digestion of the CS chains of the
placental CSPG fractions with S. dysgalactiae
hyaluronidase. CS-2a and CS-2b, obtained as shown in Fig. 3,
were treated with S. dysgalactiae hyaluronidase in 100 mM phosphate buffer, pH 6.2, containing 0.02% BSA at
37 °C for 24 h. The digests were chromatographed on Bio-Gel P-6
columns (1 × 47 cm) in 0.1 M pyridine, 0.1 M acetic acid. Fractions of 0.67 ml were collected, and
aliquots were assayed for uronic acid content (530 nm). A,
elution pattern of the oligosaccharides formed from CS-2a;
B, elution pattern of the oligosaccharides formed from
CS-2b. The oligosaccharide fractions were pooled as indicated by the
horizontal bars. The oligosaccharides obtained by
the digestion of chondroitin under the above conditions were similarly
chromatographed (not shown). The pattern was similar to that observed
for the enzyme digestion products of CS-2a. The oligosaccharides larger
than five disaccharide moieties (fractions 20-25 and 26-35) were
pooled and analyzed. The elution position of blue dextran
(BD) and glucose (Glc) and the oligosaccharides
(number of disaccharide units) formed by the digestion of bovine C4S
with testicular hyaluronidase are indicated.
The yield and disaccharide compositions of the oligosaccharides
corresponding to the sulfate group-rich regions in the CS chains of
the placental CSPGs
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Fig. 5.
Electrophoresis of the oligosaccharides
formed by the digestion of the CS chains of the placental CSPG
fractions with S. dysgalactiae
hyaluronidase. The oligosaccharides obtained by the
treatment of CS-2a and CS-2b with S. dysgalactiae
hyaluronidase (see Fig. 4) were dissolved in 100 mM Tris
base, 100 mM boric acid, 2 mM EDTA, pH 8.3, containing 5% glycerol. The solutions were electrophoresed on 10%
polyacrylamide gels (15 × 16 cm). The gels were stained with
0.03% Alcian Blue in 25% ethanol, 10% acetic acid for 4 h and
destained with 25% ethanol, 10% acetic acid and stained with silver.
Lane 1, oligosaccharide mixtures obtained by the treatment
of bovine C4S with testicular hyaluronidase as described in Ref. 51;
lane 2, the intact CS chains (CS-2a; 9 µg) of BCSPG-2a;
lane 3, the oligosaccharide FI (3 µg) from CS-2a;
lane 4, the oligosaccharide FII (4 µg) from CS-2a;
lane 5, the intact CS chains (CS-2b; 5 µg) of BCSPG-2b;
lane 6, the oligosaccharide FI (6 µg) from CS-2b;
lane 7, the oligosaccharide FII (8 µg) from CS-2b. The
numbers on the left indicate the size (the number
of disaccharide repeating units) of various oligosaccharides determined
by Bio-Gel P-6 chromatographic pattern (Fig. 4; see Ref. 67) and with
respect to the mobility of the C4S hexasaccharide purified to
homogeneity and identified by mass spectrometry. Note that the di- and
tetrasaccharides do not stain on the gel (67).
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Fig. 6.
Inhibition of the IRBC adherence by the CS
chains of placental CSPG fractions and oligosaccharides of the CS
chains. The plastic Petri plates were coated overnight with 200 ng/ml solution (15 µl) of BCSPG-2b in PBS, pH 7.2, and blocked with
2% BSA as outlined in the legend to Fig. 2. The CS chains and their
oligosaccharides were incubated at the indicated concentrations with a
2% IRBC suspension in PBS, pH 7.2, for 30 min at room temperature and
then overlaid onto the CSPG-coated spots. After a 40-min incubation at
room temperature, the unbound cells were washed. The bound cells were
fixed, stained with Giemsa, and measured using light microscopy. Shown
is the inhibition of IRBC binding to BCSPG-2b-coated plates. ,
CS-2a;
, CS-2b;
, oligosaccharide FI from CS-2b;
,
oligosaccharide FII from CS-2b;
, bovine trachea C4S/C6S copolymer;
×, C4S with 36% 4-sulfate prepared by the regioselective
6-O-desulfation of bovine trachea C4S followed by
DEAE-Sepharose fractionation;
, the CS chains obtained from total
BCSPG-2 (nonfractionated mixture of BCSPG-2a and BCSPG-2b);
and
, C4Ss with 3 and 11% 4-sulfate prepared by solvolytic partial
desulfation of sturgeon notochord C4S (51);
, chondroitin. The
inhibition capacities of oligosaccharide Fractions I and II from CS-2a
were either similar or only marginally lower compared with those from
CS-2b (not shown). Inhibition of IRBC binding to BCSPG-2a was similar
to that observed in the case of BCSPG-2b, except that a 10-20% higher
inhibition was observed for each inhibitor used. Note that the
inhibitory capacity of the 6-mer prepared by the testicular
hyaluronidase digestion of C4S with 36% 4-sulfated disaccharide was
similar to that by the intact C4S with 36% 4-sulfate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-interferon-inducible protein 10, platelet factor 4, and
liver and activation-regulated chemokine (64-66). It has been shown
that CS-dependent binding of these proteins to versican is
important in leukocyte trafficking, signal transduction to trigger
inflammatory responses, and the regulation of chemokine functions
(64-66). These proteins have been shown to bind different structural
features within the CS chains of versican. CD44, a hyaluronic
acid-binding protein, was found to bind to the nonsulfated regions of
the CS chains of versican, whereas L- and P-selectins and various
chemokines have been shown to interact with motifs consisting of
specific sulfated residues (66). Therefore, analogous to this, the
nonsulfated regions of the CS chains of placental CSPGs may bind CD44,
whereas sulfate group-clustered regions support binding of other
functional proteins. The placental CSPGs have been identified as
members of the aggrecan family of proteoglycans (50). Since versican
and aggrecan exhibit many similarities with respect to both core
protein and CS chain structural features, it is possible that the CS
chains of placental CSPGs also have diverse biological functions.
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ACKNOWLEDGEMENT |
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We thank Dr. V. P. Bhavanandan of our Department for critical reading of the manuscript.
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FOOTNOTES |
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* The study was supported by NIAID, National Institutes of Health, Grant AI45086.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Pennsylvania State University College of
Medicine, Milton S. Hershey Medical Center, 500 University Dr.,
Hershey, PA 17033. Tel.: 717-531-0992; Fax: 717-531-7072; E-mail:
gowda@psu.edu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211015200
2 R. N. Achur and D. C. Gowda, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: IRBCs, infected red blood cells; CS, chondroitin sulfate; CSPG, chondroitin sulfate proteoglycan; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; BSA, bovine serum albumin; CsBr, cesium bromide; PBS, phosphate-buffered saline; 1-mer, 2-mer, 3-mer, 4-mer, 5-mer, and 6-mer, size of C4S oligosaccharides with 1, 2, 3, 4, 5, and 6 disaccharide repeating units, respectively; EMP1, erythrocyte membrane protein 1; HPLC, high pressure liquid chromatography.
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REFERENCES |
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