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J. Biol. Chem., Vol. 277, Issue 25, 22438-22446, June 21, 2002
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From the
Received for publication, November 29, 2001, and in revised form, April 12, 2002
An important characteristic of malaria parasite
Plasmodium falciparum-infected red blood cells (IRBCs) is
their ability to adhere to host endothelial cells and accumulate in
various organs. Sequestration of IRBCs in the placenta, associated with
excess perinatal and maternal mortality, is mediated in part by
adhesion of parasites to the glycosaminoglycan chondroitin sulfate A
(CSA) present on syncytiotrophoblasts lining the placental blood
spaces. To define key structural features for parasite interactions, we isolated from CSA oligosaccharide fractions and established by electrospray mass spectrometry and high performance liquid
chromatography disaccharide composition analysis their differing chain
length, sulfate content, and sulfation pattern. Testing these defined oligosaccharide fragments for their ability to inhibit IRBC adhesion to
immobilized CSA revealed the importance of non-sulfated disaccharide units in combination with 4-O-sulfated disaccharides
for interaction with IRBCs. Selective removal of
6-O-sulfates from oligo- and polysaccharides to increase
the proportion of non-sulfated disaccharides enhanced activity,
indicating that 6-O-sulfation interferes with the
interaction of CSA with IRBCs. Dodecasaccharides with four or five
4-O-sulfated and two or one non-sulfated disaccharide units, respectively, comprise the minimum chain length for effective interaction with IRBCs. Comparison of the activities of CSA and CSB
oligo- and polysaccharides with a similar sulfation pattern and content
achieved from partial desulfation demonstrated that glucuronic acid
rather than iduronic acid residues are important for IRBC binding.
An important pathological characteristic of red blood cells
infected by the malaria parasite Plasmodium falciparum is
their ability to adhere to host endothelial cells and accumulate in various organs. During pregnancy, the accumulation of infected red
blood cells (IRBCs)1 in the
placenta is a key feature of infection and is associated with adverse
outcomes and excess perinatal and maternal mortality (1, 2). Several
studies (3-6) in the past few years suggest that sequestration of
IRBCs in the placenta is mediated in part by adhesion of parasites to
the glycosaminoglycan chondroitin sulfate A (CSA) present on
syncytiotrophoblasts lining the placental blood spaces. Recently we
have reported that hyaluronic acid (HA) can also support the adhesion
of IRBCs in vitro and appears to be another receptor for
parasite sequestration in the placenta (7, 8). HA is non-sulfated and
is the simplest member of the glycosaminoglycan family. Because there
are thought to be no other modifications in the carbohydrate chain, the
structural requirement of HA for interaction with IRBC can be readily
deduced, and the major feature is believed to be a minimum chain length of 12 monosaccharide residues (7, 8). However, understanding the
molecular interactions involved between CSA and IRBC is more difficult
due to the greater complexity of chondroitin sulfate (CS) chains.
CS chains comprise repeating disaccharide units of
4-O-substituted hexuronic acid (HexA) To further define key structural features that influence adhesion of
IRBCs, we have isolated and characterized a range of variously sized
oligosaccharide fragments from CSA polysaccharides, including fully
sulfated components and undersulfated minor components, and tested
their ability to competitively inhibit parasite adhesion to CSA.
Selective desulfation of oligo- and polysaccharides was performed to
directly compare the influence of 6-O-sulfated disaccharide units and non-sulfated units on adhesion, and undersulfated CSB oligo-
and polysaccharides were prepared to evaluate the relative importance
of the form of hexuronic acid (GlcUA or IdoUA).
Preparation and Fractionation of CS Oligosaccharide
Fragments--
Chondroitin sulfate A (from bovine trachea, Sigma;
average molecular mass: 45,500 kDa, based on low angle laser light
scattering), B (from porcine intestinal mucosa, Sigma), and C (from
shark cartilage, Sigma) were partially depolymerized by controlled
digestion with chondroitin lyase ABC (EC 4.2.2.4, from Proteus
vulgaris, Sigma) essentially as described previously (12, 13). In
brief, 1 g of CSA was incubated with 2 units of lyase. Reaction
products were desalted on a short G-10 column and fractionated on a
Bio-Gel P-6 column (1.6 × 90 cm) with elution by ammonium acetate
(0.2 M) at a flow rate of 15 ml/h. The major fractions F1
to F10 were collected and detected by electrospray mass spectrometry
(ES-MS) to be the di- to eicosaccharide fragments. CSB oligosaccharide fragments were similarly prepared but on a smaller scale (100 mg of
polysaccharide and 0.5 units of chondroitin lyase ABC in 4 ml of solution).
CSA deca-, dodeca-, tetradeca-, and hexadecasaccharide fractions F5 to
F8 were further subfractionated on a short cartridge column (HiTrap
Q-Sepharose HP, 1 ml, Amersham Biosciences) of strong anion exchange
(SAX) with detection at UV 232 nm. Elution was carried out with a
linear gradient of NaCl (solvent A, 0.2 M NaCl; solvent B,
1.5 M NaCl; pH 3.5). The deca-, dodeca-, and tetradecasaccharide fractions were chromatographed by a gradient of
0-40% B in 20 min, whereas the hexadecasaccharide fraction was from
10 to 50% B in 20 min at a flow rate of 1 ml/min. The subfractions
were collected and desalted on a short G-10 column before
lyophilization. CSB tetradecasaccharides were fractionated in a similar manner.
The undersulfated CSA dodecasaccharide fraction F6C was further
chromatographed by SAX-HPLC on a Spherisorb S5-SAX column (4.6 × 250 mm, Waters, Milford, MA). A gradient of NaCl (solvents A and B as
above), 20-60% B in 45 min, was used at a flow rate of 1 ml/min with
detection at UV 232 nm. The SAX-HPLC fractions were desalted and
freeze-dried before further fractionation by normal phase HPLC on an
aminopropyl silica (APS) column (APS-2, 4.6 × 250 mm, Hypersil,
Runcorn, England). A linear gradient of NaH2PO4
(solvent A, 0.1 M; and solvent B, 1.0 M;
30-50% B in 40 min) was used to elute SAX-HPLC fractions F6C-2 and
F6C-3. Identification of the components present in each
subfractions was carried out by ES-MS, and quantitation was by
carbazole assay for glucuronic acid content using
D-glucurono-6,3-lactone as a standard (14).
De-6-O-sulfation of CS Poly- and
Oligosaccharides--
De-6-O-sulfation was essentially as
described (15) but with some modifications. CS polysaccharides (5 mg)
or oligosaccharides (200 µg) were converted into their pyridinium
salts by passing through a short column of cation exchange resin
(AG50W-X8, H-form) and washing with H2O. The eluants were
added to a small amount of pyridine and freeze-dried. To the residue of
the ammonium salt of the polysaccharide was added 1 ml of anhydrous
pyridine and 200 µl of
N,O-bis(trimethylsilyl)acetamide (BTSA).
For oligosaccharides, 100 µl of pyridine and 20 µl of BTSA were
used. The mixture was heated to 80 °C for 4 h. After cooling,
the excess reagent and the silyl ester were decomposed by the addition
of H2O. The bulk of liquid was evaporated under reduced
pressure. The residual trimethylsilyl groups were hydrolyzed in a 2.5%
NH4F in MeOH solution and removed essentially as described
(16).2
De-6-O-sulfated CSA dodecasaccharide fraction (dF6) and the
subfractions derived from it, dF6A to dF6G containing 0 to 6 sulfates,
respectively, were obtained essentially as described above for CSA
oligosaccharide fragments.
Partial De-4-O-Sulfation of CSB Polysaccharides--
CSB
polysaccharide was converted into the pyridinium salt as described
above. The pyridinium salt was dissolved in 90% Me2SO in
H2O and heated to 80 °C (17). The reaction was stopped
at 40-, 70-, and 100-min intervals (products designated as CSB deS-1, -2, and -3, respectively) by cooling to room temperature and
neutralized with dilute NaOH solution. The mixture was freeze-dried and
co-evaporated with H2O three times. HPLC disaccharide
composition analysis found that 30.1, 45.6, and 50.8% of sulfates were
removed for CSB deS-1, -2, and -3, respectively.
Electrospray Mass Spectrometry--
Electrospray mass
spectrometry was carried out on a Micromass Q-Tof mass
spectrometer (Micromass UK Ltd, Manchester, England) in the
negative-ion mode (18). A cone voltage of 20 volts was used, and the
capillary voltage was kept at 4000 volts. The solvent, acetonitrile,
0.5 mM NH4HCO3, 1:1, was delivered
into the electrospray source by a syringe pump at a flow rate of 5 µl/min. Nitrogen was used as the nebulizing and desolvation gas.
Source temperature was maintained at 80 °C, and desolvation
temperature was maintained at 150 °C. Sample solution (5 µl), typically at a concentration of 10-20 pmol/µl, was injected
using a flow injector. The raw data were processed and transformed into
mass values using the MassLynx data system (Micromass UK Ltd) to derive
the compositions of oligosaccharide fragments in terms of GalNAc, HexA,
and sulfate.
Disaccharide Composition Analysis--
Analysis for disaccharide
composition was essentially as described previously (13). Typically,
polysaccharide (50 µg) or oligosaccharide (10 µg) was dissolved in
5 mM sodium phosphate, pH 7.0, containing 0.2 M
NaCl and digested exhaustively at 37 °C overnight with chondroitin
ABC lyase (2 µl, 5 milliunits in the same phosphate buffer). The
resulting disaccharides (0.5-1 nmol) were separated by SAX-HPLC. A
gradient of NaCl (solvents A and B as above), 0-80% B in 45 min was
used for the elution of disaccharides at a flow rate of 1 ml/min with
detection at UV 232 nm.
Parasitized Erythrocytes--
P. falciparum was
cultured in group O red blood cells suspended in RPMI-HEPES medium at
pH 7.4 supplemented with hypoxanthine (50 µg/ml), NaHCO3
(25 mM), gentamicin (25 µg/ml), and 10% human serum
(v/v) in an atmosphere of 1% O2, 4% CO2, and
95% N2, as previously described (5). Parasite cultures
were synchronized weekly by sorbitol lysis (19).
Parasite line CS2 was used in all assays examining the interaction
between IRBCs and CSA. CS2 was derived from FAF-EA8, a clone of
Brazilian P. falciparum isolate ItG2F6 (20), by selection for adhesion to CSA expressed on the surface of Chinese hamster ovary
cells (3) and to immobilized purified CSA (21). CS2 has been previously
shown to adhere in a specific manner to immobilized purified CSA, to
CSA side chains on the proteoglycan thrombomodulin, and cell surface
CSA in static assays and under conditions of physiologically relevant
flow (3, 10, 21, 22). CS2-infected red blood cells also adhere to
HA (7, 8).
Cytoadherence Assays--
Assays were performed using P. falciparum trophozoite-infected erythrocytes at a parasitemia of
3-7% and hematocrit of 1% in 150-mm diameter plastic Petri dishes
(Falcon 1058; BD PharMingen) as described (5, 10). Receptors used were
CSA from bovine trachea (Sigma) and CD36 purified from platelets (a
gift of M. Berndt, Baker Medical Research Institute, Melbourne,
Australia). Parasites from culture, suspended in RPMI-HEPES containing
10% pooled human serum, pH 6.8, were incubated with inhibitors or control (phosphate-buffered saline) for 10 min at room temperature before testing adhesion. Samples were randomized and coded, and cytoadherence assays were carried out blinded.
Minor Subfractions of CSA 12-mer, 14-mer, and 16-mer Fragments Are
the Most Effective Inhibitors of IRBC Adhesion to
CSA--
Subfractions derived from the deca-, dodeca-, tetradeca-, and
hexadecasaccharide fractions (Fig. 1)
were tested at a concentration of 50 µg/ml for inhibition of IRBC
adhesion to CSA. None of the decasaccharide subfractions (F5A to F5D)
effectively inhibited adhesion (Fig.
2a). Dodecasaccharide
subfractions F6C to F6F (Fig. 2b), tetradecasaccharide
subfractions F7B to F7E (Fig. 2c), and hexadecasaccharide
subfractions (F8B to F8G) (Fig. 2d) substantially inhibited
adhesion at this concentration.
To identify differences in inhibitory activity among these active
subfractions, they were further tested at reduced concentrations. At 10 µg/ml (ranging from 3.1 to 4.9 nmol/ml for F6A to F6F), the minor
subfraction F6C was clearly the most effective inhibitor of adhesion
among the dodecasaccharide subfractions (Fig. 2b), being
more active than the parent fraction F6. A similar pattern of
inhibitory activity was observed among larger oligosaccharides, with
the subfractions F7C, F7D, and F7E derived from tetradecasaccharides and F8C, F8D, and F8E from hexadecasaccharides the most inhibitory when
re-tested at 5 µg/ml (ranging from 1.4 to 2.3 nmol/ml for F7B to F7G)
and 2 µg/ml (ranging from 0.5 to 0.6 nmol/ml for F8B to F8G),
respectively. In both cases, minor oligosaccharide subfractions were
the most active and inhibited adhesion to a greater degree than the
parent oligosaccharide fraction or CSA polysaccharide. The inhibitory
activity of the subfractions increased with increasing chain length;
the minimum concentration of hexadecasaccharide subfraction F8C
required for near complete inhibition of adhesion was severalfold lower
than that for the corresponding dodecasaccharide subfraction F6C.
The Most Inhibitory Subfractions Are Undersulfated--
Analysis
of oligosaccharide subfractions by negative-ion ES-MS (Table
I) showed that the major
subfractions of each Bio-Gel P-6 fractions (F5D, F6E, F7F, and F8F,
Fig. 1) were the fully sulfated CSA oligosaccharides comprising one
sulfate per disaccharide unit. The subfractions eluting after the major
subfractions (F6F, F7G, and F8G) were mainly the fully sulfated higher
oligomers (Table I) that resulted from incomplete size fractionation on size exclusion chromatography. The earlier-eluting subfractions (Fig.
1) were the undersulfated analogues together with some lower oligomers.
The most active dodecasaccharide subfraction F6C contained mainly
12-mer with 4 sulfates and also 10-mer with 4 sulfates at a lower
concentration (Table I). The most effective inhibitors in the
tetradecasaccharide subfractions F7E and F7D were 14-mers with 6 and 5 sulfates, respectively, whereas F7C was analogous to F6C, and its major
component was a 12-mer with 4 sulfates. Similarly, the most active
hexadecasaccharide subfractions F8E, F8D, and F8C all contained
undersulfated 16-mers with 7, 6, and 5 sulfates, respectively. Clearly,
undersulfation is a major structural feature of the active CSA
oligosaccharide fragments in addition to
4-O-sulfation.
HPLC disaccharide composition analysis (Table
II) of selected poly- and
oligosaccharides is in agreement with this conclusion. The difference
in composition between CSA and CSC polysaccharides was not only the
content of 4-O-sulfate and 6-O-sulfate but also the degree of sulfation. CSA contained more than 5% non-sulfated disaccharide units (0S) compared with less than 1% in CSC. For CSA
tetradecasaccharides, the more active subfraction F7D contained 13.3%
0S, and the less active F7F contained only 1.3% 0S, although their 6S
contents were similar.
Twelve Monosaccharide Residues Comprise the Minimum Chain Length
Required for Activity--
When each of the size-homogeneous fractions
was used for inhibition, the CSA tetradecasaccharide fraction was found
to contain the minimum chain length able to almost completely inhibit
binding, whereas the decasaccharide fraction gave only minor
inhibition. Dodecasaccharides exhibited intermediate inhibition. As
shown above, inhibition of IRBC binding to CSA is not only influenced by oligosaccharide chain length but also by sulfate content. It is
important to compare activities of oligosaccharides of different chain
lengths with the same degree of sulfation. At 10 µg/ml, the
dodecasaccharide fraction only gave minor inhibition (<30%), but the
subfraction F6C containing mainly two-undersufated 12-mer (Table I)
showed significant inhibition (>80%, Fig. 2b). Similarly, 14-mer and 16-mer subfractions F7D and F8D, which contained mainly two-undersulfated components, showed significant inhibition even at a
concentration of 5 and 2 µg/ml (Fig. 2, c and
d), respectively. However, with the same degree of
undersulfation, the 10-mer subfraction F5B did not show any inhibitory
activity even at the higher concentration of 50 µg/ml (Fig.
1a).
A similar pattern of activity was observed with one-undersulfated
subfractions. The 14- and 16-mer subfractions F7E and F8E effectively
inhibited adhesion (Fig. 2, c and d), whereas the 12-mer subfraction F6D showed moderate activity (Fig. 2b).
The 10-mer subfraction F5C lacked any inhibitory effect. These data clearly demonstrated that CSA fragments with 12 monosaccharide residues
and an appropriate sulfation pattern comprise the minimum chain length
required for activity.
6-O-Sulfate Interferes with Interaction--
CSA and CSC
polysaccharides were selectively desulfated at the
6-O-position of GalNAc residues. Upon re-testing for
inhibition of parasite adhesion to CSA, de-6-O-sulfated CSA
polysaccharide showed an increase in its inhibitory activity (Fig.
3, a and c), whereas that of de-6-O-sulfated CSC increased to a lesser
extent (Fig. 3a). The reason for the difference in the
increases of activity may be attributed to the difference in the
contents of the disaccharide units 0S and 4S in these desulfated
polysaccharides and/or in the sequence arrangement of the 0S and
4S disaccharides. The de-6-O-sulfated CSA contained 45.3%
0S and 54.7% 4S, whereas the de-6-O-sulfated contained CSC
74.5% 0S and 15.9% 4S (Table II). In addition, the presence of a
significant amount (7.8%) of disaccharide unit 2S (
Two CSA 14-mer subfractions, the more inhibitory F7D and the less
active F7F, were also submitted to regioselective removal of
6-O-sulfates (Table II). De-6-O-sulfation
substantially increased the inhibitory activity of the less active
subfraction F7F but only slightly increased the activity of F7D (Fig.
3, b and c), suggesting that further removal of
6-O-sulfate groups does not increase the abundance of
inhibitory motifs in this fraction. Interestingly,
de-6-O-sulfated F7F was more inhibitory than the de-6-O-sulfated F7D, which again may be accounted for by the
higher ratio of disaccharide units 4S (55.0%) to 0S (44.6%) in the
de-6-O-sulfated F7F subfraction.
Dodecasaccharides with Four or Five 4-O-Sulfates Are the Most
Active--
To gain further information on the effect of sulfation
pattern without the influence of 6-O-sulfate,
de-6-O-sulfated CSA was partially depolymerized, and the
dodecasaccharide fraction was isolated. Further group separation by
anion-exchange chromatography gave seven subfractions, dF6A to dF6G,
containing mainly dodecasaccharides (except dF6A) with zero to six
4-O-sulfates, respectively, as judged by ES-MS analysis
(Table III). Inhibition of IRBC adhesion to CSA by the subfractions was tested at various concentrations (Fig.
4) and clearly demonstrated that CS
12-mers with four or five 4-O-sulfates (subfractions
dF6E and dF6F, respectively) were the most effective inhibitors. The
non-sulfated subfraction (dF6A) and those with one or two
4-O-sulfates (dF6B and dF6C, respectively) were only
partially inhibitory at the maximum concentration tested (50 µg/ml).
The fully sulfated and the trisulfated subfractions (dF6G and dF6D,
respectively) were intermediate in effect. The major component of
subfraction dF6G was fully sulfated 12-mer; however, 1-undersulfated
12-mers were also present, and these would be expected to have
contributed to its inhibitory activity.
GlcUA Is Required for Inhibitory Activity--
The oligosaccharide
inhibitors were prepared from CSA polysaccharide that was shown to
contain no IdoUA residues when analyzed by 1H NMR (Ref. 23;
data not shown). CSB, which contains IdoUA rather than GlcUA, did not
substantially inhibit parasite adhesion to CSA (Fig.
5) despite being predominantly
4-O-sulfated (Table II) as is CSA. However, a lower
concentration of disaccharide unit 0S was present in CSB than CSA
(Table II), and it was therefore not clear if either the presence of
IdoUA or the lower degree of undersulfation accounted for the lower
inhibitory activity of CSB.
To examine the possible influence of uronic acid on parasite adhesion,
CSB tetradecasaccharide fraction F7 was subfractionated according to
sulfate content, and the inhibitory activities of subfractions F7C,
F7D, and F7E were compared with the parent polysaccharide and CSA (Fig.
5a). All the subfractions were relatively non-inhibitory at
5 µg/ml, whereas the undersulfated CSA 14-mers were strongly inhibitory at the same concentration. At a higher concentration of 50 µg/ml CSB, subfraction F7C, which contained 5.3% of disaccharide 0S
(Table II), showed considerable inhibition (82.6%), whereas CSB F7D
(1.9% 0S) and F7E (0.3% 0S) gave moderate inhibition (51 and 46%,
respectively; Fig. 5a).
To enable a more accurate comparison between the inhibitory effects of
GlcUA and IdoUA residues, CSB polysaccharides were partially
de-4-O-sulfated and compared with de-6-O-sulfated
CSA. The three partially desulfated CSB preparations deS-1, -2, and -3 had 0S disaccharide contents of 30.1, 45.6, and 50.8% (Table II),
respectively. When tested at 10 µg/ml, none of the desulfated CSB
samples significantly inhibited parasite adhesion (Fig. 5b), whereas CSA inhibited adhesion by >90%. De-6-O-sulfated
CSA (0S, 45.3%; 4S, 54.7%) almost completely inhibited parasite
adhesion and had a very similar composition to CSB deS-2 and deS-3,
which were not inhibitory. These experiments clearly demonstrate that GlcUA is required for maximum inhibitory activity.
HPLC-fractionated Undersulfated 12-mer Fragments Differ in
Activity--
To investigate if a specific undersulfated sequence is
required for adhesion to IRBCs, the most active CSA dodecasaccharide subfraction F6C, containing mainly 2-undersulfated 12-mers, was further
fractionated by HPLC. Incomplete separation was obtained with a SAX
column (Fig. 6a), and further
chromatography of fractions F6C-2 and F6C-3 was then carried out on an
APS column (Fig. 6, b and c). Due to the high
degree of complexity and despite two different HPLC columns being used
sequentially, the final fractions still contained mixed components.
Fractions with sufficient amounts of oligosaccharides were then tested
for inhibitory activity at 10 and 20 µg/ml. F6C-3e was clearly the
most inhibitory at both concentrations, with the other fractions
demonstrating only a modest inhibitory effect (Fig.
7). Disaccharide composition analysis (Table II) revealed a similar content of 4S disaccharide unit among the
different fractions, but the most active subfraction F6C-3e had the
highest proportion of non-sulfated 0S and a correspondingly lower
proportion of 6S units. This reaffirmed that undersulfation together
with 4-O-sulfation has an important influence on IRBC adhesion. However, it has not yet been possible to determine the sequence order of disaccharide units in each oligosaccharide fragment in fractions to derive a key sequence determinant(s) for activity.
The importance of 4-O-sulfation of CS for the adhesive
interaction with P. falciparum-IRBCs has been previously
demonstrated by comparing the activity of CSA, CSB, and CSC
polysaccharides, CSA and CSC oligosaccharide mixtures, and desulfated
chondroitin (3, 9-11). However, because of the heterogeneous
composition of CS chains, particularly regarding the sulfate content
and sulfation pattern, it remained possible that other structural
features could influence the interaction. The present study indicates
that the minimum motif of CSA for interaction with IRBCs is a sequence of 12 monosaccharide residues formed by 4-O-sulfated and
non-sulfated disaccharide units, that 6-O-sulfation
interferes with the interaction, and that the form of the uronic acid
residues is important, with GlcUA being required for high inhibitory activity.
Initial testing of size-homogenous fractions isolated from CSA digests
indicated the presence of inhibitory sequences among the 12-mer and
higher oligosaccharide fractions (10). Fractionation, on the basis of
sulfate content, of different-sized oligosaccharide fragments together
with determination of sulfate content and sulfation pattern by ES-MS
and HPLC disaccharide-composition analysis revealed the importance of
non-sulfated disaccharide units for the interaction with IRBCs. Among
the subfractions tested for inhibitory activity, clearly 1- and
2-undersulfated oligosaccharides of 12 monosaccharide residues or
larger exhibited substantial inhibitory activity. Higher degrees of
undersulfation were not detected in oligosaccharide fragments from
bovine trachea CSA.
The importance of non-sulfated disaccharides in forming a part of the
parasite adhesive motif was clearly confirmed by selective removal of
6-O-sulfates to increase the proportion of 0S disaccharide units. This increased the inhibitory activity of CSA polysaccharides and oligosaccharides. Inhibition experiments of dodecasaccharide subfractions derived from de-6-O-sulfated CSA further
supported the conclusion that a combination of 4-O-sulfated
and non-sulfated disaccharide units is important, the most active
oligosaccharides being among the one- and two-undersulfated
oligosaccharide sequences.
Our data show that the minimum chain length required for inhibitory
activity is 12 monosaccharide residues, but only when combined with an
appropriate sulfate content and sulfation pattern. None of the
undersulfated CSA 10-mer subfractions showed any inhibition, confirming
the importance of size. Reflecting the significance of size and
sulfation, fully sulfated 12-, 14-, and 16-mer fractions derived from
bovine trachea CSA were relatively non-inhibitory when compared with
their undersulfated equivalents. The inhibition activities of
oligosaccharide subfractions increased with increasing chain length
when measured using mass concentrations (Fig. 2). If molar
concentrations are taken into account, this trend is still apparent and
indicates that additional recognition elements are present in extended
sequences. CSA polysaccharide has an average molecular mass of 45,500 Da, equivalent to 200 monosaccharide residues in length. It is not
surprising that, on a molar basis, the polysaccharide is 13-17 times
more active in inhibition than the 12-, 14-, and 16-mer fractions
(average molecular masses 2675, 3135, and 3594 Da, respectively, Table
I) due to the presence of multiple recognition motifs and possible
polyvalent interactions.
In an attempt to isolate specific sequences present in the active CSA
dodecasaccharide subfraction F6C, sequential SAX- and APS-HPLC was used
(13). Due to the highly heterogeneous characteristics of CSA, the HPLC
fractions obtained were still mixtures. Despite small differences in
composition, the 12-mer HPLC fractions demonstrated significantly
different inhibitory activities. From these results it is not possible
to determine whether a specific sequence(s) is required for interaction
with P. falciparum IRBCs or to establish the recognition
epitope(s). An alternative strategy is needed to achieve the separation
and preparation of different homogeneous sequences of undersulfated CSA dodecasaccharides.
The present studies have demonstrated the relevance of different forms
of hexuronic acid residue (GlcUA/IdoUA) in parasite adhesive
interactions. Undersulfated CSB 14-mers were isolated and shown to have
a slightly higher inhibitory activity than fully sulfated analogues;
however, the extent of undersulfation was less than observed with the
more active CSA 14-mers. Partial de-4-O-sulfation of CSB and
de-6-O-sulfation of CSA polysaccharides was carried out to
produce CSB and CSA with a similar proportion of 0S and 4S disaccharide
elements. Significantly higher inhibitory activity was shown from
partially desulfated CSA. Because the major difference between the
partially desulfated CSA and CSB preparations was the sole presence of
GlcUA in CSA and IdoUA in CSB, our results strongly point to specific
requirement for GlcUA.
Our experiments unambiguously demonstrate the importance of
non-sulfated disaccharide units and the negative effect of
6-O-sulfates for the interaction between CSA and IRBCs.
After removal of the 6-O-sulfates from CSA and CSC
polysaccharides and CSA tetradecasaccharide subfractions, the
inhibitory activities increased. The removal of 6-O-sulfates
from CSA had a modest effect on its activity, probably because CSA is
already a potent inhibitor of adhesion. The negative effect of
sulfation at the 6-O-position was most clearly demonstrated
with the tetradecasaccharide subfraction F7F, which had only modest
inhibitory activity and is composed of approximately equal amounts of
disaccharide unit 4S and 6S. Removal of 6-O- sulfate
increased its inhibitory activity by some 10-fold. Using a different
approach and a different immobilized receptor for comparing variously
sulfated CS polysaccharides, Alkhalil et al. (24) recently
reported the importance of 4-O-non-sulfated disaccharides in
determining parasite-CS interactions but suggested that
6-O-sulfation neither interacts nor interferes with IRBC binding and that the parasite adhesive motif could be formed by combinations of 4-O-sulfated disaccharides with 0S or 6S
disaccharides. This view contrasts with earlier findings suggesting
that 4-O-sulfation is required for binding and that
6-O-sulfation of GalNAc interferes with binding (3, 10, 11).
Consistent with our findings, Alkhalil et al. (24) conclude
that 12-mer was the minimum length for full inhibitory activity,
although lower oligomers also showed substantial inhibitory activity.
The importance of 4S and 0S disaccharide units for interactions between
CSA and IRBCs is consistent with what is known about the likely CS
receptors for parasite sequestration that occurs in the vasculature
in vivo. CS proteoglycans isolated from placental blood and
tissue support parasite adhesion in vitro and appear to
comprise predominantly non-sulfated and 4-O-sulfated
disaccharide units (25). The proteoglycan thrombomodulin contains CS
chains comprised of 4S disaccharides with GlcUA (26), and is present in
placental and other vascular beds (27) where parasite sequestration occurs, supports parasite adhesion in vitro (22), and may be an important receptor in vivo. Undersulfated chondroitin
sulfate is also prominent in normal human plasma (28) and may influence parasite sequestration in vivo.
In our studies, CSA from bovine trachea was used as the immobilized
receptor, and the derived oligosaccharide fragments were used as
inhibitors in adherence assays as they contain predominantly GlcUA with little or no IdoUA (data not shown) and can support high
levels of parasite adhesion (7). Although this CSA is different from
that of human placenta, it is highly effective at inhibiting parasite
adhesion to placental CS proteoglycans (24), placental tissue sections
(4), and CSA present on endothelial cells (29). Chondroitin lyase ABC,
used in our studies to generate different sized fragments, resulted in
oligosaccharides with modified hexuronic acid P. falciparum erythrocyte membrane protein 1 (PfEMP1) has
been identified as the parasite protein mediating adhesion of IRBCs to
the glycosaminoglycans CSA (32) and heparan sulfate (33, 34) and other
host molecules (35). It is clear that dodecasaccharide is the minimum
chain length of CSA to effectively interact with P. falciparum IRBCs. The minimum chain length requirement for interaction between HA or HS and P. falciparum IRBCs is also
a dodecamer sequence (8, 34). Carbohydrate-protein interactions are
generally weak and are enhanced by the cooperative effect of
multivalent binding sites, which may explain the apparent requirement for longer chain structures of glycosaminoglycans for parasite binding.
Alternatively, it may be that longer CS chains are required because
important basic amino acid residues for CS binding occur in
discontiguous segments of the PfEMP1 protein. The key amino acid
residues could be present in either We thank Graham Brown for valuable
comments on the study and for reviewing the manuscript, Stephen
Rogerson for helpful comments and discussion, and Colin Herbert for
technical assistance.
*
The Glycosciences Laboratory is supported by United Kingdom
Medical Research Council Program Grant G9601454.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. Tel.: 44-20-8869-3252;
Fax: 44-20-8869-3253; E-mail: w.chai@ic.ac.uk.
Published, JBC Papers in Press, April 15, 2002, DOI 10.1074/jbc.M111401200
2
S. Hara and R. Takano, personal communication.
The abbreviations used are:
IRBC, P.
falciparum-infected red blood cells;
CS, chondroitin sulfate;
HA, hyaluronic acid;
HexA, hexuronic acid;
GlcUA, glucuronic acid;
IdoUA, iduronic acid;
GalNAc, N-acetylgalactosamine;
SAX, strong
anion exchange;
APS, aminopropyl silica;
ES-MS, electrospray mass
spectrometry;
0S,
The Structural Motif in Chondroitin Sulfate for Adhesion of
Plasmodium falciparum-infected Erythrocytes Comprises
Disaccharide Units of 4-O-Sulfated and Non-sulfated
N-Acetylgalactosamine Linked to Glucuronic Acid*
§,
, and
Medical Research Council Glycosciences
Laboratory, Imperial College School of Medicine, Northwick Park
Hospital, Harrow, Middlesex HA1 3UJ, United Kingdom and
¶ Department of Medicine, University of Melbourne, Royal
Melbourne Hospital, Parkville, Victoria 3050, Australia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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1-3-linked to
N-acetylgalactosamine (GalNAc), i.e.
4HexA1-3GalNAc1. CS chains show heterogeneity in sulfation patterns
and hexuronic acid compositions (glucuronic acid
(GlcUA)/iduronic acid (IdoUA)) due to different sulfate content and substitution and differing degrees of isomerization of GlcUA to
IdoUA. Typically, GalNAc is mono-O-sulfated at either the 4- or the 6-O-position, and this differentiates the principal
CSA and CSC disaccharide units, respectively. CSB (or dermatan sulfate) is similar in sulfation to CSA, but the hexuronic acid is predominantly IdoUA. In addition, other variations in sulfation pattern frequently occur, such as GalNAc being non-sulfated or
4,6-di-O-sulfated, and GlcUA can be 2-O-sulfated.
Several studies demonstrate the importance of 4-O-sulfation
for binding of IRBC (3, 9-11). Parasite adhesion to purified or cell
surface CSA can be effectively inhibited by CSA but not by CSB, CSC,
heparin, or desulfated chondroitin (3, 9-11). We previously defined
the minimum chain length of CSA required for parasite adhesion to be
12-14 monosaccharide residues and identified that undersulfation was a
structural feature of the active CSA, and oversulfation was a feature
of the inactive CSC (10). This suggested that non-sulfated disaccharide
units may also be important elements of the structural motif required for parasite adhesion in addition to 4-O-sulfate groups of
GalNAc.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
Subfractionation of CSA deca- (F5), dodeca-
(F6), tetradeca- (F7), and hexadecasaccharide (F8) fractions. The
oligosaccharide fractions F5-F8 were subfractionated by an
anion-exchange cartridge column with elution by a gradient of NaCl.
Subfractions were collected at the peaks indicated by
A-G.
View larger version (23K):
[in a new window]
Fig. 2.
Inhibition of binding of P. falciparum-infected red blood cells to immobilized CSA by CS
oligosaccharide subfractions. Subfractions (Fig. 1) of CSA deca-
(a), dodeca- (b), tetradeca- (c), and
hexadecasaccharides (d) together with their parent fractions
and CSA and CSC polysaccharides were tested as competitive inhibitors
of adhesion of P. falciparum-IRBCs to immobilized CSA. All
samples were tested at 50 µg/ml (open bars) in addition to
10 µg/ml (filled bars; ranging from 3.1 to 4.9 nmol/ml for
F6A to F6F) for dodecasaccharides (b), 5 µg/ml
(filled bars; ranging from 1.4 to 2.3 nmol/ml for F7B to
F7G) for tetradecasaccharides (c), and 2 µg/ml
(filled bars; ranging from 0.5 to 0.6 nmol/ml for F8B to
F8G) for hexadecasaccharides (d). All values represent the
mean ± S.E. from two experiments performed in duplicate or
triplicate.
ES-MS analysis of subtractions of CSA deca-, dodeca-, tetradeca- and
hexadecasaccharides
HPLC disaccharide composition analysis of CS poly- and oligosaccharides
UA(2S)1-3GalNAc) in the de-6-O-sulfated CSC may
also have a negative effect on inhibition.
View larger version (12K):
[in a new window]
Fig. 3.
Comparison of inhibitory activities of CS
polysaccharides and CSA tetradecasaccharide subfractions with their
de-6-O-sulfated analogues. a, CSA and
CSC together with de-6-O-sulfated CSA and CSC
(CSA-de6S and CSC-de6S) were used at 10 µg/ml
as competitive inhibitors of binding of P. falciparum-IRBCs
to immobilized CSA. b, CSA tetradecamer subfractions F7D and
F7F and corresponding de-6-O-sulfated samples
(F7D-de6S and F7F-de6S) were tested at 5 µg/ml
for competitive inhibition of P. falciparum-IRBCs binding to
immobilized CSA. c, CSA polysaccharide and
tetradecasaccharide subfraction F7F together with their
de-6-O-sulfated products were tested for competitive
inhibition at a range of concentrations using non-sulfated chondroitin
as the negative control. All values represent the mean ± S.E.
from two experiments performed in duplicate or triplicate.
ES-MS analysis of dodecasaccharide subfractions derived from
de-6-O-sulfated CSA
View larger version (20K):
[in a new window]
Fig. 4.
Comparison of inhibitory activities of
dodecasaccharide subfractions derived from
de-6-O-sulfated CSA. Seven dodecasaccharide
subfractions were tested at various concentrations for competitive
inhibition of P. falciparum-IRBCs binding to immobilized
CSA. All values represent the mean ± S.E. from two experiments
performed in duplicate.
View larger version (20K):
[in a new window]
Fig. 5.
The inhibitory activities of CSB oligo- and
polysaccharides with different degrees of undersulfation.
a, three tetradecasaccharide subfractions F7C, F7D, and F7E
of CSB were compared with CSA polysaccharides as competitive inhibitors
of adhesion of P. falciparum-IRBCs to immobilized
CSA at a concentration of 5 µg/ml (filled bars) and 50 µg/ml (open bars). b, CSA,
de-6-O-sulfated CSA (CSA-de6S), CSB, and three
de-4-O-sulfated CSB samples (CSB-deS1,
CSB-deS2, and CSB-deS3) were tested at 10 µg/ml
for inhibition of parasite adhesion to immobilized CSA. The 0S content
of CSB-deS1 to 3 was 30, 46, and 51% respectively. All values
represent the mean ± S.E. from two experiments performed in
duplicate or triplicate.
View larger version (16K):
[in a new window]
Fig. 6.
HPLC separation of CSA decasaccharide
subfraction F6C. Separation of F6C was carried out on a SAX-HPLC
column (a), and further chromatography on an APS-HPLC column
was performed for fractions F6C-2 (b) and F6C-3
(c).
View larger version (16K):
[in a new window]
Fig. 7.
Comparison of inhibitory activities of HPLC
fractions from CSA undersulfated dodecasaccharide F6C. HPLC
fractions F6C-2c and -2d and F6C-3b, -3c, -3d, and -3e (Fig 5) were
compared with CSA and CSC polysaccharides as competitive inhibitors of
adhesion of P. falciparum -infected red blood cells to
immobilized CSA. Samples were tested at 10 µg/ml, and all values
represent mean ± S.E. from three experiments performed in
duplicate.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
UA at the non-reducing
terminal. Because oligosaccharides prepared from testicular
hyaluronidase digestion of CSA (24) have unmodified uronic acid at the
terminal also showed that 12-mers were required for inhibitory
activity, the form of non-reducing terminal hexuronic acid is not a
crucial element in the interaction. Caution should be applied in using
hyaluronidase, an endo-type glycosidase (30), because its relatively
high transglycosylation activity can result in artificial
sequences generated by reconstruction of CS chains from the cleaved
oligosaccharide fragments (31).
-helices or -strands, and the
folding of the protein brings together two or more sequences required
for binding and activity. Definition of the structural motifs on
host-cell chondroitin sulfate involved in the adhesion of
malaria-infected erythrocytes will increase our understanding of the
pathogenesis of placental infection at the molecular level and
facilitate the development of novel therapeutic approaches to inhibit
this adhesion.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by grants from the National Health and
Medical Research Council of Australia and a Cottrell Research
Fellowship of the Royal Australasian College of Physicians.
ABBREVIATIONS
UA-GalNAc;
4S, UA-GalNAc(4S);
6S, UA-GalNAc(6S);
HexA, hexuronic acid;
HPLC, high performance liquid
chromatography.
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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