Glycobiology and Extracellular Matrices:

Structural Basis for Binding of Plasmodium

falciparum Erythrocyte Membrane Protein
1 to Chondroitin Sulfate and Placental
Tissue and the Influence of Protein
Polymorphisms on Binding Specificity

Downloaded from http://www.jbc.org/ at CHULALONGKORN UNIVERSITY on June 15, 2014

James G. Beeson, Katherine T. Andrews,
Michelle Boyle, Michael F. Duffy, Ee Ken
Choong, Tim J. Byrne, Joanne M. Chesson,
Alexander M. Lawson and Wengang Chai
J. Biol. Chem. 2007, 282:22426-22436.
doi: 10.1074/jbc.M700231200 originally published online June 11, 2007

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 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 31, pp. 22426 –22436, August 3, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Structural Basis for Binding of Plasmodium falciparum

Erythrocyte Membrane Protein 1 to Chondroitin Sulfate and
Placental Tissue and the Influence of Protein Polymorphisms
on Binding Specificity*□ S

Received for publication, January 9, 2007, and in revised form, June 6, 2007 Published, JBC Papers in Press, June 11, 2007, DOI 10.1074/jbc.M700231200
James G. Beeson‡1, Katherine T. Andrews§, Michelle Boyle‡, Michael F. Duffy¶, Ee Ken Choong¶, Tim J. Byrne¶,
Joanne M. Chesson‡, Alexander M. Lawson储, and Wengang Chai储2
From ‡The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia, §Clinical Tropical Medicine
Laboratory and the Griffith Medical Research College, Queensland Institute of Medical Research, Queensland 4066, Australia,
¶
Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Victoria 3050, Australia, and 储Glycosciences
Laboratory, Imperial College London, Northwick Park and St. Mark’s Campus, Harrow, Middlesex HA1 3UJ, United Kingdom

Downloaded from http://www.jbc.org/ at CHULALONGKORN UNIVERSITY on June 15, 2014

Chondroitin sulfate (CS) A is a key receptor for adhesion of sis and biology of malaria, particularly during pregnancy, and
Plasmodium falciparum-infected erythrocytes (IEs) in the pla- the development of targeted interventions.
centa and can also mediate adhesion to microvascular endothe-
lial cells. IEs that adhere to CSA express var2csa-type genes,
which encode specific variants of the IE surface antigen P. fal- Adhesion of parasite-infected erythrocytes (IEs)3 to the
ciparum erythrocyte membrane protein 1 (PfEMP1). We report microvascular endothelium of various organs is an important
direct binding of native PfEMP1, isolated from IEs and encoded pathogenic feature of Plasmodium falciparum (1, 2), the major
by var2csa, to immobilized CSA. Binding of PfEMP1 was cause of malaria accounting for up to 3 million deaths annually
dependent on 4-O-sulfated disaccharides and glucuronic acid (3). Sequestration of IEs is thought to contribute to the survival
rather than iduronic acid, consistent with the specificity of of parasites by aiding replication and evasion of splenic clear-
intact IEs. Using immobilized CS oligosaccharides as neoglyco- ance and can lead to severe consequences when large numbers
lipid probes, the minimum chain length for direct binding of of IEs accumulate in vital organs. Several cell adhesion mole-
PfEMP1 was eight monosaccharide units. Similarly for IE adhe- cules have been reported for P. falciparum-IEs, including CD36
sion to placental tissue there was a requirement for 4-O-sulfated (4), intercellular adhesion molecule 1 (ICAM-1) (5), and the
GalNAc and glucuronic acid mixed with non-sulfated disaccha- glycosaminoglycans (GAGs) chondroitin sulfate A (CSA) (6),
rides; 6-O-sulfation interfered with the interaction between pla- heparan sulfate (7), and hyaluronic acid (8).
cental CSA and IEs. The minimum chain length for maximal Evidence from several studies demonstrates that CSA is an
inhibition of adhesion was 10 monosaccharide residues. Par- important receptor for sequestration of IEs in the placenta
tially 4-O-sulfated CS oligosaccharides (45–55% sulfation) were (9 –11). P. falciparum isolates from the placenta typically
highly effective inhibitors of placental adhesion (IC50, 0.15 adhere to CSA, and IEs can adhere to ex vivo placental tissue in
␮g/ml) and may have potential for therapeutic development. a CSA-dependent manner in vitro (10, 12). CSA has also been
We used defined P. falciparum isolates expressing different identified as a cell surface receptor for adhesion to endothelial
variants of var2csa in adhesion assays and found that there were cells (13) and may therefore contribute to parasite sequestra-
isolate-specific differences in the preferred structural motifs for tion in other organs (14). IEs can also bind via CSA side chains
adhesion to CSA that correlated with polymorphisms in to the proteoglycan thrombomodulin (15) that is present on
PfEMP1 encoded by var2csa-type genes. This may influence endothelial cells throughout the vasculature (16). Other CSPGs
sites of IE sequestration or parasite virulence. These findings that support IE adhesion in vitro have been isolated from the
have significant implications for understanding the pathogene- placenta, Saimiri brain endothelium, and other cells (supple-
mental Table S1) (17–20).
* This work was supported by funding from the National Health and Medical P. falciparum erythrocyte membrane protein 1 (PfEMP1) has
Research Council, Australia (project grant and program grant; Career been identified as the principle adhesive ligand of IEs and is
Development Award (to J. G. B.)); the Miller Fellowship of The Walter and encoded by the var multigene family (up to 60 copies per
Eliza Hall Institute (to J. G. B.); the Medical Research Council (Program Grant
G9601454), UK; and a Griffith University research grant (to K. T. A.). The
3
costs of publication of this article were defrayed in part by the payment of The abbreviations used are: IE, infected erythrocyte; GAG, glycosaminogly-
page charges. This article must therefore be hereby marked “advertise- can; CSPG, chondroitin sulfate proteoglycan; PfEMP1, P. falciparum eryth-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rocyte membrane protein 1; CS, chondroitin sulfate; NGL, neoglycolipid;
□S
The on-line version of this article (available at http://www.jbc.org) contains ICAM-1, intercellular adhesion molecule-1; HexUA, hexuronic acid; GlcUA,
supplemental Tables S1 and S2. glucuronic acid; IdoUA, iduronic acid; de6S, de-6-O-sulfated; de4S, de-4-O-
1
To whom correspondence may be addressed: The Walter and Eliza Hall Inst. sulfated; ATS, acidic terminal segment; BSA, bovine serum albumin; ⌬UA,
of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. 4,5-unhydrohexuronic acid; 0S, ⌬UA-GalNAc; 4S, ⌬UA-GalNAc4S; 6S, ⌬UA-
Tel.: 61-3-9345-2555; Fax: 61-3-9347-0842; E-mail: [email protected]. GalNAc6S; HPLC, high pressure liquid chromatography; MES, 4-morpho-
2
To whom correspondence may be addressed. Tel.: 44-20-8869-3255; Fax: lineethanesulfonic acid; CSA-PR, CSA from porcine rib cartilage; RPMI, RPMI
44-20-8869-3455; E-mail: [email protected]. 1640 medium; PBS, phosphate-buffered saline.

22426 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 31 • AUGUST 3, 2007

 Interactions between P. falciparum and Chondroitin Sulfates
genome) (1, 2). var genes are highly diverse both within and human immunodeficiency virus (45), have been reported, and
between different genomes, and different variants encode pro- P. falciparum also interacts with heparan sulfate during inva-
teins with different receptor binding properties. IEs selected for sion of hepatocytes (46). Despite its widespread distribution
adhesion to CSA up-regulate var2csa-type genes, which appear and expression on many cell surfaces there is relatively little
to be the predominant var gene encoding CSA-binding known about the structure-function relationship of CS. Fur-
domains (21–24) and show increased expression in placental thermore it is not known how polymorphisms, which are com-
malaria (25). Recombinant domains of var2csa bind CSA (22). mon among antigens of microbial pathogens, influence the
However, binding of full-length or native PfEMP1 to CSA or specificity and function of interactions with GAGs, which may
inhibition of IE adhesion by antibodies to var2csa domains has influence the clinical manifestations of infection.
not been demonstrated to confirm a direct role of var2csa in In this study, we aimed to further define the molecular basis
adhesion. Some Duffy-binding-like ␥-type domains from other of the interactions among IEs, CSA, and placental tissue and to
var genes have also been shown to bind CSA in vitro when identify key features common to different CSA-binding vari-
expressed as recombinant proteins (26 –28), but their signifi- ants. To date, the structural requirements of CS for interactions
cance in mediating IE adhesion to CSA remains unclear (29, with P. falciparum have been defined only by inhibition of IE
30). Although var2csa genes are relatively conserved, there is adhesion rather than through direct PfEMP1 binding studies
considerable polymorphism among different variants (21, and have predominantly used purified immobilized CSA prep-

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31–33). These polymorphisms can substantially influence the arations for adhesion. Here we examined direct binding of
binding and adhesion-inhibitory activity of acquired antibodies native PfEMP1 to oligosaccharides and polysaccharides, as neo-
suggesting that they have evolved to facilitate immune evasion glycolipid (NGL) (47) and biotin probes, respectively; adhesion
(34). Polymorphisms also influence sensitivity of var2csa of IEs from different P. falciparum isolates to polysaccharides
PfEMP1 to trypsin (34, 35), but it is not known whether they with different degrees of sulfation; and inhibition of IE adhesion
influence specificity of interactions with CSA. to CSA and placental tissue using various types of defined oligo-
CS comprises repeating disaccharide units of hexuronic and polysaccharides. Additionally we hypothesized that the
acid (HexUA) ␤1–3 linked to GalNAc, i.e. -(4HexUA␤1– requirement for P. falciparum to interact with diverse CS struc-
3GalNAc1)n-. Typically GalNAc is mono-O-sulfated at either tures in the vasculature will have favored the evolution of CS-
the 4- or the 6-O position and this differentiates the principal binding PfEMP1 variants with differing fine specificities for CS
CSA and CSC disaccharide units, respectively. CSB (or derma- structural motifs. More broadly, we used PfEMP1 as a model to
tan sulfate) is similar in sulfation to CSA but has iduronic acid investigate strain-specific differences in microbial interactions
(IdoUA) rather than glucuronic acid (GlcUA) as the predomi- with GAGs and whether polymorphisms in GAG-binding pro-
nant uronic acid. Other variations in sulfation pattern also fre- teins may alter the fine specificity of interactions with struc-
quently occur. CS chains show heterogeneity in sulfation pat- tural motifs.
terns and uronic acid compositions, and these features
determine their biological properties. EXPERIMENTAL PROCEDURES
Several studies have demonstrated the importance of 4-O- Preparation of Desulfated and Biotinylated CS Poly-
sulfation for IE binding to CS (6, 36). The minimum chain saccharides—CSA (from bovine trachea), CSB (from porcine
length for inhibition of adhesion to CSA is 12 monosaccharide intestinal mucosa), and CSC (from shark cartilage) were
residues (36 –38), and the optimal motif for interaction with IEs obtained from Sigma. Chondroitinase ABC (EC 4.2.2.20, from
is formed by mixed 4-O-sulfated and non-sulfated GalNAc Proteus vulgaris; Sigma) digestion and strong anion-exchange
alternating with GlcUA (37, 38). IdoUA and 6- or 2-O-sulfation HPLC disaccharide composition analysis (38) indicated mixed
interfere with the interaction (38). In the vasculature, the sulfation patterns in all three CS preparations. They all con-
CSPGs, to which IEs may bind, are present as diverse structures tained three disaccharide units: ⌬UA-GalNAc (0S), ⌬UA-
that differ significantly in their level and pattern of sulfation GalNAc4S (4S), and ⌬UA-GalNAc6S (6S). CSA contained 4.9%
(supplemental Table S1) (17–20, 39 – 41). CSPGs isolated from 0S, 55.3% 4S, and 39.8% 6S; CSB contained 0.5% 0S, 85.6% 4S,
placental intervillous blood are particularly low in sulfate con- and 4.8% 6S; and CSC contained 0.8% 0S, 15.5% 4S, and 76.7%
tent (17, 42), containing 2– 8% 4-O-sulfation; there are also 6S (38) (Table 1). Selective de-6-O-sulfation of CS poly- and
regions of higher levels of sulfation (20 –28%) (43). Cell-associ- oligosaccharides and partial de-4-O-sulfation of CSB were per-
ated placental CSPGs present in detergent extracts have a formed as described previously (38). De-6-O-sulfation involved
higher level of sulfation with ⬃30% 4-O- and 13–15% 6-O-sul- conversion of poly- and oligosaccharides to pyridinium salts
fation (17). Thrombomodulin, which is widely expressed in the and desulfation in a mixture of anhydrous pyridine and N,O-
vasculature, is typically composed of ⬃87% 4-O-sulfated disac- bis(trimethylsilyl)acetamide. CSB polysaccharide was con-
charides balanced with non-sulfated disaccharides (39), and verted to pyridinium salt, dissolved with 90% Me2SO in H2O,
CSPGs from Saimiri brain endothelial cells were composed and heated to 80 °C. The reaction was stopped at 70- and 100-
predominantly of 4-O-sulfated disaccharides with ⬃37% non- min intervals by cooling and neutralization with NaOH. The
sulfated disaccharides (18). desulfation products were confirmed by strong anion-exchange
GAGs play essential roles in a range of biological functions and HPLC disaccharide composition analysis as described previ-
have been increasingly recognized as important molecules for ously (38) (Table 1). Results indicated that all 6-O-sulfate
attachment, adhesion, and invasion of infectious pathogens groups were selectively removed from CSA and CSC poly- and
(44). Interactions between CS and other microbes, such as oligosaccharides, and 30 and 46% of sulfates were removed

AUGUST 3, 2007 • VOLUME 282 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 22427

Interactions between P. falciparum and Chondroitin Sulfates
from the two partially de-4-O-sulfated (de4S) CSB prepara- sion to immobilized CSA to generate isolate 3D7-CSA (34, 35).
tions, CSB-de4S-1 and CSB-de4S-2, respectively (Table 1). Isolates CS2, HCS3, and 3D7-CSA are genetically distinct and
Biotinylated CSA and CSC (biotin-CSA and biotin-CSC, were reselected for adhesion to CSA prior to use in assays. The
respectively) were prepared and purified as described previ- specificity of adhesion of the different isolates to CSA has been
ously (48) with some modification. The level of biotin intro- established elsewhere (35, 36). Isolate 3CI was generated from
duced was one for every 50 carboxyl groups.4 In brief, 50 mg of 3D7 by selection for adhesion to ICAM-1 (52). The phenotypes
CSA or CSC polysaccharide were dissolved in 10 ml of 0.1 M and properties of the different isolates are shown in supplemen-
MES buffer (pH 5.5) before addition of 109 ␮l of 25 mM biotin- tal Table S2.
LC-hydrazide (Pierce, Perbio Science, Tattenhall, UK) solution Parasite Adhesion Assays—Adhesion assays were performed
in Me2SO and 130 ␮l of 25 mg/ml 1-ethyl-2-(2-dimethylamino- using trophozoite-infected erythrocytes at 3–5% parasitemia
propyl)carbodiimide solution in 0.1 M MES buffer. The reaction and 1% hematocrit, resuspended in RPMI-HEPES with 5%
mixtures were stirred at room temperature for 24 h and then pooled serum from non-exposed donors (9). Adhesion recep-
dialyzed extensively against deionized water. A short Sephadex tors used were CS polysaccharides and various desulfated ana-
G-10 column (1.6 ⫻ 30 cm) was used to remove remaining logs described above together with CSA from porcine rib carti-
biotin and other reagents. Quantitation of CS poly- and oligo- lage (CSA-PR; Sigma), CD36, and recombinant human
saccharides and their modified forms was carried out by carba- ICAM-1 (Bender MedSystems). Oligo- and polysaccharides

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zole assay (38). were tested for inhibitory activity by preincubating with para-
Preparation of CS Oligosaccharide Fragments and Their NGL site suspensions for 15 min prior to testing for adhesion (36, 38).
Probes—Oligosaccharides were prepared as described previ- Receptor-bound IEs were stained with Giemsa stain and
ously (36, 38) from CSA, CSB, CSC, and de-6-O-sulfated CSA counted by microscopy. Parasite adhesion was also examined
(CSA-de6S) by partial depolymerization with chondroitinase using receptor-coated beads. Streptavidin-coated beads (M288,
ABC (49) followed by size fractionation using a Bio-Gel P-6 Dynal, Melbourne, Australia) were incubated with biotin-CSA
column (1.6 ⫻ 90 cm) with elution by ammonium acetate (0.2 or biotin-CSC (100 ␮g/ml in PBS) for 30 min and then blocked
M). The 12-mer and 14-mer fractions isolated from partially with 1% bovine serum albumin (BSA) or 1% casein in PBS for 1 h
digested CSA-de6S were each further fractionated by strong or overnight. IEs, labeled with 10 ␮g/ml ethidium bromide,
anion-exchange HPLC into six subfractions. All six 12-mer were incubated with receptor-coated beads at 3–5% parasite-
subfractions (containing zero to six 4-O-sulfates, designated mia and 5% hematocrit in RPMI-HEPES with 5% pooled human
as subfractions CSA-12-0S to -6S, respectively) and two 14-mer serum on a rotating wheel for 60 –120 min at room tempera-
subfractions containing five and seven sulfates (designated as ture. Cell suspensions (10 ␮l) were examined by combined light
CSA-14D and CSA-14F, respectively) were used in inhibition and fluorescence microscopy.
assays after quantitation by carbazole assay. The chain length Adhesion assays with ex vivo placental tissue were performed
and sulfate content of the fractions and subfractions were as described previously (12). Briefly trophozoite stage IEs were
determined by electrospray mass spectrometry using opti- purified using gelatin and resuspended at 5 ⫻ 106 IEs/ml in
mized conditions to minimize potential sulfate loss (38, 50). RPMI (pH 6.8) containing 10% human serum. Test oligosaccha-
HPLC disaccharide composition analysis was performed as rides and polysaccharides were preincubated with IE suspen-
described previously (38). sions for 15 min before incubating on unfixed sets of three
NGL probes of CSA and CSC oligosaccharide fragments consecutive 5-␮m cryosections of normal human placenta. IEs
were prepared by conjugation to 1,2-dihexadecyl-sn-glycero-3- were allowed to adhere to placental sections for 1 h with agita-
phosphoethanolamine (Fluka, Dorset, UK) as described pre- tion every 20 min followed by gentle washing in PBS to remove
viously (47). In brief, lyophilized oligosaccharide fragments unbound IEs. Following fixing in 2% glutaraldehyde and stain-
(typically 100 nmol) were mixed with 5 ␮l of water, 100 ␮l of ing with Giemsa the average number of adherent IEs (⫾S.E.)
1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine stock was determined for at least three independent cryosections.
solution (7 nmol/␮l in CHCl3/MeOH, 1:3, v/v), and 10 ␮l of Binding was compared with untreated controls present on each
freshly prepared tetrabutylammonium cyanoborohydride solu- slide. Placental tissue was obtained after informed written con-
tion (20 ␮g/␮l in MeOH). The mixture was incubated at 60 °C sent and with approval from the Mater Human Research Ethics
for 96 h. NGLs of CS oligosaccharides were purified on a mini- Committee, Brisbane, Australia (approval number 751M).
silica column and analyzed by high performance TLC and mass Extraction of Parasite Protein PfEMP1 and Its Detection by
spectrometry (47). Western Blotting—PfEMP1 was extracted from pigmented tro-
Parasite Culture—P. falciparum was maintained in continu- phozoite stage IEs in culture at 10 –15% parasitemia. IEs were
ous culture (9, 36) using human group O positive erythrocytes. lysed with cold 1% Triton X-100 in PBS containing protease
Parasite isolate E8B (or FAF-EA8) (51) is a clone of Brazilian inhibitors. Insoluble proteins were resuspended in 2% SDS in
isolate ItG2. CS2 was derived from E8B by selection for adhe- PBS by vigorous mixing over several minutes to achieve a satu-
sion to CSA (6). HCS3 was originally isolated from a traveler to rating concentration of proteins (34, 53). After centrifugation at
Asia and was generated by selection for adhesion to immobi- high speed, the supernatant containing PfEMP1 (confirmed by
lized CSA (35, 36). 3D7 was selected multiple times for adhe- Western blotting) was diluted 1:20 in cold RPMI-HEPES with
0.5% Triton X-100, 1% BSA (or 0.1% casein), and protease
inhibitors and then passed through a 0.45-␮m-pore size filter
4
A. J. Day, personal communication. before use in binding assays.

22428 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 31 • AUGUST 3, 2007

 Interactions between P. falciparum and Chondroitin Sulfates
Polysaccharides used were CSA,
CSB, CSC, and heparan sulfate
(from bovine kidney; Sigma). Oligo-
saccharide NGLs of 2–20 monosac-
charide units prepared from CSA
and CSC were used. After coating,
wells were washed and blocked for
2 h at 37 °C or overnight at 4 °C with
4% BSA in PBS with 0.05% Tween 20
or with 1% casein in PBS (Pierce).
After washing, wells were incubated
with IE membrane extract for 2 h at
37 °C and then washed with RPMI-
HEPES containing 0.5% Triton
X-100 and 1% BSA. To detect bound
PfEMP1, wells were incubated with

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affinity-purified rabbit antibodies or
a mouse monoclonal antibody gen-
erated against the ATS of PfEMP1,
diluted in RPMI-HEPES with 1%
BSA and 0.5% Triton X-100, for 1 h
FIGURE 1. Binding of PfEMP1 isolated from CS2 IEs to CSA. A, beads coated with biotinylated CSA, CSC, or at room temperature; then washed;
BSA were incubated with protein extracts from CS2 IEs. Bead-bound proteins were eluted and probed for
PfEMP1 on Western blots. PfEMP1 was detected in eluates from CSA-coated beads (lane CSA) but not CSC- and incubated with horseradish
or BSA-coated beads (lanes CSC and BSA, respectively). The left lane shows antibody labeling of CS2 IE peroxidase-conjugated anti-rabbit
protein extract that was not incubated with beads. Molecular mass markers (kDa) are shown to the left of
the blot. B, adhesion of CS2 IEs to beads coated with biotinylated CSA (arrows). Uninfected erythrocytes did not
or anti-mouse IgG (1:500 in the
bind. C, there was no adhesion of CS2 IEs to control beads. D, PfEMP1 bound to CSA immobilized on the plastic same buffer) for 45 min. After wash-
surface of 96-well microtiter plates but not to immobilized CSB or CSC. Results represent mean ⫾ S.E. OD is ing with RPMI-HEPES with 0.5%
expressed in arbitrary units where levels for CSA binding were designated a value of 100 (three experiments
performed in duplicate). Binding of PfEMP1 was detected using anti-ATS antibodies. E, proteins bound in Triton X-100, color was developed
receptor-coated wells were eluted, and PfEMP1 was detected by Western blot in eluates from CSA-coated wells with azino-bis(3-ethylbenthiazo-
(lane CSA) but at much lower levels among control wells coated with heparan sulfate (lane HS). Molecular mass line-6-sulfonic acid) liquid sub-
markers (kDa) are shown to the left of the blot. PfEMP1 was detected using antibodies raised against the
conserved ATS of PfEMP1. F, PfEMP1 was captured in microtiter wells coated with an anti-ATS monoclonal strate system (Sigma-Aldrich), and
antibody and tested for binding of biotinylated CS. Biotin-CSA bound at much higher levels than biotin-CSC. absorbance was read by spectropho-
Values represent mean ⫾ S.E. (three experiments performed in duplicate). OD is expressed in arbitrary units
where levels for CSA binding were designated a value of 100. tometry. Binding was measured as
absorbance less background levels
Western blotting for detection of PfEMP1 was performed in control wells that were coated with BSA or PBS only, and
as described previously (34, 53) using Triton X-100-insolu- results from each experiment were standardized and expressed
ble proteins isolated from pigmented trophozoite stage IEs in arbitrary units where levels for CSA binding were designated
and resolubilized in 2% SDS. Proteins on nitrocellulose a value of 100. Prior studies have established the specificity of
membranes were probed with an affinity-purified antiserum anti-ATS antibody for PfEMP1 and that the antibody does not
raised in rabbits against a conserved sequence in the acid label other parasite proteins or erythrocyte proteins by West-
terminal segment (ATS) of PfEMP1 or a monoclonal anti- ern blot (26, 34). To confirm that PfEMP1 is responsible for the
body against ATS (26, 34). interaction, bound proteins were eluted from the surface of
Binding of PfEMP1 to CS Polysaccharides and Oligosaccha- multiple CSA-coated wells by sequential incubation with 50 ␮l
ride NGL Probes—For PfEMP1 binding to receptor-coated of SDS-PAGE sample buffer. Heparan sulfate-, CSB-, and BSA-
beads, biotin-CSA or biotin-CSC (prepared as described above) coated wells were also used and eluted in the same way as con-
or biotinylated BSA (Sigma) were incubated at 100 –200 ␮g/ml trols. The eluted proteins were fractionated by SDS-PAGE fol-
in PBS with streptavidin-coated beads (Dynal) for 1 h at room lowed by Western blotting for detection of PfEMP1.
temperature and then blocked with 1% casein in PBS overnight In alternate assays, microtiter plates (96-well, Maxisorb,
at 4 °C. Beads were incubated with PfEMP1 extract for 1 h at Nunc) were incubated with an anti-ATS monoclonal antibody
room temperature or 4 h at 4 °C and then washed three times (2 ␮g/ml) overnight at 4 °C. Wells were washed and blocked for
with RPMI-HEPES with 0.5% Triton X-100 and three times 1–2 h with 1% casein in PBS (Pierce). After washing, wells were
with RPMI-HEPES. Bound proteins were eluted with SDS- incubated with the IE membrane extract containing PfEMP1
PAGE sample buffer at 100 °C and detected by SDS-PAGE and diluted in PBS with 0.1% casein and 0.5% Triton X-100 for 1 h at
Western blotting with PfEMP1-specific anti-ATS antibody. room temperature. After washing with 0.5% Triton X-100 in
To perform plate-based binding assays, 96-well microtiter PBS, wells were blocked with 1% casein in PBS for 30 min and
plates (Falcon 3072, BD Biosciences; or Maxisorb, Nunc) were then incubated with biotin-CSA or biotin-CSC (10 ␮g/ml) or
incubated with polysaccharides (50 ␮g/ml in PBS) or NGLs of buffer only followed by streptavidin-horseradish peroxidase (1
oligosaccharides (1–5 ␮M in water or PBS) overnight at 4 °C. ␮g/ml; Dako) in PBS with 0.1% casein and 0.05% Tween 20 for

AUGUST 3, 2007 • VOLUME 282 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 22429

Interactions between P. falciparum and Chondroitin Sulfates
1 h at room temperature for each step. After washing with PBS,
color was developed with azino-bis(3-ethylbenthiazoline-6-
sulfonic acid), and absorbance was read by spectrophotometry.

RESULTS
Extraction and Identification of Native PfEMP1 and Its
Specific Binding to Immobilized CSA Polysaccharides—We
first sought to determine whether native PfEMP1 isolated
from parasite cultures binds specifically to immobilized CSA
in a manner similar to the characteristics of IE adhesion.
Membrane-bound proteins were isolated from mature stage
IEs of the CSA-binding isolate CS2 and tested for binding to
biotinylated CSA, CSC, and BSA immobilized on streptavidin-
coated beads. Proteins eluted from the beads were probed with
anti-ATS antibodies to detect PfEMP1 by Western blotting. A
single PfEMP1 band was observed in proteins eluted from CSA-

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coated beads, but only a faint band from CSC-coated beads was
observed (Fig. 1A). No band from BSA-coated beads was
detected. The size of the eluted PfEMP1 (⬃300 kDa) corre-
sponded to the dominant PfEMP1 band detected in Western
blots of CS2 IE extracts. When we probed membranes with pooled
serum from exposed pregnant women, no additional bands were
found that were specific to the CSA-coated beads (data not
shown). We confirmed that intact CS2 IEs bind to biotin-CSA
immobilized on beads (Fig. 1B). Most (⬎90%) CS2 IEs bound
CSA-coated beads, whereas there was little or no binding of IEs to
CSC- or BSA-coated beads or to uncoated beads (Fig. 1C) or of
uninfected erythrocytes to CSA-coated beads (Fig. 1B).
We next aimed to develop an assay that would allow further
investigations to define the specificity of interactions between
PfEMP1 and CS. Direct binding assays were performed with
CSA and other molecules coated in wells of microtiter plates,
and bound PfEMP1 was detected by enzyme immunoassays
with anti-ATS antibodies. Across repeated experiments,
PfEMP1 extracted from CS2 IEs bound to immobilized CSA
(Fig. 1D) but not to CSB or CSC in agreement with the adhesion
characteristics of intact IEs (6, 36). Positive binding to CSA, but
not CSC, was observed when using affinity-purified polyclonal
anti-ATS or monoclonal anti-ATS antibodies. Specific binding
FIGURE 2. Binding of PfEMP1 isolated from CS2 IEs to CSA oligosaccha-
of PfEMP1 was also observed with CSA covalently linked to ride NGL probes immobilized in plastic wells. A, PfEMP1 isolated from CS2
plastic surfaces using Nunc CovaLink or amino Immobilizer IEs bound at a substantially higher level to immobilized CSA 20-mers com-
pared with CSC 20-mers. Values represent mean ⫾ S.E. of three experiments
plates (data not shown). PfEMP1 extracted from isolate E8B did performed in duplicate. B, PfEMP1 from CS2 IEs bound to immobilized CSA
not show significant binding to CSA (data not shown); E8B IEs 18-mers and 20-mers, whereas there was no binding of PfEMP1 isolated from
do not adhere to CSA (35). To further validate the assay and the ICAM-1-adherent isolate 3CI. Values represent mean ⫾ range OD from
one experiment performed in duplicate. C, PfEMP1 isolated from CS2 IEs
establish that PfEMP1 was the protein detected in the assays, bound in a size-dependent manner to immobilized CSA oligosaccharide
proteins bound in wells were eluted and examined by Western NGLs. Oligosaccharides from 4 –16 monosaccharide units in length were
blotting. PfEMP1 was detected in elutes from CSA-coated tested. Values represent mean ⫾ S.E. of two experiments performed in dupli-
cate. In all experiments, PfEMP1 was detected using antibodies raised against
wells, whereas little or no protein was detected from control the conserved ATS of PfEMP1. Values in A and C are OD expressed in arbitrary
wells coated with heparan sulfate (Fig. 1E), CSB, or BSA (data units where the maximum level of binding was designated a value of 100.
not shown). Using an alternate approach, PfEMP1 was cap-
tured in wells coated with an anti-ATS monoclonal antibody probes to determine the specificity of CS binding to PfEMP1,
and tested for binding of biotinylated CS in solution. We including the chain length and sulfation pattern. Oligosaccha-
observed specific binding of biotin-CSA to PfEMP1 compared ride fragments of CSA and CSC were converted into NGL
with biotin-CSC (Fig. 1F). probes by conjugation to an aminolipid for immobilization on
Direct Binding of Native PfEMP1 to CS Oligosaccharides solid matrices. The importance of sulfation pattern of CS for
Depends on Their Chain Length and Sulfation Pattern—We interaction with PfEMP1 isolated from CS2 IEs was demon-
further examined interactions between PfEMP1 and CS oligo- strated with the largest NGLs (20-mers) derived from CSA and
saccharides by direct binding assays using oligosaccharide NGL CSC (Fig. 2A). We observed higher levels of binding of PfEMP1

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 Interactions between P. falciparum and Chondroitin Sulfates
TABLE 1
Disaccharide compositions and inhibitory activities of CS polysaccharides and oligosaccharides
Disaccharide composition analysis was performed by strong anion-exchange HPLC (38).
Compositiona
Activity in parasite assaysb
0S 2S 4S 6S 2,6DiS 4,6DiS 2,4DiS
%
CSAc 4.9 — 55.3 39.8 — — — ⫹
CSB 0.5 — 85.6 4.8 — 6.4 2.3 ⫺
CSC 0.8 — 15.5 76.7 7.0 ⬍0.5 — ⫺
CSA-PR 4.2 — 66.8 29.0 — — — ⫹
CSA-de6Sd 45.3 — 54.7 ⬍0.5 — — — ⫹⫹
CSC-de6S 74.5 7.8 15.9 1.8 — — — ⫹/⫺
CSB-de4S-1d 30.1 2.1 64.7 3.1 — ⬍0.5 — ⫺
CSB-de4S-2 45.6 2.2 50.1 2.2 — ⬍0.5 — ⫺
CSA-14F 1.3 — 54.9 43.7 — — — ⫹
CSA-14F-de6S 44.6 — 55.0 0.4 — — — ⫹⫹
CSA-14D 13.3 — 45.6 41.1 — — — ⫹
CSA-14D-de6S 53.5 — 44.9 1.6 — — — ⫹⫹
CSB-14 5.3 — 88.4 6.2 — — — ⫺
a
2S, ⌬UA(2S)1–3GalNAc; 2,6DiS, ⌬UA(2S)1–3GalNAc(6S); 4,6DiS, ⌬UA1–3GalNAc(4,6S); 2,4DiS, ⌬UA(2S)1–3GalNAc(4S). Trisulfated disaccharides were not

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detected. —, not detected.
b
Compounds inhibited adhesion of P. falciparum-infected erythrocytes to immobilized CSA and/or placental tissue. ⫺, little or no inhibition; ⫹/⫺, some inhibition with some
P. falciparum isolates only; ⫹, inhibitory; ⫹⫹, highest inhibitory activity. See text for details.
c
CSA and CSC contained undetectable levels of IdoUA; CSB contained predominantly IdoUA (36, 38).
d
de6S, prepared by regioselective de-6-O-sulfation (38); de4S, prepared by partial solvolytic desulfation (38).

from CS2 IEs to the CSA NGLs compared with CSC NGLs. In of the extent and pattern of sulfation of CS for parasite adhe-
additional experiments, we observed similar findings using sion to placental tissue (Fig. 3B and Table 1). CSA-derived
CSA and CSC 18-mer NGLs (data not shown). As a reflection of 14-mers effectively inhibited adhesion. CSA 14-mer subfrac-
specificity, PfEMP1 isolated from CS2 IEs bound CSA 18-mer tion CSA-14D was composed of 13% 0S, 46% 4S, and 41% 6S,
and 20-mer NGLs, whereas PfEMP1 from the ICAM-1-binding and CSA-14F was composed of 1% 0S, 55% 4S, and 44% 6S
isolate 3CI did not bind to CSA NGLs (Fig. 2B). (38). Their inhibitory activity was enhanced by selective
Testing NGLs of CSA oligosaccharides with 2–16 monosac- de-6-O-sulfation (Fig. 3B), indicating that 6-O-sulfation
charide units in length suggests significant binding occurs with inhibits parasite-CS interactions even when a sufficient
8-mer or longer chain oligosaccharide NGLs (Fig. 2C). This number of 4-O-sulfate groups are present. CSA-14F-de6S
pattern is similar to size-dependent inhibition of IE adhesion to (55% 4S) was somewhat more inhibitory than CSA-14D-
CSA by oligosaccharides with which 12-mer or larger frag- de6S (45% 4S). CSC-de6S (16% 4S) was not inhibitory,
ments were required for maximum inhibition of adhesion to whereas CSA-de6S (55% 4S and 45% 0S) was highly inhibi-
immobilized CSA (36 –38). tory, indicating the significance of the extent of 4-O-sulfa-
Structural Requirements for IE Adhesion to Placental Tissue— tion for placental adhesion.
Having defined structural requirements of CS for binding To evaluate the importance of GlcUA versus IdoUA, we com-
PfEMP1 and confirming that PfEMP1 is a ligand for binding CS, pared the inhibitory activity of CSA and CSB. However, CSB
we next investigated the specificity of IE adhesion to placental has a much higher level of 4-O-sulfation than CSA (Table 1).
tissue to further define the molecular basis of parasite seques- Therefore, CSB was partially de-4-O-sulfated to enable a more
tration. This was studied by inhibition of the interaction using direct comparison with CSA. CSB-de4S was composed of 50%
various sequence-defined CS oligo- and polysaccharides (Table 4S and 46% OS disaccharides, similar to the sulfation level of
1) (38) that may have potential for therapeutic development. CSA-de6S, but has IdoUA rather than GlcUA (36, 38). CSB-
Oligosaccharide fractions of 4 –18 monosaccharide residues de4S was not inhibitory compared with CSA-de6S, indicating
in length derived from either CSA or CSC were tested for inhi- the importance of uronic acid type for adhesion (Fig. 3B). Fur-
bition of binding of IEs to fresh placental cryosections using the thermore a CSB 14-mer was relatively non-inhibitory com-
parasite line CS2 (Fig. 3A) to determine the minimum chain pared with CSA 14-mers. CSB with or without various degrees
length for interaction. The inhibitory effects of the CSA oligo- of desulfation (30 –50% 0S) did not inhibit adhesion of IEs to
saccharides were highly dependent on length. Maximum inhi- immobilized CSA (data not shown).
bition of adhesion equivalent to that observed with the polysac- In earlier experiments we found that partially 4-O-sulfated
charide was found with the CSA-derived 10-mer or larger CSA oligosaccharides, generated by selective de-6-O-sulfation,
oligosaccharides at 10 ␮g/ml. Substantial inhibition (83%) was were the most effective inhibitors of adhesion to immobilized
also observed with CSA 8-mer at 20 ␮g/ml (data not shown) but CSA polysaccharide (38). We demonstrated here that these are
not at 10 ␮g/ml. By comparison, inhibition by the 10-mer was also highly effective inhibitors of placental adhesion. The inhib-
79% (Fig. 3A) and 98% (data not shown) at 10 and 20 ␮g/ml, itory activity of the partially sulfated CSA 14-mers, CSA-14F-
respectively. Little inhibition was observed with shorter CSA de6S, was equivalent to that shown with CSA-de6S (Fig. 3C);
oligosaccharides or CSC-derived oligosaccharides of the same both preparations (CSA-de6S and CSA-14F-de6S) contained
lengths. around 55% 4S and 45% 0S disaccharides, which is similar to the
The inhibitory activities of partially desulfated polysac- level of 4-O-sulfation of optimal 12-mer inhibitors (Fig. 4A).
charides and oligosaccharides demonstrated the significance The IC50 of CSA-14F-de6S was 0.15 ␮g/ml.

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Interactions between P. falciparum and Chondroitin Sulfates

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FIGURE 4. Inhibition of IE adhesion to immobilized CSA by CSA 12mers
and polysaccharides using parasite isolates CS2 (A), HCS3 (B), and 3D7-
CSA (C). Values represent mean adhesion (⫾S.E.; two to three experiments
performed in duplicate) relative to control. The 12-mer fractions 0S to 6S were
derived from de-6-O-sulfated CSA and contained, on average, zero to six 4-O-
sulfated disaccharides, respectively. CSB-deS, partially de-4-O-sulfated CSB
(CSB-deS-1, 70% sulfation; CSB-deS-2, 50% sulfation).
FIGURE 3. Inhibition of IE adhesion to placental cryosections by oligo-
saccharides and polysaccharides using the CSA-binding parasite iso-
late CS2. A, CSA- and CSC-derived oligosaccharides ranging from 4 to 18 real time PCR demonstrated that the three isolates express
monosaccharide units in length, together with the parent polysacchar- var2csa-type genes as the dominant var gene transcripts (23,
ides, were tested for inhibition of parasite adhesion at a concentration of
10 ␮g/ml. Values represent mean adhesion (⫾S.E.) relative to control 54), and Western blots showed that each expresses a single
(three experiments performed in duplicate). B, inhibition of adhesion of surface-exposed PfEMP1 variant of apparently the same Mr
IEs by partially desulfated CS polysaccharides and 14-mer oligosaccha- (⬃300,000) (34). Isolates were recognized in a parity- and gen-
rides. Samples were tested for inhibition at 1 and 2.5 ␮g/ml. Values represent
mean adhesion (⫾S.E.; four experiments performed in duplicate) relative to der-associated manner by sera from exposed donors (34, 55),
control. C, concentration-dependent inhibition of IE adhesion by CSA-14F- suggesting that they are representative of CSA-binding variants
de6S (containing 55% 4S) and CSA-de6S (containing 55% 4S). Values repre-
sent mean adhesion (⫾S.E.; four experiments) relative to control. CSA-de6S
expressed in malaria during pregnancy.
contained 55% 4S and 45% 0S, CSC-de6S contained 16% 4S and 77% 0S, and By comparison of the inhibitory activities of defined CS
CSB-de4S contained 50% 4S and 46% 0S. CSA-14D/F, CSA 14-mer fractions D oligo- and polysaccharides (Table 1) on adhesion of the differ-
or F; CSB-14, CSB 14mer fraction; polysacc, polysaccharides.
ent isolates, several common features were observed (Fig. 4).
For all three isolates inhibition of adhesion was dependent on
Strain-specific Variation in the Specificity of Interactions 4-O-sulfated GalNAc and GlcUA as the predominant uronic
Relates to Polymorphisms in PfEMP1—We sought to identify acid rather than iduronic acid, whereas 6-O-sulfation interfered
oligosaccharides with optimal inhibitory activity against differ- with the binding interaction. Additionally using defined
ent isolates and evaluate differences between strains in the 12-mers with varying levels of 4-O-sulfation, we demonstrated
specificity for CS structural motifs for adhesion that relate to that inhibition was greater with oligosaccharides containing
polymorphisms in var2csa PfEMP1. We used three genetically mixed 4-O-sulfated and non-sulfated disaccharides for all three
distinct CSA-binding isolates, CS2, HCS3, and 3D7-CSA (sup- isolates. CSA-de6S (54.7% 4S and 45.3% 0S) more effectively
plemental Table S2) (6, 35, 36). Analysis by Northern blots and inhibited adhesion of all three isolates than did unmodified

22432 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 31 • AUGUST 3, 2007

 Interactions between P. falciparum and Chondroitin Sulfates
CSA (composition: 4.9% 0S, 55.3% 4S, and 39.8% 6S). CSC and
partially de-4-O-sulfated CSB had much less inhibitory activity.
Partially desulfated CSB (64.7 or 50.1% 4-O-sulfation for CSB-
de4S-1 and CSB-de4S-2, respectively) were used for compari-
son with the activity of CSA-de6S; the limited inhibitory activ-
ity of CSB-de4S can therefore be attributed to the presence of
IdoUA compared with GlcUA in CSA-de6S.
The isolates differed in the pattern and extent of inhibition by
defined oligo- and polysaccharides and varied in the level of
sulfation of test inhibitors needed for optimal inhibition of
adhesion. The level of sulfation required for optimal inhibition
by oligo- and polysaccharides was greater for CS2 than HCS3
and 3D7-CSA and somewhat higher for HCS3 than 3D7-CSA
(Fig. 4). The optimal sulfate content of 12-mers for inhibition of
IE adhesion was four or five sulfate groups (67– 83% sulfation)
for CS2, three or four sulfate groups (50 – 67% sulfation) for

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HCS3, and three sulfate groups for 3D7-CSA. The 12-mer frac-
tion with an average of two sulfate groups (33% sulfation) was
relatively inhibitory against 3D7-CSA but not CS2 or HCS3.
Those with zero or one sulfate group (0 –17% sulfation), on
average, were relatively non-inhibitory for all isolates. The
12-mer fraction with five sulfate groups was most effective with FIGURE 5. A, inhibition of adhesion of CS2 IEs to immobilized CSA or de-6-O-
CS2 IEs but had less activity with HCS3 or 3D7-CSA. Some sulfated CSA by CS 12-mers with differing sulfate content (0S– 6S) or by CSA
inhibition was also observed with fully 4-O-sulfated 12-mers, polysaccharide. Values represent mean ⫾ S.E. adhesion relative to control
inhibitor (two experiments in duplicate). The 12-mer fractions 0S to 6S were
but this was less than the inhibition with the partially 4-O- derived from de-6-O-sulfated CSA and contained, on average, zero to six 4-O-
sulfated 12-mers for all three isolates. Additionally HCS3 and sulfated disaccharides, respectively. B, adhesion of CS2 or 3D7-CSA IEs to
3D7-CSA IEs were substantially inhibited by CSC-de6S immobilized CS polysaccharides with differing sulfation levels and patterns.
Values are mean relative adhesion ⫾S.E. (for multiple experiments) where the
polysaccharide, which has a lower level of 4-O-sulfation (16%) maximum level of adhesion for each isolate is 100%. Polysaccharides were
than CSA-de6S (55%), whereas adhesion of CS2 IEs was not coated onto plastic surfaces at 10 ␮g/ml except for CSA-PR, which was coated
at 100 ␮g/ml. CSA and CSA-de6S were from bovine trachea. CSB-deS, partially
substantially inhibited by CSC-de6S. We found no major dif- desulfated CSB (50% 4-O-sulfated).
ferences between isolates in the minimum oligosaccharide
chain length for maximum inhibition of IE adhesion to CSA CSA-de6S. These findings reflect the preference for higher lev-
(data not shown). els of CS sulfation by CS2 versus 3D7-CSA that were observed
The activity of different inhibitors was not influenced by the in inhibition assays (Fig. 4) and indicate that CS2 is less affected
degree and pattern of sulfation of the CS receptor used in adhe- by the presence of 6-O-sulfation. CS2 IEs also bound well to
sion assays. To enable the most direct evaluation, we used CSA CSA-PR (64% of CSA-de6S binding), whereas 3D7-CSA bound
compared with CSA-de6S as the receptors. Inhibition of adhe- very poorly to this preparation (2% of CSA-de6S levels). The
sion of CS2 IEs to both receptors was maximal with 12-mers disaccharide composition of CSA-PR was determined as 4.2%
composed of four or five 4-O-sulfated disaccharide units bal- 0S, 66.8% 4S, and 29% 6S, which is very similar to the composi-
anced with non-sulfated disaccharide units (Fig. 5A). There was tion of CSA from bovine trachea used in our assays (4.9% 0S,
no substantial difference in the pattern of inhibitory activity of 55.3% 4S, and 39.8% 6S). However, the 4-O-sulfated disacchar-
the oligosaccharides when using CSA compared with CSA- ides of CSA-PR contain equal amounts of GlcUA and IdoUA,
de6S. Furthermore inhibition of adhesion to CSPGs isolated whereas CSA from bovine trachea has no detectable IdoUA (38,
from placental blood (17), which have very low levels of sulfa- 56). None of the isolates adhered to CSC, CSB, or partially de-4-
tion, was also maximal with 12-mers having four or five sulfate O-sulfated CSB (Fig. 5B and data not shown).
groups (data not shown). These findings suggest that the spec-
ificity of parasite-CS interactions is determined by parasite fac- DISCUSSION
tors such as the PfEMP1 type expressed. These studies establish several important principles regard-
Differences between isolates in their fine specificity for CS ing the molecular basis of interactions among PfEMP1, CSA,
interaction were also observed in direct adhesion assays of IEs and placental tissue that are highly relevant to understanding
to immobilized CSA polysaccharides (Fig. 5B). For all isolates, malaria pathogenesis and biology. We demonstrated that
adhesion was substantially higher with CSA-de6S than the par- native PfEMP1, isolated from CS2 IEs and encoded by var2csa,
ent CSA, consistent with observations of greater inhibitory can bind to immobilized CSA but not CSC or CSB, consistent
activity of partially 4-O-sulfated oligosaccharides. There was a with the specificity observed with intact IEs. We successfully
large difference between CS2 and 3D7-CSA IEs in binding to adapted this to an assay method using a 96-well plate format
unmodified CSA compared with CSA-de6S. Adhesion of 3D7- that can be used to probe CS-PfEMP1 interactions in further
CSA to CSA was only 12% of adhesion to CSA-de6S, whereas detail. Although recombinant var2csa PfEMP1 domains have
adhesion of CS2 IEs to unmodified CSA was 58% of adhesion to been shown to bind to CSA (22), it was important to establish

AUGUST 3, 2007 • VOLUME 282 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 22433

Interactions between P. falciparum and Chondroitin Sulfates
that native PfEMP1 also binds in a specific manner because to immobilized CSA and was similar to results from PfEMP1
expression of single recombinant domains may not accurately binding assays.
reflect the binding activities of full-length PfEMP1 (30, 57, 58). The structural basis of placental adhesion has not been
This is highlighted by studies reporting that several recombi- defined previously. Prior placental adhesion studies showed
nant Duffy-binding-like ␥ domains can bind CSA, but their role that CSA, but not CSC, polysaccharides inhibited adhesion (10,
in mediating IE adhesion or PfEMP1 binding to CSA remains 12). Overall results from placental adhesion studies were
unclear (30, 59). Our findings further support var2csa PfEMP1 remarkably similar to our previous results obtained by testing
as the key ligand for adhesion to CSA and its role in placental inhibition of adhesion to immobilized purified CSA (38). How-
malaria. It remains possible that other proteins could be ever, the difference in minimum chain length for inhibition
involved with PfEMP1 as part of a receptor-binding complex highlights the need for studies using placental tissue, or other
(60); however, no other genes have yet been identified. Our cell types, to confirm results obtained using purified CS prepa-
approach using native PfEMP1 may be valuable for comple- rations. Our findings that IEs adhere better to CSA-de6S than
menting future studies of the CSA binding activity of recombi- to unmodified CSA and that there is variation among isolates in
nant PfEMP1 domains expressed in other systems (22, 27) and the preference for undersulfated motifs may partly explain
to identify effective inhibitors. prior observations that placental isolates vary in the efficiency
We demonstrated direct binding of native PfEMP1 to CS with which they adhere to immobilized CSA preparations (9).

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oligosaccharides as NGL probes, which were derived from con- We identified partially 4-O-sulfated CSA 14-mers and
jugation of CS oligosaccharide fractions to an aminophospho- polysaccharides, generated by de-6-O-sulfation of commer-
lipid. The NGLs were immobilized on solid matrices and cially available CSA, as highly effective inhibitors of placental
probed with PfEMP1. The NGL approach has been used to adhesion that may have potential for therapeutic development.
study many other carbohydrate-protein interactions (61, 62) The IC50 values are the lowest reported for any placental adhe-
but has not been applied previously either to protein-GAG sion inhibitor to date and are within a range that has been
interactions or to malaria research. In previous investigations, achieved previously in vivo by administration of CS to humans
the minimum motif for interactions between IEs and CSA was (63). CSA polysaccharide has an average molecular mass of 45
determined by inhibition of IE adhesion to purified immobi- kDa, equivalent to ⬃200 monosaccharide residues in length. It
lized CSA or CSPGs (36 –38). In the present study, we found is not surprising that, on a molar basis, CSA polysaccharides are
that the minimum chain length for direct binding of PfEMP1 severalfold more active in inhibition assays than 14-mers (aver-
was eight monosaccharide units, whereas prior studies found age molecular mass, 3.135 kDa) due to multiple recognition
that 12-mers were required for inhibition of IE adhesion. These motifs present along the polysaccharide chain. Although there
differences may reflect real differences in requirements for were differences between isolates, we identified common fea-
PfEMP1 binding compared with requirements for inhibition of tures of CS-parasite interactions. These include a consistent
cellular adhesion. Alternatively the clustered presentation of preference or requirement for 4-O-sulfated mixed with non-
oligosaccharides immobilized as NGLs (62) may have increased sulfated GalNAc together with GlcUA rather than IdoUA,
the protein binding activity of shorter chain oligosaccharides. whereas 6-O-sulfation of GalNAc inhibited CS interaction with
Furthermore we demonstrated the importance of 4-O-sulfated IEs. These requirements correspond with the composition of
GalNAc and GlcUA rather than IdoUA for PfEMP1 binding. known CSPGs available for adhesion in the vasculature (sup-
PfEMP1 bound CSA oligosaccharide NGLs at higher levels than plemental Table S1). Oligosaccharides with 50 – 67% sulfation
NGLs derived from CSC; the difference between oligosaccha- would be effective against many isolates as they are active
ride NGLs from CSA and CSC of the same length was their against the three isolates tested here. Variation between isolates
pattern of sulfation (predominantly 4-O-sulfated versus 6-O- in the level of 4-O-sulfation required for maximum inhibition
sulfated for CSA and CSC, respectively) (36). The lack of may limit the ability to design and synthesize a single oligosac-
binding to CSB indicated the importance of GlcUA rather charide sequence that effectively inhibits adhesion of all iso-
than IdoUA for the interaction. These findings have impor- lates; oligosaccharide mixtures may be required.
tant implications for understanding the molecular basis of Isolate-specific variation among P. falciparum IEs in the pre-
adhesion, including structural studies that may identify crit- ferred structural motifs for adhesion to CSA correlates with
ical residues for binding. polymorphisms in PfEMP1 encoded by var2csa-type genes.
The structural features for IE adhesion to placental tissue Three different isolates expressing polymorphic variants of
were similar to those determined for direct binding of PfEMP1. var2csa varied in their sensitivity to inhibition of adhesion by
There was a requirement for 4-O-sulfated GalNAc mixed with oligo- and polysaccharides with different levels of sulfation and
non-sulfated GalNAc, GlcUA rather than IdoUA was impor- varied in their levels of direct binding to immobilized CS
tant, and 6-O-sulfation inhibited the interaction with IEs. Can- polysaccharides with differing disaccharide compositions.
didate CSA receptors in the placenta, including CSPGs of the These findings have major implications for the potential devel-
intervillous blood, cell-associated CSPGs, and thrombomodu- opment of therapies or vaccines against maternal malaria and
lin, vary in their level of sulfation, but all contain substantial for structural studies of receptor-ligand interactions. Structural
amounts of non-sulfated disaccharides in addition to 4-O-sul- and functional studies established with a single variant will
fated disaccharides (supplemental Table S1). The minimum need to be extended to different variants. The apparent effect of
chain length for maximal inhibition of adhesion was 10-mer. ligand polymorphism on specificity is likely to apply to other
This is shorter than that required for inhibition of IE adhesion pathogens that use GAGs for adhesion and host cell invasion

22434 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 31 • AUGUST 3, 2007

 Interactions between P. falciparum and Chondroitin Sulfates
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ble that the evolution of polymorphisms in CSA-binding 9. Beeson, J. G., Brown, G. V., Molyneux, M. E., Mhango, C., Dzinjalamala, F.,
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