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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2121-2127
CD36 Peptides That Block Cytoadherence Define the CD36 Binding
Region for Plasmodium falciparum-Infected Erythrocytes
By
Dror I. Baruch,
Xin C. Ma,
Brittan Pasloske,
Russell J. Howard, and
Louis H. Miller
From the Laboratory of Parasitic Diseases, National Institute of
Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, MD; and Affymax Research Institute, Santa Clara,
CA.
 |
ABSTRACT |
Mature Plasmodium falciparum parasitized erythrocytes (PE)
sequester from the circulation by adhering to microvascular endothelial cells. PE sequestration contributes directly to the virulence and
severe pathology of falciparum malaria. The scavenger receptor, CD36,
is a major host receptor for PE adherence. PE adhesion to CD36 is
mediated by the malarial variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1), and particularly by its cysteine-rich interdomain region 1 (CIDR-1). Several peptides from the
extended immunodominant domain of CD36 (residues 139-184), including
CD36 139-155, CD36 145-171, CD36 146-164, and CD36 156-184 interfered
with the CD36-PfEMP1 interaction. Each of these peptides affected
binding at the low micromolar range in 2 independent assays. Two
peptides, CD36 145-171 and CD36 156-184, specifically blocked PE
adhesion to CD36 without affecting binding to the host receptor
intercellular adhesion molecule-1 (ICAM-1). Moreover, an adhesion
blocking peptide from the ICAM-1 sequence inhibits the PfEMP1-ICAM-1
interaction without affecting adhesion to CD36. These results confirm
earlier observations that PfEMP1 is also a receptor for ICAM-1. Thus,
the region 139-184 and particularly the 146-164 or the 145-171 regions
of CD36 form the adhesion region for P. falciparum PE.
Adherence blocking peptides from this region may be useful for modeling
the PE/PfEMP1 interaction with CD36 and for development of potential
anti-adhesion therapeutics.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SEQUESTRATION OF MATURE
stage parasitized erythrocytes (PE) is central for the survival and the
pathology of P. falciparum parasites.1-4 Adherence
of P. falciparum PE to endothelial cells in various blood
vessels can result in local microvascular occlusion contributing
directly to the pathology of P. falciparum
malaria.1,5,6
Adherence of PE to endothelial cells is mediated by the binding of the
infected erythrocyte to host receptors expressed on endothelial cells
including CD36, thrombospondin (TSP), intercellular adhesion molecule-1
(ICAM-1), and chondroitin sulfate A.2,7-10 Other molecules
such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and
CD31 mediate adherence of a minority of P. falciparum parasites.11-13 CD36 may play a predominant role in PE
adherence, as almost all adherence positive P. falciparum
strains and isolates bind to CD36.13 There is a significant
correlation between PE sequestration and expression of CD36 (and
ICAM-1) in various organs and vascular endothelium.5,14
Under flow conditions, which mimic in vivo blood flow, CD36 supports
stable stationary adherence of PE, while ICAM-1 mediates PE rolling and
adherence to TSP appears to be unstable.15 Recently, a
recombinant protein from P. falciparum erythrocyte membrane
protein 1 (PfEMP1), named rC1-2, located in the first cysteine-rich
interdomain region (CIDR-1) immediately after the first Duffy
binding-like (DBL) domain was shown to mediate PE adherence to
CD36.16,17
CD36 is an 88-kD glycoprotein scavenger receptor expressed on the
surface of various cells including platelets, adipocytes, monocytes,
macrophages, leukocytes, megakaryocytes, erythroblasts, melanoma cells,
and endothelial cells.5,18,19 Several CD36 genes cloned
from different organisms display high sequence
conservation.20 In the last years, several other proteins
were found to have sequence and structural homology with CD36. This
includes the type B scavenger receptor SR-BI/CLA-1, the lysosomal
protein LIMP II, 2 Drosophila proteins, and a
Caenorhabditis elegans protein, all members of the CD36 gene
family.20-22 CD36 is implicated in uptake of oxidized low-density lipoproteins (ox-LDL), long chain fatty acids, anionic phospholipids, phagocytosis of apoptotic neutrophils, and in signal transduction.23-30 CD36 can interact with a wide range of
different molecules including ox-LDL, the extracellular matrix proteins collagen and TSP, and the malarial protein
PfEMP1.17,20,27,29,31-35
Several anti-CD36 monoclonal antibodies (MoAbs) (8A6, OKM5, FA6-152,
and 10/5) that block binding of ox-LDL and recognition of apoptotic
neutrophils react with the CD36 immunodominant domain, defined by
residues 155-183.33,34,36 These MoAbs also efficiently block adhesion of PE to human CD36.8 However, it was
recently shown37 that the binding domain for ox-LDL resides
elsewhere (residues 28-93) on CD36. Thus, these MoAbs block a function
that resides outside the immunodominant region (155-183) of CD36,
either by steric hindrance or by changing the conformation of CD36
required for binding. The binding domains for collagen and TSP were
ascribed to the 415-427 and the 93-120 regions,
respectively.31,32 Residues 139-155, located just before
the immunodominant domain, were shown to induce a conformational
change in TSP, which leads to higher affinity interaction between TSP
and the 93-110 region of CD36.27,38
The inhibitory effect of MoAbs specific for the 155-184 region suggests
that the binding domains for the inhibitory MoAbs and for PE overlap.
However, PE bind to both human and mouse CD36, although only the human
CD36 is recognized by the inhibitory MoAbs.39,40 This
significant difference indicates that inhibitory MoAbs and infected
erythrocytes recognize different residues of the CD36 multifunctional
domain or that residues outside this region contribute significantly to
the PE-CD36 interaction.
To study the region(s) of CD36 involved in binding of PfEMP1 and PE
adhesion, we made a set of peptides from different regions of CD36 and
tested their ability to inhibit the PfEMP1-CD36 interaction and to
block PE adhesion.
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MATERIALS AND METHODS |
Parasites.
Malayan Camp (MC R+) and the FCR3-C5 P. falciparum
strains adhere to the host receptors CD36 and TSP, but not ICAM-1. The
ItG2-ICAM strain and clone A4ultra adhered to all 3 host
receptors.16,35
Peptides.
CD36-derived peptides CD36 62-75 (residues 62-75), CD36 233-250 (residue 238 (Lys) was replaced by a Tyr residue), CD36 358-370, CD36
397-409, and CD36 413-426 (with an additional Tyr residue) were custom
synthesized by Bio-Synthesis Inc, (Lewisville, TX). Peptides CD36
139-155 and CD36 93-110 were synthesized both by Neuros Corp (San Jose,
CA) and by Affymax Research Institute (Santa Clara, CA).
Peptides CD36 93-110C and CD36 C139-155 with additional terminal
cysteines at the carboxyl- or amino-terminus, respectively, were
purchased from Bachem California (Torrance, CA) and also synthesized at
Affymax Research Institute. Peptides CD36 139-149, CD36 142-152, and
CD36 146-155 were synthesized by SynPeP Corporation (Dublin, CA). A
scrambled version of the CD36 139-155 was also synthesized, but was
largely insoluble even at 100% dimethyl sulfoxide (DMSO).
Peptides CD36 145-171, CD36 146-164, and CD36 156-184 were synthesized
by the support facility, NIAID, NIH. The ICAM 15-20 was synthesized at
Affymax Research Institute. The amino acid sequences of the peptides
are described in Table 1. All peptides were synthesized by the Fmoc
method, purified to homogeneity by high-performance liquid
chromatography (HPLC) and confirmed structurally by mass
spectrometry. Peptide concentration was determined by BCA protein assay
(Pierce, Rockford, IL).
Soluble receptors.
Soluble CD36 extracellular domain was obtained in the form of harvest
supernatant (approximately 1 to 5 µg/mL) after phospholipase C
treatment of cultured cells as described before.35 Soluble ICAM-1 extracellular domain carrying the Protein A Ig binding domain
(ZZ-ICAM) was a gift from Dr Andrew Hutchinson (Glaxo-Wellcome, Stevange, UK).
Antibodies.
Mouse MoAb 179 (Affymax Research Institute) recognizes an epitope
sequence incorporated into the carboxy terminus of CD36. Mouse MoAb 141 (Affymax Research Institute) recognizes GST.17,35 Rabbit
IgG was purchased from Jackson Immunoresearch Labs (West Grove, PA).
Affinity purification of surface radiolabeled PfEMP1.
PE were surface radioiodinated and sequentially extracted with triton
X-100 (TX100) and sodium dodecyl sulfate (SDS) as
described.35 Inhibition of binding of PfEMP1 to sCD36 or
ICAM-1 was as described.35 Briefly, 5 to 10 µL of PE SDS
extracts were reconstituted in 1 mL of 25 mmol/L HEPES, 1% bovine
serum albumin (BSA) 0.5% TX100 in RPMI-1640 (BM-T, binding medium
containing TX100) at pH 6.7 for CD36 and pH 7.3 for ICAM-1. Peptides
dissolved in DMSO were added to the appropriate concentration. DMSO was
added to give a final concentration of 5% DMSO. The reconstituted SDS
extracts were incubated, 16 hours at 4°C, with CD36-derived
peptides or, 1 hour at 21°C, with peptide ICAM-1 15-20 followed by
incubation, 3 hours at 21°C, with immobilized receptor, and
processed as described.35
Affinity-purification of CD36 with immobilized recombinant fragments
of PfEMP1.
Affinity-purification of CD36 with immobilized rC1-2 [1-179]
(GST-fusion) was described earlier.17 Briefly, 25 µL of
GammaBind Plus Sepharose beads (Pharmacia Biotech Inc, Piscataway, NJ)
were precoated, 90 minutes 21°C, with 10 µg of MoAb 141 (anti-GST), then incubated with 2.5 µg of rC1-2 [1-179]. The coated
beads were incubated 60 minutes at 21°C, with 450 µL of BM
containing peptides and 5% DMSO final concentration. A total of 50 µLl of soluble CD36 (0.5 to 1 µg/mL) was added and incubated 2 hours at 21°C. The beads were washed twice with BM, once with BM
without BSA, and solubilized in 40 µL of SDS sample buffer. One to
2.5-µL samples were fractionated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), immunoblotted, and probed with a 1:10,000
dilution of biotinylated MoAb 179 as primary antibody followed with
horseradish peroxidase (HRP)-conjugated streptavidin
(Jackson Immunoresearch Inc) at 1:5,000 dilution.
Densitometry.
Autoradiographs were scanned and individual bands were quantified after
background subtraction using the NIH Image 1.61 standard program.
Cytoadherence microassay.
Adherence of PE to immobilized proteins was performed by standard
methods.17,41,42 For ICAM-1 binding, plates were spotted with 50 µg/mL of rabbit IgG and incubated with 25 µg/mL of ZZ-ICAM. Blockade of PE adherence by the different peptides was tested by
preincubating, 1 hour at 37°C, PE in BM media (0.2% BSA)
containing peptides and 1% DMSO final concentration. PE were added to
the spotted receptors, incubated, 1 hour at 37°C, washed 4 times
with BM, fixed, stained, and counted.42
| |
RESULTS |
CD36 and ICAM-1-derived peptides inhibit the binding of PfEMP1 to CD36
and ICAM-1, respectively.
A panel of CD36 peptides27 (Fig
1) were assayed at 500 µmol/L (300 µmol/L for peptide CD36 233-150)
for their effects on the binding of 125I-PfEMP1 from the
Malayan Camp (MC R+) strain (Fig 1A) and clone
FCR3-C5 (Fig 1B) to CD36. Addition of DMSO (5%) had very
little effect (Fig 1B), but in some of the assays, a 50% reduction in
binding was observed (Fig 1A). Therefore, in each assay, the effect of
each peptide was measured and compared with the binding of the DMSO
control (100% binding). Peptides CD36 139-155, CD36 62-75, and CD36
233-250 blocked the CD36-PfEMP1 interaction by more than 50% for MC
and 40% for FCR3-C5 (Fig 1). Peptide CD36 139-155 was the
most active, affecting binding by 70% to 82%. Other peptides either
increased binding or had very limited effect (up to 20%). The
different effects of peptides on specific PfEMP1 bands is attributed to
the variant sequence of different PfEMP1 molecules.17,41,43
Three partial peptides from the 139-155 region (CD36 139-149, CD36
142-152, and CD36 145-155, Table 1) had no
effect on binding (unpublished data).
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| Fig 1.
CD36 peptides inhibit the binding of
125I-PfEMP1 to CD36. TX100 insoluble material from surface
iodinated PE was extracted with SDS (SDS extract) and reconstituted in
RPMI containing 1% BSA and 0.5% TX100.35 Peptides
dissolved in DMSO were added to the sample, incubated overnight, and
allowed to bind to CD36-coated beads for 3 hours. The beads were washed
3 times and processed for SDS-PAGE as before.35 Peptides
were tested at 500 µmol/L, except for peptide CD36 233-255, which was
tested at 300 µmol/L. All samples except No DMSO, had a 5% final
concentration of DMSO. The percent inhibition of binding from the DMSO
control is indicated at the bottom of the figure. Negative numbers
means higher binding than the DMSO control. (A) MC R+ SDS
extract; (B) FCR3-C5 SDS extract. The percent inhibition of
peptide CD36 358-370 with FCR3-C5 extract was calculated without the
specific background subtraction.
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Dose response assays with the 3 active peptides demonstrated that
peptides CD36 62-75 and CD36 233-250 were active only at high
concentrations (250 to 300 µmol/L) and actually increased binding at
concentrations of 100 µmol/L or lower
(Fig 2A). Thus, the activity of these
peptides may not be highly specific for the PfEMP1-CD36 interaction. On
the other hand, peptide CD36 139-155 had an apparent 50% inhibition
(IC50) of  5 µmol/L (Fig 2A and Table 2). The peptide had no effect on the
binding of ItG-ICAM PfEMP1 to ICAM-1 (not shown), indicating that its
blocking activity is specific for the PfEMP1-CD36 interaction. To
verify these results, 3 preparations of peptide CD36 139-155 derived
from independent sources were assayed and found to consistently block
binding of PfEMP1 to CD36.
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| Fig 2.
Concentration-dependent inhibition of binding of
125I-PfEMP1 to CD36 and ICAM-1. (A) Inhibition of binding
to CD36 using MC R+ SDS extract was performed as in Fig
1. Peptide concentration is given in µmol/L. The concentration of
peptide CD36 397-409 was 500 µmol/L. (B) Inhibition of binding to
ICAM-1 using ItG-ICAM SDS extract was performed as in Fig 1, except
that the peptides were preincubated with the extract for 1 hour at room
temperature. Peptide concentration is given in µmol/L and all samples
(except No DMSO) had a 5% final concentration of DMSO. The percent
inhibition of binding from the DMSO control is indicated at the bottom
of the figure.
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Table 2.
Approximate IC50 Values of Peptides
Inhibiting the Interaction of PfEMP1 or Infected Erythrocytes With CD36
or ICAM-1
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Peptide ICAM-1 15-20 blocks adherence of intact PE (ItG2-ICAM strain)
to ICAM-1.44 This peptide also blocked the binding of
ItG2-ICAM PfEMP1 to ICAM-1 at a concentration of 100 µmol/L or
higher with an IC50 of about 200 µmol/L (Fig 2B). The
peptide did not block the interaction of MC PfEMP1 with CD36 (not
shown). These results are consistent with independent binding domains for CD36 and ICAM-1 on PfEMP1.35,45
CD36-derived peptides block binding of rC1-2 [1-179] to CD36.
A region from the CIDR-1 domain of MC-PfEMP1, represented by the
recombinant protein rC1-2, was recently demonstrated to bind CD36 and
to mediate PE adherence to CD36.17 Peptide CD36 139-155 blocked the interaction of CD36 with rC1-2 [1-179] with apparent IC50 of about 2 µmol/L (Fig 3
and Table 2). At 50 µmol/L, peptides CD36 62-75 and CD36 233-250, which blocked binding of PfEMP1 to CD36 at high concentrations (Fig 2),
had little (23.1%) to no effect (6.3%) on this interaction compared
with 78% to 92% inhibition with peptide CD36 139-155 (Fig 3).
Peptides CD36 139-149 and CD36 142-152, but not peptide CD36-145-155,
blocked the binding of CD36 to rC1-2 (1-179) (Fig 3A). Some of
this activity may be attributed to the highly hydrophobic nature of the
first 2 peptides. Also, these assays are not truly quantitative, but
demonstrate the range of effective inhibition of binding of each
peptide.
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| Fig 3.
CD36 peptides inhibit the binding of rC1-2 [1-179] to
CD36. Peptides were prepared in 0.45 mL of binding media pH 6.7 containing 1% BSA and 5% DMSO and microfuged for 5 minutes before
addition to the coated beads to remove insoluble peptide material.
Peptides were incubated, 1 hour at room temperature with beads coated
with rC1-2 [1-179] before addition of sCD36. After 3 hours
incubation, the beads were washed and solubilized for
SDS-PAGE.17 The binding of CD36 was assayed by Western blot
using biotinylated MoAb 179 recognizing a tag incorporated to the
sequence of sCD36.17 All samples (except No DMSO) had a 5%
final concentration of DMSO. Percent inhibition of binding (from the
DMSO control) is indicated at the bottom of the figure. Negative
numbers means higher binding than the DMSO control. (A) Various
CD36-derived peptides tested at 50 µmol/L. (B)
Concentration-dependent inhibition of binding of CD36 to rC1-2
[1-179]. Peptides concentration is in µmol/L. Peptide CD36 139-155 was largely insoluble at 100 µmol/L and peptide CD36 146-164 was only
partially soluble at 50 µmol/L. In both cases, most of the peptide
was removed by the centrifugation step resulting in low to no
inhibition (data not shown).
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Earlier studies suggested that the adherence blocking MoAb OKM5
recognizes the 139-155 region of CD36.27 Human-mouse
hybrids of CD36 demonstrated that OKM5, 15.2, and other adherence
blocking MoAbs actually recognize the adjacent region (155-183) of
CD36.39 Therefore, additional peptides from the 155-184 and
139-184 regions were synthesized (see Table 1) and measured for their
effect on binding of CD36 to the rC1-2 [1-179] GST fusion protein
(Fig 3 and Table 2). Peptides CD36 146-164 and CD36 156-184 inhibited the CD36-rC1-2 [1-179] interaction at 50 µmol/L (Fig 3A) and were active even at a concentration of 0.1 µmol/L (Fig 3B). Peptide CD36
156-184 was also active at 0.01 µmol/L (only partial inhibition), while peptide CD36 146-164 appeared to be inactive at this
concentration (data not shown). Peptide CD36 145-171 inhibition profile
for the rC1-2 [1-179]-CD36 interaction was similar to that of CD36 146-164 (not shown). Thus, peptides from both regions, CD36 139-155 and
CD36 156-184, represent sites of CD36, which interact with the PE
receptor, PfEMP1.
The inhibitory effect of different peptides is influenced by their
solubility.
The relative solubility of the various peptides was determined by the
appearance and the size of a precipitate after centrifugation. Although
this does not give an accurate measurement of solubility and of the
actual concentration of soluble material, it gives a good indication if
a peptide is only partially or completely soluble at the conditions of
each assay. Peptide CD36 156-184 was soluble even at high
concentrations (>250 µmol/L). However, several of the active
peptides, mainly CD36 139-155 and CD36 146-164, were not completely
soluble at concentrations higher than 50 to 100 µmol/L, as large
precipitates were observed and removed by centrifugation. This
observation explains the reduction of inhibition associated with high
peptide concentrations for some peptides (D. Baruch, unpublished
observations). Peptide CD36 145-171 solubility was higher than CD36
146-164, but not as good as CD36 155-184. Thus, at the high µmol/L
range, the actual final concentration of the peptide in the assay
solution may be lower, depending on the relative solubility of the
peptide in the assay solution. This is particularly important in
blockade of adherence assays. In this assay, the amount of DMSO must be
restricted to 1% (final) compared with 5% DMSO or 5%
DMSO/0.5%TX100/0.1% SDS used in the affinity purification assays.
Moreover, significantly higher peptide concentration is needed to block
the interaction of the multivalant PE with CD36. Thus, marginally
soluble peptides may not be soluble at the concentration required for
blockade of PE adherence, although they may effectively block the
PfEMP1-CD36 interaction. However, differences in the IC50
(Table 2), measured at the low µmol/L range are not affected by
peptide solubility and represent true differences in the inhibitory
activity of the different peptides.
CD36 and ICAM-1-derived peptides block PE adhesion.
Peptide CD36 156-184 at a concentration of 100 µmol/L blocked PE
adhesion to CD36 of strain A4ultra by more than 70%
(Fig 4A), with an IC50 of about
25 µmol/L (Fig 4C and Table 2). Another peptide, CD36 145-171, with
higher solubility than CD36 146-164 (but lower than CD36 156-184)
blocked PE adhesion to CD36 with an IC50 of about 86 µmol/L (Fig 4C and Table 2). Other peptides tested, CD36 413-426, CD36 139-155, CD36 146-164, and ICAM-1 15-20 had no inhibitory effect
on PE binding (Fig 4A). However, peptides CD36 139-155 and CD36 146-164 were only partially soluble at the 50 to 100 µmol/L concentration and
failed to reach the high concentration necessary to block PE adhesion.
This is particularly true for peptide CD36 139-155 that had much higher
IC50 than the other peptides (Fig 3B and Table 2). The
higher binding found with some of the peptides is attributed to the
combined effect of both peptide and DMSO resulting in elevated
adherence of PE or higher binding of PfEMP1 (see Figs 1 through 4 and
D. Baruch, unpublished observation). In each case, the percent
inhibition of adherence was calculated from the DMSO control (no
peptide).
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| Fig 4.
Blockade of PE adherence to immobilized CD36 and ICAM-1.
PE from clone A4ultra were incubated for 1 hour in binding media
containing the appropriate peptide and final concentration of 1% DMSO,
then added to the spotted receptor for 1 hour and processed as
described in Materials and Methods. Results are given as percent
binding from the control (no peptide). (A) Blockade of adhesion to CD36
tested at 100 µmol/L. MoAb 8A6 was tested at 10 µg/mL. (B) Blockade
of adherence to ICAM-1. Peptide CD36 156-184 was tested at 100 µmol/L, peptide ICAM-1 15-20 was tested at 100 and 500 µmol/L. (C)
Concentration-dependent blockade of adherence to CD36. Peptide
inhibition was tested at 1 to 100 µmol/L range as described in (A).
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Peptide CD36 156-184 had no inhibitory effect on PE adherence to ICAM-1
(Fig 4B). The ICAM-1 15-20 peptide completely blocked adhesion to
ICAM-1 at 500 µmol/L and more than 60% at 100 µmol/L (Fig 4B and
Table 2) with an IC50 of about 97 µmol/L, but had no
effect on PE adhesion to CD36 (Fig 4A). These results demonstrate that
the activity of the CD36 peptides and in particular CD36 156-184 is
specific for PE adherence to CD36 and that the ICAM-1 15-20 peptide was
specific for PE adherence to ICAM-1 (Fig 4).
| |
DISCUSSION |
Binding to CD36 plays a significant role in adherence of P. falciparum-infected erythrocytes.2,13-15 Antibodies
that are able to block this interaction, such as 8A6, 10/5, and OKM5,
recognize a single immunodominant domain (155-183) in human
CD36.39,40 However, the inhibitory MoAbs do not bind to
mouse CD36 while PE do.40 The MoAbs also block the binding
of ox-LDL residing well outside the immunodominant
domain,37 indicating a possible conformational change in
CD36 mediated by Ab binding. This suggests that the regions of CD36
recognized by PE and the inhibitory MoAbs may not be the same.
Therefore, we tested peptides from the sequence of CD36 to identify the
PE binding domain of CD36.
Three independent assays were applied to assess the ability of
different peptides to block the PE or PfEMP1 interaction with CD36. We
identified several peptides CD36 139-155, CD36 146-171, CD36 145-171, and CD36 156-184 to have blocking effect on binding to CD36 in 2 different assays. These peptides had no effect on binding to the host
receptor ICAM-1, indicating that the inhibition is specific for CD36.
These peptides derive from the extended immunodominant region of CD36
(139-183), as residues 139-155 are believed to contribute to the
binding of the OKM5 and similar antibodies to CD36.27,39
Several other peptides CD36 62-75, CD36 233-250, CD36 139-149, and CD36
142-152 also gave partial blocking activity (Figs 1 and 3). However,
these peptides were positive for inhibition only in 1 of the 3 assays
used and were active at much higher concentrations (IC50
>200 µmol/L) than the CD36 139-184 peptides (IC50 <5
µmol/L). In addition, none of these peptides blocked PE adhesion.
Peptides CD36 97-110 and CD36 8-21 reported by Asch et al46
to block PE adhesion did not show any inhibition in our assays (CD36
97-110) or are lacking (CD36 8-21) from the soluble CD36 used here.
Nevertheless, the possibility that the above residues contribute to the
PE interaction with CD36 cannot be excluded.
Attempts to define the minimal binding domain were partially
successful. Both peptides CD36 145-171 and CD36 146-164 were active,
although only CD36 145-171 was able to block PE adhesion. Our attempts
were complicated by the lack of solubility of the different peptides.
Apart from CD36 156-184 and ICAM-1 15-20, which were readily soluble in
aqueous solution, other peptides were much less soluble and could not
be properly tested at high concentrations required for efficient
inhibition. This is especially true in the PE blockade of adhesion
assay where the DMSO content is restricted to maximum of 1% (compare
with 5% in other assays). However, it is clear that although peptide
CD36 139-155 was active, it displayed a much lower inhibition than the
other 3 peptides (Table 2). Thus, we submit that the CD36 binding
domain for PfEMP1 and infected erythrocyte is located at residues
139-184 of CD36 and in particular residues 145-171 or residues 146-164.
The PfEMP1 region corresponding to the rC1-2 [1-179] fragment of the
CIDR-1 domain display significant variability among var genes including those originated from CD36 adherent
parasites.17 Variation in the sequence of the CD36 binding
domain, CIDR-1, and in binding domains for other host receptors, may
result in qualitative and quantitative differences in adhesion of
individual PE expressing PfEMP1 variants. These variations are expected
to play a significant role governing the specific organ (eg, lung, heart, brain, placenta etc) and site of sequestration of individual PE.
For example, PE sequestered in placenta binds to chondroitin sulfate A,
but not to CD36.47 Although alternative recognition of
residues of CD36 is possible, it appears that infected erythrocytes recognize the same CD36 domain, regardless of their variant sequences. The recombinant protein rC1-2 [1-179] blocked adhesion of all antigenically variant CD36-adherent PE tested.17 Also, the
adhesion of various P. falciparum strains is blocked by Abs to
the immunodominant domain of CD36.8,17,40 The peptides
tested here provide additional support for a single adhesion region.
These peptides inhibit the PE/PfEMP1 interaction with CD36 of 3 different strains, known to express different CIDR-1
sequences.16,17
Peptides from the CD36 139-184 region may have an anti-adhesion
therapeutic potential. However, the high concentration needed for
effective blockade of adherence and the relative low solubility of
these peptides must be improved significantly for any possible anti-adherence therapeutics. In addition, several functions of CD36 may
reside in this region or are influenced by Ab binding to this region,
including binding of Ox-LDL, binding of fatty acids, phagocytosis of
apoptotic neutrophils, and signal transduction properties.20,25,33,34,37 Adhesion of PE to this region may
cause local inhibition or activation of some of the CD36-dependent processes on endothelial cells. Individuals who lack CD36 expression on
monocytes are at risk of developing anti-CD36 antibodies
(anti-NaKa) directed against the 155-183 region.36 The finding that infected erythrocytes bind to
the CD36 multifunctional immunodominant domain raises the question of
the effect of PE sequestration on CD36-dependent functions.
Malaria-infected erythrocytes were found to activate monocytes and
platelets, presumably by binding to CD36.25 Injection of
peptides from the CD36 139-184 region may also have some adverse effect. However, it is conceivable that only a brief treatment will be
required to affect PE adhesion resulting in release of sequestrating
parasites to the circulation, without any prolonged adverse effect on
the host.
In summary, we identified the extended immunodominant multifunctional
domain of CD36 (residues 139-184) as the PE binding domain and in
particular residues 145-171 and 146-164. Peptides from this domain
blocked PE adherence in vitro and may be of therapeutic value. These
findings expand our knowledge about the molecular interactions between
infected erythrocytes and CD36.
| |
ACKNOWLEDGMENT |
The authors thank Dr John Barnwell for providing MoAb 8A6 and Andrew
Hutchinson for the ZZ-ICAM-1 recombinant protein.
| |
FOOTNOTES |
Submitted November 3, 1998; accepted May 12, 1999.
Supported by Affymax Research Institute and Grant No.
(HRN-6001-A-00-2043-00) to R.J.H from the United States Agency for
International Development Malaria Vaccine Development Program.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dror I. Baruch, MD, Laboratory
of Parasitic Diseases, NIAID, NIH Bldg 4, Room B1-37, 4 Center Dr, MSC
0425, Bethesda, MD 20892; e-mail: dbaruch{at}niaid.nih.gov.
| |
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