Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 326-332
Inhibitory Activity of Human Lactoferrin and Its Peptide on Chondroitin
Sulfate A-, CD36-, and Thrombospondin-Mediated Cytoadherence of
Plasmodium falciparum-Infected Erythrocytes
By
Shigetoshi Eda,
Keiko Eda,
Jacques G. Prudhomme, and
Irwin W. Sherman
From Department of Biology, University of California, Riverside, CA.
 |
ABSTRACT |
Lactoferrin (LF), a human serum protein, strongly inhibited the
adherence of Plasmodium falciparum-infected erythrocytes (PE) to immobilized chondroitin sulfate A (CSA)-conjugated albumin at a
concentration of 100 µg/mL and blocked the PE binding to CD36-expressing Chinese hamster ovary (CHO) cells, as
well as immobilized CD36 at concentrations of 5 µg/mL and 100 µg/mL, respectively. Biotinylated LF bound to CD36 in a saturable
manner, and such binding was inhibited by unlabeled LF and the
anti-CD36 monoclonal antibody, 8A6, suggesting specificity of binding.
Additionally, LF inhibited PE binding to immobilized thrombospondin
(TSP) at a concentration of 100 µg/mL, and specific binding of LF to
TSP was confirmed using biotinylated LF. LF inhibited PE binding to C32
amelanotic melanoma cells in a dose-dependent manner. A peptide of LF,
Arg-Asn-Met Arg-Lys-Val Arg-Gly-Pro-Pro-Val-Ser-Cys (amino acid
residues 25-37 of LF), which has been suggested to contribute to LF
binding to various materials, including CSA, inhibited PE binding to
immobilized CSA-conjugated albumin, immobilized CD36, CD36-expressing
CHO cells, immobilized TSP, and C32 amelanotic melanoma cells, as well
as LF itself. These results suggest that LF peptide may provide the
basis for developing agents that are able to inhibit CSA-, CD36-, and
TSP-mediated cytoadherence of PE.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HUMAN MALARIA is caused by four species
of parasites (Plasmodium falciparum, P vivax, P ovale, and
P malariae). Five hundred million people are infected with
malaria each year, resulting in 1.5 to 2.7 million deaths (mainly
children under the age of 5 and a number of pregnant women)
annually.1 P falciparum is responsible for most
human deaths. P falciparum-parasitized erythrocytes (PE) bind
to postcapillary endothelial cells (EC) of the brain and other
organs.2 Several EC receptors, including CD36,3 intercellular cell adhesion molecule-1 (ICAM-1),4
thrombospondin (TSP),5 chondroitin sulfate A
(CSA),6,7 vascular cell adhesion molecule-1 (VCAM-1),
E-selectin,8 and CD31,9 have been reported to
contribute to the cytoadherence of PE. One of the first steps in
developing antiadhesive agents is to find substances that can block
cytoadherence. For instance, it was reported that a peptide,
Arg-Gly-Asp (RGD), first identified in fibronectin, has the potential
to reduce experimental metastasis (ie, adhesion of cancer cells to
endothelial cells) in vivo.10 We previously reported that
infusion of a synthetic peptide, His-Pro-Leu-Gln-Lys-Thr-Tyr (HPLQKTY), based on a sequence found in an erythrocyte-membrane protein
(band 3), into monkeys infected with P falciparum malaria resulted in the appearance of PE (bearing mature stage parasites) in
the peripheral blood, suggesting an antisequestering
effect.11
The human serum protein, lactoferrin (LF), binds to
cell-surface chondroitin sulfate12 and has been shown to
inhibit sporozoite invasion of hepatocytes.13 We examined
the effects of LF on PE adherence, first focusing on CSA as a receptor
and extending this to other PE receptors, ie, CD36 and TSP. The
N-terminus peptidic sequence of LF was suggested to be involved in LF
binding to CSA and inhibition of sporozoite invasion. Therefore, we
also examined the inhibitory effects of an N-terminus peptide of LF,
Arg-Asn-Met-Arg-Lys-Val-Arg-Gly-Pro-Pro-Val-Ser-Cys (RNMRKVRGPPVSC;
amino acid residues 25-37 of LF),14 on CSA-, CD36-, and
TSP-mediated PE binding.
| |
MATERIALS AND METHODS |
Materials.
LF (from human milk), transferrin (TF; human), CSA (from bovine
trachea; molecular weight of 20 to 30 kD), mouse anti-CSA monoclonal
antibody (MAb), and bovine serum albumin (BSA; fraction V) were
obtained from Sigma (St Louis, MO). The biotinylation reagent,
biotin-BMCC, and alkaline phosphatase-conjugated
streptavidin were obtained from Pierce (Rockford, IL). A peptide,
RNMRKVRGPPVSC (LF peptide), was obtained from Research Genetics
(Huntsville, AL). Other peptides
(Lys-Pro-Ser-Glu-Lys-Ile-Gln-Val-Leu-Lys-Asn-Leu-Lys-Arg-Asn-Tyr [KPSE
peptide; amino acid residues 394-409 of CD36] and
Phe-Ala-Ser-Pro-Val-Glu-Asn-Pro-Asp-Asn-Tyr-Gly-Phe [FASP peptide;
amino acid residues 300-312 of CD36]) were obtained from Coast
Scientific (San Diego, CA). CSA-conjugated BSA (CSA-BSA) was prepared
to facilitate immobilization as described previously.15 Briefly, a mixture of CSA (final concentration 80 mg/mL), BSA (final
concentration 13.6 mg/mL), and sodium cyanoborohydride (final
concentration 20 mg/mL) in 0.2 mol/L potassium phosphate (pH 8.0) were
incubated at 37°C for 96 hours. The mixture was dialyzed against 1 L
of distilled H2O (dH2O) before
use. Conjugation of CSA to BSA was confirmed by checkerboard titration
with anti-CSA antibody in an enzyme-linked immunosorbant assay, because
unconjugated CSA will not bind to plastic. CSA-BSA conjugate or BSA (2 mg/mL) was serially diluted in dH2O in precleaned
Costar vinyl microtiter plates (Corning, Acton, MA) at
50 µL/well and allowed to bind at 4°C overnight. Plates were
blocked with 2% BSA in 10 mmol/L Tris-buffered saline (TBS; pH 7.4) at
100 µL/well at room temperature for 1 hour. After rinsing with TBS,
anti-CSA MAb diluted 1:200 in TBS was added at 50 µL/well at room
temperature for 3 hours. Wells were rinsed with TBS, and alkaline
phosphatase-conjugated antimouse IgM (1:500 dilution in TBS) was added
at 50 µL/well at room temperature for 1 hour. Wells were washed with
TBS and color developed with p-nitrophenyl phosphate reagent
(Sigma) and read at 405 nm on a BioRad 450 microplate
reader (BioRad, Hercules, CA). The anti-CSA MAb did not bind to
BSA-coated wells. The concentration of BSA in CSA-BSA solution was
determined by Protein Assay (BioRad) using BSA as a
standard, and the concentrations of CSA-BSA were expressed as the
concentration of BSA (µg BSA/mL). The Protein Assay was not
interfered with by the presence of CSA in solution. The amount of CSA
was quantified according to the method of Enobakhare et
al.16 To immobilize CSA-BSA, 50 µL of CSA-BSA solution (2 mg BSA/mL) was added to wells of the microtiter plate and incubated at
37°C for 4 hours. Free CSA was completely removed by several washings
with dH2O. The amount of BSA and CSA of immobilized CSA-BSA was determined as described above. It was determined that 12 molecules (average) of CSA bound to 1 molecule of BSA by assuming the molecular weights of CSA and BSA are 25 kD and 67 kD, respectively. (Molecular weight of CSA ranges from 20 to 30 kD, according to the manufacturer.) Chinese hamster ovary (CHO) cells transfected with genes coding for
CD36 (CD36-CHO cells)17 were grown in RPMI
1640/10% fetal calf serum. The C32 amelanotic melanoma cell line
(American Type Culture Collection No. CRL 1585; ATCC,
Manassas, VA) was maintained in culture as described
earlier.18 The CD36-CHO cells and C32 amelanotic melanoma
cells were deposited on multiwell glass slides (Cel-line Associates,
Inc, Newfield, NJ) for cytoadherence assays as described
previously.19 Partially purified CD36 (CD36 fraction) was
obtained from human platelets essentially by the method of Tandon et
al.20 However, the last purification step using
Ultrogel AcA-44 (LKB, Rockville, MD) was omitted. TSP
was purified as described previously.21 Anti-CD36 MAbs,
FA6, and 8A6 were kindly provided by Dr John Barnwell (New York
University Medical Center, New York, NY) and Dr Lena
Edelman (Institut Pasteur, Paris, France), respectively.
Parasites.
All P falciparum lines (FCR-3 from The Gambia,
Brazil; and the CSA-preferring line FAF-EA8CHO5,
designated CS2,22 derived from the Brazilian Ituxi isolate)
were cultured in O+ human erythrocytes as described
previously.23 Cultures of FCR-3 and CS2 were synchronized at the mature stage by gelatin flotation.24
Immobilization of proteins on slides.
CSA-BSA was diluted to 5 µg BSA/mL in dH2O, and 3 µL
was spotted on plastic slides (Nunc, Inc, Naperville, IL). After drying at room temperature, the surface of the CSA-BSA-bound spot was blocked
with 0.1% BSA in phosphate-buffered saline (PBS) at room temperature
for 2 hours. In the case of CD36, 5 mL of CD36 solution containing
0.1% Triton X-100 (Sigma) (initial concentration 268 µg protein/mL) was incubated with 0.5 g of SM-2 beads (Bio-Rad) at
room temperature for 2 hours and dialyzed against 500 mL of distilled
water to remove most of the detergent. Five microliters of the dialyzed
solution was mounted onto multiwell glass slides, and incubated at
4°C overnight. To immobilize TSP, a TSP solution (50 µg/mL in 50 mmol/L Bis-Tris, 100 mmol/L NaCl, 25 mmol/L calcium lactate, 1 mmol/L
MnCl2, pH 7.4 [BTC]) was spotted on plastic slides and
incubated at 4°C overnight. CD36- and TSP-immobilized spots were
blocked with 0.5% BSA in PBS at room temperature for 1 hour.
Cytoadherence assay.
Cytoadherence assays were performed as described
previously.19,25 The spots of fixed target cells or
immobilized protein were pretreated with 5 µL of inhibitor (LF, LF
peptide, and control proteins/peptides) in 10 mmol/L PBS (pH 7.0) at
room temperature for 2 hours. After removal of inhibitor solution, the
slides were placed in individual compartments of a partitioned plastic
box containing 7 mL Bis-Tris buffer (50 mmol/L Bis-Tris, 100 mmol/L NaCl, 2 mmol/L CaCl2, pH 6.6 for CSA and CD36, BTC for
TSP). One to two hundred microliters of packed erythrocytes (5 to 10%
parasitemia) was added to each compartment of the plastic box. After
incubating under a heat lamp for 90 minutes with gentle rocking, the
erythrocyte suspension was carefully aspirated. The slides were rinsed
by adding 7 mL of the buffer and rocking for 5 minutes, after which time the buffer was aspirated. The slides were washed three times, fixed, and stained as described previously.19 The number of bound erythrocytes/target cells or square millimeters
area of immobilized protein was counted under a light microscope.
LF binding to immobilized CD36 and TSP.
Ten microliters of ascites fluid of the anti-CD36 MAb, FA6, was diluted
1:100 in distilled water and dried on microtiter wells. To immobilize
CD36 protein on the FA6-bound wells, the solution of CD36 (5.36 µg
protein/mL) was added to the wells and incubated at 4°C overnight.
The wells were blocked with 100 µL of 1% casein at room temperature
for 1 hour. In the case of TSP, TSP was immobilized by incubating the
wells with 5 µL of 50 µg/mL TSP at 4°C overnight. The wells were
blocked with 100 µL of 1% casein at room temperature for 1 hour.
Twenty microliters of biotinylated LF (prepared according to the
manufacturer's [Pierce] instructions using biotin-BMCC) was added to
the wells and incubated at room temperature for 2 hours. Unbound
biotinylated LF was removed by several washes. The amount of bound
biotinylated LF to the immobilized CD36 or TSP was determined using
alkaline phosphatase-conjugated streptavidin (50 µL of 1:1,000
dilution). Sigma 104 phosphatase substrate (Sigma) was
used for color development, and the plate was read at 405 nm.
| |
RESULTS |
P falciparum PEs have been shown to bind to cell-surface CSA,
and it has been suggested that this binding is implicated in the
development of malarial infections of the placenta.26
Recently, it was reported that LF binds to cell-surface
CSA12; so first, we examined the effect of LF on the
binding of CS2 (CSA-preferring P falciparum line)-PE to CSA-BSA
immobilized on plastic slides. Binding of PE to CSA was inhibited in a
dose-dependent manner when immobilized CSA-BSA was pretreated with
LF-containing solutions; 70% inhibition was obtained at a
concentration of 100 µg/mL (Fig 1). We
used human TF as a negative control, because LF and TF have 59%
homology in their primary amino acid sequence.14
Pretreatment of immobilized CSA-BSA with TF was without effect at a
concentration of 1 mg/mL. TF was without effect in all cytoadherence
assays (described below), even at concentrations in which LF exerted a
maximum inhibitory effect (data not shown).
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| Fig 1.
Inhibition of CS2-PE binding to immobilized CSA-BSA by
LF. Spots of CSA-BSA immobilized on plastic slides were pretreated with
indicated concentrations of LF. After removal of the LF solution, the
slides were subjected to the cytoadherence assay. Each point represents
the mean ± standard deviation (SD) of triplicate
determinations. The average of the number of CS2 bound to immobilized
CSA-BSA in the absence of LF was 298.8 PEs/mm2.
|
|
Because CD36-mediated cytoadherence has been claimed to be implicated
in cerebral malaria, we next attempted to see whether LF would also
inhibit PE binding to CD36. Pretreatment of immobilized CD36 with LF
inhibited the binding of FCR-3 (CD36-preferring P falciparum
line)-PE to immobilized CD36 in a dose-dependent manner (Fig
2A). Maximal inhibition (~70%
inhibition) was obtained at 100 µg/mL. The FCR-3-PE binding to
CD36-expressing CHO cells was also inhibited (~90%) by pretreatment
with LF (Fig 2B) at a concentration of 5 µg/mL. These results
indicated that LF had the ability to bind to CD36 protein. To confirm
this, the binding of biotinylated LF to CD36 was examined as described
in Materials and Methods. MoAb FA6-immobilized microtiter plates were
used to specifically immobilize CD36 protein on the wells. The binding
of biotinylated LF to immobilized CD36 was observed and reached a
plateau at a concentration of 15 µg/mL (Fig
3). Binding was considerably lower when FA6
or CD36 was omitted, showing the specificity of binding. Furthermore,
binding was inhibited by pretreatment of immobilized CD36 by LF (Fig
4A) or the anti-CD36 monoclonal antibody,
8A6 (Fig 4B), but not by TF or an irrelevant monoclonal antibody (data not shown). This, again, showed the specificity of the LF binding and
confirmed that the observed inhibitory activity of LF was caused by its
binding to CD36. The PE binding of FCR-3 to immobilized TSP was also
inhibited by pretreatment of TSP with LF in a dose-dependent manner
(Fig 5), reaching 30% of the control at a
concentration of 200 µg/mL. Binding of biotinylated LF to immobilized
TSP was also observed (Fig 6A), and such
binding was inhibited by the presence of unlabeled LF (Fig 6B), but not
by TF (data not shown), showing that the inhibitory activity of LF was
a result of its binding to TSP. The effect of LF as an antiadhesive for
PE binding to the amelanotic melanoma cell, C32 was also examined:
binding was inhibited by pretreatment of C32 cells with LF (Fig
7). Fifty percent inhibition was obtained
at a concentration of 200 µg/mL; higher concentrations were without
further effect. Therefore, we next examined inhibitory effect of LF
peptide (RNMRKVRGPPVSC) on PE binding to target cells or immobilized
protein used above. Surprisingly, LF peptide inhibited PE binding in a
dose-dependent manner to all targets we tested (Table
1). Maximum inhibition of PE binding to
immobilized CSA-BSA, immobilized CD36, CD36-CHO cells, immobilized TSP,
and C32 amelanotic melanoma cells by LF peptide was 20.7%, 34.9%,
49.0%, 20.1%, and 39.5% of control at concentrations of 500, 1,000, 100, 200, and 100 µg/mL, respectively. Two irrelevant peptides, FASP
and KPSE peptide, were without effect even at a concentration of 1 mg/mL.
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| Fig 2.
Inhibition of FCR-3-PE binding to (A) immobilized CD36
and (B) CD36-CHO cells. The immobilized CD36 or CD36-CHO cells on glass
slides were pretreated with LF at the indicated concentrations. After
removal of the LF solution, the slides were subjected to the
cytoadherence assay. Each point represents the mean ± SD of
triplicate determinations. The averages of the number of FCR-3 bound to
CD36-CHO cell and immobilized CD36 in the absence of LF were 3.85 PEs/CHO cell and 1,415.9 PEs/mm2, respectively.
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| Fig 3.
The binding of biotinylated LF to immobilized CD36. CD36
was immobilized to microtiter wells via FA6 anti-CD36 antibody. The
biotinylated-LF binding to the immobilized CD36 was examined ( ). The
same assays were conducted, omitting immobilization of FA6 ( ) or
CD36 ( ). Each point represents the mean ± SD of triplicate
determinations. A405, absorbance at 405 nm.
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| Fig 4.
Inhibition of biotinylated-LF binding to immobilized CD36
by (A) unlabeled LF or (B) anti-CD36 antibody, 8A6. The
CD36-immobilized wells were pretreated with (A) unlabeled LF or 8A6 at
the indicated concentration. After removal of pretreatment solution,
the microtiter plates were subjected to biotinylated-LF-binding assay.
Absorbance at 405 nm (A405) obtained in the absence of FA6 was
subtracted from data, and the results are expressed as a percentage of
the binding in the absence of inhibitor. Each point represents the mean ± SD of triplicate determinations. The averages of A405 obtained by
biotinylated LF binding to CD36 in the absence of unlabeled LF were (A)
0.126 and (B) 0.115, respectively.
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| Fig 5.
Inhibition of FCR-3-PE binding to immobilized TSP by LF.
The spots of TSP immobilized on plastic slides were pretreated with the
indicated concentrations of LF. After removal of the LF solution, the
slides were subjected to the cytoadherence assay. Each point represents
the mean ± SD of triplicate determinations. The average of the number
of FCR-3 bound to immobilized TSP in the absence of LF was 1,757.5 PEs/mm2.
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| Fig 6.
(A) The binding of biotinylated LF to immobilized TSP.
TSP was immobilized to microtiter wells. The biotinylated-LF binding to
the immobilized TSP () was examined. The same assay was conducted
omitting TSP (). Each point represents the mean ± SD of triplicate
determinations. (B) Inhibition of biotinylated-LF binding to
immobilized TSP by unlabeled LF. Biotinylated LF binding to immobilized
TSP was examined in the presence of the indicated concentrations of
unlabeled LF. Absorbance at 405 nm obtained in the absence of TSP was
subtracted, and the results are expressed as a percentage of the
binding in the absence of inhibitor. The average A405 obtained by
biotinylated LF binding to TSP in the absence of unlabeled LF was 0.26. Each point represents the mean ± SD of triplicate determinations.
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| Fig 7.
Inhibition of FCR-3-infected cell binding to C32 cells.
The C32 cells were pretreated with LF at the indicated concentrations.
After removal of the LF solution, the slides were subjected to the
cytoadherence assay. Each point represents the mean ± SD of
triplicate determinations. The average number of FCR-3 bound to C32
amelanotic melanoma cells in the absence of LF was 6.66 PEs/melanoma
cell.
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| |
DISCUSSION |
LF, an iron-binding glycoprotein with molecular weight of 80 kD, exists
in milk, other body fluids, serum, and specific granules of
neutrophils. Several roles for LF have been suggested, such as
bactericidal and immunomodulatory effects.27 It was also reported that LF inhibited the invasion of P falciparum
sporozoites into host hepatocytes.13 Cytoadherence of PE to
postcapillary EC is believed to relate to pathogenesis and death in
falciparum malaria. As a possible antiadhesion protein, we focused on
LF, because it was reported that LF bound to CSA,12 a
receptor for PE. Herein we have shown that LF inhibited PE binding to
immobilized CSA (Fig 1), immobilized CD36, CD36-CHO cells (Fig 2),
immobilized TSP (Fig 5), and C32 amelanotic melanoma cells (Fig 7), and
the effective concentrations of LF ranged from 5 to 100 µg/mL. These concentrations are much higher than the physiological level of LF in
serum (0.2 µg/mL).28 However, because LF is released from neutrophils activated by tumor necrosis factor (TNF)-
29
and this cytokine is increased in serum during a malarial
infection,30 it is possible that higher concentrations of
LF do occur during infection. Increased concentrations of LF (0.9 µg/mL) were found in the serum of patients with sepsis.28
Furthermore, other investigators have observed 12 µg/mL LF in serum
of relapsing chronic myeloid leukemia.31 Possibly in the
microenvironment, where infected cells are sequestered, the
concentration of LF released from neutrophils can be sufficient to
partially inhibit cytoadherence.
We showed that the inhibitory effects of LF on PE binding were a result
of specific binding of LF to CD36 and TSP. Recently, we found that the
PE binding site on TSP is the RGD sequence-containing motif in the
type 3 repeat region; LF also bound to the type 3 repeat region (S. Eda, unpublished results). This region of TSP has been
reported to be involved in the binding of many cell types, including
human endothelial cells, smooth muscle cells, C32 amelanotic melanoma
cells, U937 monocytic cell line, etc, to TSP.32 This raises
the possibility that LF inhibits not only PE binding, but also the
binding of many cell types via the type 3 repeat region of TSP. In the
case of CD36, Asch et al reported that the amino acid sequences 8-21 and 97-110 were responsible for PE binding.33 Later,
Serghides et al claimed that the amino acid sequence 243-333 was
responsible for PE binding.34 The LF binding site on CD36 is presently unknown; however, it is possible that LF binds to the CD36
binding site for the PE, or to adjacent sites. It will be of interest
to examine this possibility, because PE-binding sites on CD36 have been
located to overlapping or adjacent binding sites for various CD36
ligands, such as TSP (amino acid residues 87-9933 and
93-12035 of CD36), oxidized low density lipoprotein (LDL;
residues 155-183 of CD36),36 and fatty acid (residues 127-279 of CD36).37
LF binds to a wide range of materials (such as lysozyme,
lipopolysaccharide (LPS), oxidized LDL, DNA, etc) and cell surface receptors (intestinal brush border, phytohemagglutinin-activated lymphocyte, monocyte/macrophage, hepatocyte, platelet,
etc).27 Our present results add CD36 and TSP to the list of
LF-binding materials. This feature of LF may be favorable to block PE
binding in the malarial patient, because as mentioned above many EC
receptors are thought to contribute to PE binding. Indeed, the binding
of PE to C32 cells (which express CD36, ICAM-1, CSA, and
TSP5,38,39) was blocked by LF in a dose-dependent manner
(Fig 7). This result was supported by the fact that the binding of
FCR-3 to immortalized human brain endothelial cells, BB19 (which
express CD36, ICAM-1, VCAM-1, and E-selectin40), was also
inhibited by LF in a static microtiter cytoadherent assay, which we
have recently developed (J.G. Prudhomme, unpublished results).
These results suggested that it is possible and important to find an
active peptide from LF for the future chemical design and synthesis of
antiadhesion agents. We focused on amino acid residues 25-37 of LF
(RNMRKVRGPPVSC,14 termed LF peptide in this paper), because
this region was suggested to bind to CSA41 and to inhibit
sporozoite invasion of the hepatocyte.13 Moreover, this
region has been suggested to contribute to a multifunctional property
of LF, such as LF binding to LF receptors on hemagglutinin-activated lymphocytes42 and platelets,43 remnant
receptor,44 and lipopolysaccharide.45 Synthetic
LF peptide inhibited PE binding to immobilized CSA-BSA, CD36 and TSP,
and C32 amelanotic melanoma cells, as well as LF protein per se. FASP
and KPSE peptide did not inhibit PE binding, showing the specificity of
the inhibitory effect of LF peptide. Furthermore, the inability of KPSE
peptide to inhibit PE binding indicates the inhibitory effect of LF
peptide on PE binding was not simply because of the cationic feature (4 basic amino acids out of 13 amino acids) of LF
peptide, because KPSE peptide also contains a net 4 positive charges in
15 amino acids (5 basic amino acids and 1 acidic amino acid).
Several surface proteins on PE, such as PfEMP-1, sequestrin, and band
3-related adhesin, were implicated in PE cytoadherence.2 In the case of PfEMP-1 and the band 3-related adhesin, the region(s) that contribute to PE binding to their receptor have been identified, and antiadhesive peptides have been developed. Baruch et al reported that the fusion protein (rC1-2) that lies in cysteine-rich interdomain region of PfEMP1 mediated binding to CD36, but not to TSP nor CSA.46 Amino acid residues 1-179 of rC1-2 were able to
inhibit PE binding to CD36, but not to CSA under both static and flow conditions.47 We have found that the peptidic sequence
HPLQKTY, a exofacial loop 3 region of band 3, is involved in
TSP-mediated PE binding, and the synthetic peptide inhibited PE binding
to immobilized TSP, but not to CD36.48 It should be noted
that the combination of the relatively small size (tridecapeptide) and
the multi-inhibitory effects of the LF peptide could be of considerable
advantage as a lead compound for developing potent antiadhesive agents.
Further studies on the binding sites of CD36 and TSP for LF may provide
valuable information on the molecular mechanisms of the inhibitory
action of LF peptide, as well as the possible effects of the peptide on
the functions of CD36 and/or TSP.
| |
FOOTNOTES |
Submitted November 30, 1998; accepted March 9, 1999.
Supported by the National Institute of Allergy and Infectious Diseases,
National Institutes of Health (AI21251).
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 Irwin W. Sherman, PhD,
Department of Biology, University of California, Riverside, CA 92521;
e-mail: sherman{at}mail.ucr.edu.
| |
REFERENCES |
1.
Trigg P, Kondrachine A:
The current global malaria situation, in
Sherman IW
(ed):
Malaria: Parasite Biology, Pathogenesis and Protection. Washington, DC, ASM Press, 1998, p 11.
2.
Land KM, Sherman IW, Gysin J, Crandall IE:
Anti-adhesive antibodies and peptides as potential therapeutics for Plasmodium falciparum malaria.
Parasitol Today
11:19, 1995
3.
Ockenhouse CF, Tandon NN, Magowan C, Jamieson GA, Chulay JD:
Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor.
Science
243:1469, 1989[Abstract/Free Full Text]
4.
Berendt AR, Simmons DL, Tansley J, Newbold CI, Marsh K:
Intercellular adhesion molecule 1 is an endothelial cell adhesion receptor for Plasmodium falciparum.
Nature
341:57, 1989[Medline]
[Order article via Infotrieve]
5.
Roberts DD, Sherwood JA, Spitalnik SL, Panton LJ, Howard R, Dixit VM, Frazier WA, Miller LH, Ginsburg V:
Thrombospondin binds falciparum malaria parasitized erythrocytes and mediate cytoadherence.
Nature
318:64, 1985[Medline]
[Order article via Infotrieve]
6.
Robert C, Pouvelle B, Meyer P, Muanza K, Fujioka H, Aikawa M, Scherf A, Gysin J:
Chondroitin-4-sulfate (proteoglycan), a receptor for Plasmodium falciparum-infected erythrocyte adherence on brain microvascular endothelial cells.
Res Immunol
146:383, 1995[Medline]
[Order article via Infotrieve]
7.
Fried M, Duffy PE:
Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta.
Science
272:1502, 1996[Abstract]
8.
Ockenhouse CF, Tegoshi T, Maeno Y, Benjamin C, Ho M, Kan K, Thway Y, Win K, Aikawa M, Lobb LL:
Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: Roles for endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule 1.
J Exp Med
176:1183, 1992[Abstract/Free Full Text]
9.
Treutiger CJ, Heddini A, Fernandez V, Muller WA, Wahlgren M:
PECAM/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes.
Nat Med
3:1405, 1997[Medline]
[Order article via Infotrieve]
10.
Ruoslahti E:
RGD and other recognition sequences for integrins.
Ann Rev Cell Dev Biol
12:697, 1996[Medline]
[Order article via Infotrieve]
11.
Crandall I, Collins WE, Gysin J, Sherman IW:
Synthetic peptides based on motifs present in human band 3 protein inhibit cytoadherence/sequestration of the malaria parasite Plasmodium falciparum.
Proc Natl Acad Sci USA
90:4703, 1993[Abstract/Free Full Text]
12.
Ziere GJ, Kruijt JK, Bijsterbosch MK, van Berkel CJ:
Recognition of lactoferrin and aminopeptidase M-modified lactoferrin by the liver: Involvement of proteoglycans and the remnant receptor.
Biochem J
313:289, 1996
13.
Sinnis P, Willnow TE, Briones MRS, Herz J, Nussenzweig V:
Remnant lipoproteins inhibit malaria sporozoite invasion of hepatocytes.
J Exp Med
184:945, 1996[Abstract/Free Full Text]
14.
Metz-Boutigue M-H, Jollés J, Mazurier J, Schoentgen F, Legrand D, Spik G, Montreuil J, Jollés P:
Human lactoferrin: Amino acid sequence and structural comparisons with other transferrins.
Eur J Biochem
145:659, 1984[Medline]
[Order article via Infotrieve]
15.
Gray RG:
Antibodies to carbohydrate: Preparation of antigens by coupling carbohydrate to proteins by reductive amination with cyanoborohydride.
Methods Enzymol
50:155, 1978[Medline]
[Order article via Infotrieve]
16.
Enobakhare BO, Bader DL, Lee DA:
Quantification of sulfated glycosaminoglycans in chondroitin/alignate cultures, by use of 1,9-dimethylmethylene blue.
Anal Biochem
243:189, 1996[Medline]
[Order article via Infotrieve]
17.
Hasler T, Albrecht GR, Van Schravendijk MR, Aguiar JC, Morehead KE, Pasloske BL, Ma C, Barnwell JW, Greenwood B, Howard RJ:
An improved microassay for P. falciparum cytoadherence using stable transformations of Chinese hamster ovary cells expressing CD36 or intracellular adhesion molecule-1.
Am J Trop Med Hyg
48:332, 1993
18.
Sherman IW, Valdez E:
In vitro cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells: Factors affecting adhesion.
Parasitology
98:359, 1989
19.
Crandall I, Smith H, Sherman IW:
Plasmodium falciparum: The effect of pH and Ca2+ concentration on the in vitro cytoadherence of infected erythrocytes to amelanotic melanoma cells.
Exp Parasitol
73:362, 1991[Medline]
[Order article via Infotrieve]
20.
Tandon NN, Lipsky RH, Burgress WH, Jamieson GA:
Isolation and characterization of platelet glycoprotein IV (CD36).
J Biol Chem
264:7570, 1989[Abstract/Free Full Text]
21.
Lawler JW, Slayter HS, Coligan JE:
Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.
J Biol Chem
253:8609, 1978[Abstract/Free Full Text]
22.
Cooke BM, Rogerson SJ, Brown GV, Coppel RL:
Adhesion of malaria-infected red blood cell to chondroitin sulfate A under flow conditions.
Blood
88:4040, 1996[Abstract/Free Full Text]
23.
Trager W, Jensen JB:
Human malaria parasites in continuous culture.
Science
193:673, 1976[Abstract/Free Full Text]
24.
Pasvol G, Wilson RJ, Smalley ME, Brown J:
Separation of viable schizont-infected cells of Plasmodium falciparum from human blood.
Ann Trop Med Parasitol
72:87, 1978[Medline]
[Order article via Infotrieve]
25.
Udeinya IJ, Schmidt JA, Aikawa M, Miller LH, Green I:
Falciparum malaria-infected erythrocytes specifically bind to cultured human endothelial cells.
Science
213:555, 1981[Abstract/Free Full Text]
26.
Rogerson SJ, Brown GV:
Chondroitin sulfate A as an adhesion receptor for Plasmodium falciparum-infected erythrocytes.
Parasitol Today
13:70, 1997[Medline]
[Order article via Infotrieve]
27.
Brock JH:
Lactoferrin structure-function relationship, in
Hutchens TH,
Lönnerdal B
(eds):
Lactoferrin: Interactions and biological functions. Totowa, NJ, Humana, 1997, p 3.
28.
Nuijens JH, Abbink JJ, Wachtfogel YT, Colman RW, Eerenberg JM, Dors D, Kamp AJM, Strack van Schijndel RJM, Thijs LG, Hack CE:
Plasma elastase 1-antitrypsin and lactoferrin in sepsis: Evidence for neutrophils as mediators in fatal sepsis.
J Lab Clin Med
119:159, 1992[Medline]
[Order article via Infotrieve]
29.
Balazovich KJ, Suchard SJ, Remick DG, Boxer LA:
Tumor necrosis factor- and FMLP receptors are functionally linked during FMLP-stimulated activation of adherent human neutrophils.
Blood
88:690, 1996[Abstract/Free Full Text]
30.
Kwiatkowski D, Hill AVS, Sambou I, Twumasi P, Castracane J, Manogue KR, Cerami A, Brewater DR, Greenwood BM:
TNF concentration in fatal cerebral, non-fatal cerebral and uncomplicated Plasmodium falciparum malaria.
Lancet
336:1201, 1990[Medline]
[Order article via Infotrieve]
31.
Bennett RM, Mohla C:
A solid-phase radioimmunoassay for the measurement of lactoferrin in human plasma: Variation with age, sex, and disease.
J Lab Clin Med
88:156, 1976[Medline]
[Order article via Infotrieve]
32.
Lahav J:
The functions of thrombospondin and its involvement in physiology and pathophysiology.
Biochim Biophys Acta
1182:1, 1993[Medline]
[Order article via Infotrieve]
33.
Asch AS, Liu I, Briccetti FM, Barnwell JW, Kwakye-Berko F, Dokun A, Goldberger J, Pernambuco M:
Analysis of CD36 binding domains: Ligand specificity controlled by dephosphorylation of an ectodomain.
Science
262:1436, 1993[Abstract/Free Full Text]
34.
Serghides L, Crandall I, Hull E, Kain KC:
The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36.
Blood
92:1814, 1998[Abstract/Free Full Text]
35.
Pearce SFA, Wu J, Silverstein RL:
Recombinant GST/CD36 fusion proteins define a thrombospondin binding domain.
J Biol Chem
270:2981, 1995[Abstract/Free Full Text]
36.
Nicholson AC, Pearce SFA, Silverstein RL:
Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Evidence implicating the lipid moiety of the lipoprotein as the binding site.
Arterioscler Thromb Vasc Biol
15:269, 1995[Abstract/Free Full Text]
37.
Baillie AGS, Coburn CT, Abumrad NA:
Reversible binding of long-chain fatty acids to purified FAT, the adipose CD36 homolog.
J Membr Biol
153:75, 1996[Medline]
[Order article via Infotrieve]
38.
Smith H, Nelson JA, Gahmberg CG, Crandall I, Sherman IW:
Plasmodium falciparum: Cytoadherence of malaria-infected erythrocytes to human brain capillary and umbilical vein endothelial cells
A comparative study of adhesive ligands.
Exp Parasitol
75:269, 1992[Medline]
[Order article via Infotrieve]
39.
Bumol TF, Walker LE, Reisfeld RA:
Biosynthetic studies of proteoglycans in human melanoma cells with a monoclonal antibody to a core glycoprotein of chondroitin sulfate proteoglycans.
J Biol Chem
259:12733, 1984[Abstract/Free Full Text]
40.
Prudhomme JG, Sherman IW, Land KM, Moses AV, Stenglein S, Nelson JA:
Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells.
Int J Parasitol
26:647, 1996[Medline]
[Order article via Infotrieve]
41.
Mann DM, Romm E, Migliorini M:
Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.
J Biol Chem
269:23661, 1994[Abstract/Free Full Text]
42.
Legrand D, Mazurier J, Elass A, Rochard E, Vergoten G, Maes P, Montreuil J, Spik G:
Molecular interactions between human lactotransferrin and the phytohemagglutinin-activated human lymphocyte lactotransferrin receptor lie in two loop-containing regions of the N-terminal domain I of human lactotransferrin.
Biochemistry
31:9243, 1992[Medline]
[Order article via Infotrieve]
43.
Leveugle B, Mazurier J, Legrand D, Mazurier C, Montreuil J, Spik G:
Lactotransferrin binding to its platelet receptor inhibits platelet aggregation.
Eur J Biochem
213:1205, 1993[Medline]
[Order article via Infotrieve]
44.
Ziere GJ, Bijsterbosch MK, van Berkel TJC:
Removal of 14 N-terminal amino acids of lactoferrin enhances its affinity for parenchymal liver cells and potentiates the inhibition of 
-very low density lipoprotein binding.
J Biol Chem
268:27069, 1993[Abstract/Free Full Text]
45.
Elass-Rochard E, Roseanu A, Legrand D, Trif M, Salmon V, Motas C:
Lactoferrin-lipopolysaccharide interaction: Involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide.
Biochem J
312:839, 1995
46.
Baruch DI, Ma XC, Singh HB, Bi X, Pasloske BL, Howard RJ:
Identification of a region of PfEMP1 that mediates adherence of Plasmodium falciparum infected erythrocytes to CD36: Conserved function with variant sequence.
Blood
90:3766, 1997[Abstract/Free Full Text]
47.
Cooke BM, Nicoll CL, Baruch DI, Coppel RL:
A recombinant peptide based on PfEMP-1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow.
Mol Microbiol
30:83, 1998[Medline]
[Order article via Infotrieve]
48.
Lucas JZ, Sherman IW:
Plasmodium falciparum: Thrombospondin mediates parasitized erythrocyte band 3-related adhesin binding.
Exp Parasitol
89:78, 1998[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION