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.

HUMAN MALARIA is caused by four species of parasites (Plasmodium falciparum, P vivax, P ovale, andP 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 CD36,3intercellular cell adhesion molecule-1 (ICAM-1),4thrombospondin (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.

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.15Briefly, 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.

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.26Recently, 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.14Pretreatment 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).

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.

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.

Close modal

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 falciparumline)–PE to immobilized CD36 in a dose-dependent manner (Fig2A). 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 (Fig3). 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 (Fig4A) 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 (Fig7). 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 (Table1). 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.

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.

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.

Close modal
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.

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.

Close modal
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.

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.

Close modal
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.

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.

Close modal
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.

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.

Close modal
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.

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.

Close modal
Table 1.

Effect of Peptides on PE Binding to Immobilized Proteins and Cells

Peptides Concentration (μg/mL)PE Binding (% of control)
Targets
CSA-BSA CD36 CD36-CHO Cell TSP C32 Cell
None  —  100  100  100 100  100  
LF peptide  10  76.1 ± 11.3 ND  ND  75.2 ± 13.5  ND 
 20  42.6 ± 1.2  ND  ND 68.0 ± 8.7  ND  
 50  41.2 ± 20.9 ND  77.3 ± 10.7  30.8 ± 2.6 68.1 ± 28.9  
 100  38.0 ± 14.4  85.8 ± 32.7 49.0 ± 5.0  38.2 ± 4.4  39.5 ± 14.6  
 200 ND  79.6 ± 26.4  ND  20.1 ± 6.7 ND  
 500  20.7 ± 1.9  58.0 ± 5.6 47.8 ± 8.1  ND  43.7 ± 8.8  
 1,000 21.9 ± 10.1  34.9 ± 20.3  50.6 ± 10.4 ND  37.2 ± 10.9  
FASP peptide  1,000 93.2 ± 16.9  107.6 ± 13.3  107.6 ± 30.0 98.4 ± 2.3  93.7 ± 12.4  
KPSE peptide  1,000 90.0 ± 18.7  106.6 ± 10.1  98.2 ± 14.7 95.5 ± 10.7  108.3 ± 27.4 
Peptides Concentration (μg/mL)PE Binding (% of control)
Targets
CSA-BSA CD36 CD36-CHO Cell TSP C32 Cell
None  —  100  100  100 100  100  
LF peptide  10  76.1 ± 11.3 ND  ND  75.2 ± 13.5  ND 
 20  42.6 ± 1.2  ND  ND 68.0 ± 8.7  ND  
 50  41.2 ± 20.9 ND  77.3 ± 10.7  30.8 ± 2.6 68.1 ± 28.9  
 100  38.0 ± 14.4  85.8 ± 32.7 49.0 ± 5.0  38.2 ± 4.4  39.5 ± 14.6  
 200 ND  79.6 ± 26.4  ND  20.1 ± 6.7 ND  
 500  20.7 ± 1.9  58.0 ± 5.6 47.8 ± 8.1  ND  43.7 ± 8.8  
 1,000 21.9 ± 10.1  34.9 ± 20.3  50.6 ± 10.4 ND  37.2 ± 10.9  
FASP peptide  1,000 93.2 ± 16.9  107.6 ± 13.3  107.6 ± 30.0 98.4 ± 2.3  93.7 ± 12.4  
KPSE peptide  1,000 90.0 ± 18.7  106.6 ± 10.1  98.2 ± 14.7 95.5 ± 10.7  108.3 ± 27.4 

Spots of immobilized proteins and cells were pretreated with the indicated concentrations of peptide solutions, and the pretreatment solutions were removed before cytoadherence assay. Cytoadherence assays were conducted as described under Materials and Methods and in the legends of Figs 1, 2, 5, and 7. Each value represents the mean ± SD of triplicate determinations.

Abbreviation: ND, not determined.

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 falciparumsporozoites 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)-α29and 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.28Furthermore, 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.2In 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.

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.

1
Trigg
 
P
Kondrachine
 
A
The current global malaria situation
Malaria: Parasite Biology, Pathogenesis and Protection.
Sherman
 
IW
1998
11
ASM Press
Washington, DC
2
Land
 
KM
Sherman
 
IW
Gysin
 
J
Crandall
 
IE
Anti-adhesive antibodies and peptides as potential therapeutics for Plasmodium falciparum malaria.
Parasitol Today
11
1995
19
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
1989
1469
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
1989
57
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
1985
64
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
1995
383
7
Fried
 
M
Duffy
 
PE
Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta.
Science
272
1996
1502
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
1992
1183
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
1997
1405
10
Ruoslahti
 
E
RGD and other recognition sequences for integrins.
Ann Rev Cell Dev Biol
12
1996
697
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
1993
4703
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
1996
289
13
Sinnis
 
P
Willnow
 
TE
Briones
 
MRS
Herz
 
J
Nussenzweig
 
V
Remnant lipoproteins inhibit malaria sporozoite invasion of hepatocytes.
J Exp Med
184
1996
945
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
1984
659
15
Gray
 
RG
Antibodies to carbohydrate: Preparation of antigens by coupling carbohydrate to proteins by reductive amination with cyanoborohydride.
Methods Enzymol
50
1978
155
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
1996
189
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
1993
332
18
Sherman
 
IW
Valdez
 
E
In vitro cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells: Factors affecting adhesion.
Parasitology
98
1989
359
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
1991
362
20
Tandon
 
NN
Lipsky
 
RH
Burgress
 
WH
Jamieson
 
GA
Isolation and characterization of platelet glycoprotein IV (CD36).
J Biol Chem
264
1989
7570
21
Lawler
 
JW
Slayter
 
HS
Coligan
 
JE
Isolation and characterization of a high molecular weight glycoprotein from human blood platelets.
J Biol Chem
253
1978
8609
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
1996
4040
23
Trager
 
W
Jensen
 
JB
Human malaria parasites in continuous culture.
Science
193
1976
673
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
1978
87
25
Udeinya
 
IJ
Schmidt
 
JA
Aikawa
 
M
Miller
 
LH
Green
 
I
Falciparum malaria-infected erythrocytes specifically bind to cultured human endothelial cells.
Science
213
1981
555
26
Rogerson
 
SJ
Brown
 
GV
Chondroitin sulfate A as an adhesion receptor for Plasmodium falciparum-infected erythrocytes.
Parasitol Today
13
1997
70
27
Brock
 
JH
Lactoferrin structure-function relationship
Lactoferrin: Interactions and biological functions.
Hutchens
 
TH
Lönnerdal
 
B
1997
3
Humana
Totowa, NJ
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 α1-antitrypsin and lactoferrin in sepsis: Evidence for neutrophils as mediators in fatal sepsis.
J Lab Clin Med
119
1992
159
29
Balazovich
 
KJ
Suchard
 
SJ
Remick
 
DG
Boxer
 
LA
Tumor necrosis factor-α and FMLP receptors are functionally linked during FMLP-stimulated activation of adherent human neutrophils.
Blood
88
1996
690
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
1990
1201
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
1976
156
32
Lahav
 
J
The functions of thrombospondin and its involvement in physiology and pathophysiology.
Biochim Biophys Acta
1182
1993
1
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
1993
1436
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
1998
1814
35
Pearce
 
SFA
Wu
 
J
Silverstein
 
RL
Recombinant GST/CD36 fusion proteins define a thrombospondin binding domain.
J Biol Chem
270
1995
2981
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
1995
269
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
1996
75
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
1992
269
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
1984
12733
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
1996
647
41
Mann
 
DM
Romm
 
E
Migliorini
 
M
Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.
J Biol Chem
269
1994
23661
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
1992
9243
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
1993
1205
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 β-very low density lipoprotein binding.
J Biol Chem
268
1993
27069
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
1995
839
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
1997
3766
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
1998
83
48
Lucas
 
JZ
Sherman
 
IW
Plasmodium falciparum: Thrombospondin mediates parasitized erythrocyte band 3-related adhesin binding.
Exp Parasitol
89
1998
78

Author notes

Address reprint requests to Irwin W. Sherman, PhD, Department of Biology, University of California, Riverside, CA 92521; e-mail: sherman@mail.ucr.edu.

Sign in via your Institution