Plasmodium Falciparum: The Adherence of Erythrocytes Infected with Human Malaria Can Be Mimicked Using Pfalhesin-Coated Microspheres

Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542-555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by irnmunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells tTansfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546-553 and 820-829 and called pfalhesin) were produced. The pfalhesin-coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546-553 had little binding capacity for melanoma cells.

These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel 'artificial' P. falciparum-infected erythrocytes for use in in vitro and in vivo studies.     

Plasmodium falciparum: CD36 Dependent Cytoadherence or Rosetting of Infected Erythrocytes is Modulated by Knobs

A knobless (K-) line of the FCR-3 isolate of Plasmodium falciparum was obtained by gelatin flotation. Immunofluorescent staining and immunoblots indicated that both the K-line and the K+ (knobby) line from which it was derived contained similar forms of potentially adhesive modified band 3 protein. When the K+ and K-lines were assayed for their cytoadherent and rosetting abilities the K+ line showed a high level of CD36 dependent cytoadherence, whereas the K-line demonstrated a marked pH dependent increase in rosetting. Rosetting was inhibited by the addition of peptides based on band 3 motifs, suggesting that cytoadherence and rosetting involve the same adhesin but that the presence of knobs affects whether the adherent preference of the infected erythrocyte is uninfected red cells or endothelial/C32 amelanotic melanoma cells.     

Plasmodium falciparum: CD36 Dependent Cytoadherence or Rosetting of Infected Erythrocytes is Modulated by Knobs

A knobless (K-) line of the FCR-3 isolate of Plasmodium falciparum was obtained by gelatin flotation. Immunofluorescent staining and immunoblots indicated that both the K-line and the K+ (knobby) line from which it was derived contained similar forms of potentially adhesive modified band 3 protein. When the K+ and K-lines were assayed for their cytoadherent and rosetting abilities the K+ line showed a high level of CD36 dependent cytoadherence, whereas the K-line demonstrated a marked pH dependent increase in rosetting. Rosetting was inhibited by the addition of peptides based on band 3 motifs, suggesting that cytoadherence and rosetting involve the same adhesin but that the presence of knobs affects whether the adherent preference of the infected erythrocyte is uninfected red cells or endothelial/C32 amelanotic melanoma cells.     

Plasmodium falciparum: cytoadherence of malaria-infected erythrocytes to human brain capillary and umbilical vein endothelial cells--a comparative study of adhesive ligands.

The cytoadherence of Plasmodium falciparum-infected erythrocytes (FCR-3 line) to human brain capillary endothelial cells (HBEC), C32 amelanotic melanoma cells, and human umbilical vein endothelial cells (HUVEC) was studied. The adhesion of infected red cells was HBEC > amelanotic melanoma > HUVEC. The presence or absence of the adhesive ligands ICAM-1 (CD54 or intercellular adhesion molecule 1), ICAM-2, and CD36 (= glycoprotein IV) was determined for each of these cells by indirect immunofluorescence using the monoclonal antibodies RR1/1, 6D5, and OKM 5/OKM 8, respectively. It appeared that a major ligand for the FCR-3 line of P. falciparum with amelanotic melanoma cells and HBECs was CD36. Binding to HUVECs was very low, presumably due to their lack of expression of CD36. HBECs, because of their ease of in vitro propagation, long-term maintenance of cytoadherent properties, and their high degree of adhesiveness, will be useful for in vitro studies of adherence.     

An in vitro assay for sequestration: binding of Plasmodium falciparum-infected erythrocytes to formalin-fixed endothelial cells and amelanotic melanoma cells

Erythrocytes infected with Plasmodium falciparum bind specifically to cultured endothelial cells and to a line of amelanotic melanoma cells. We have fixed endothelial cells and amelanotic melanoma cells in various ways and determined whether the fixed cells were still able to bind infected erythrocytes. Only cells fixed with 1.0-2.5% formalin in phosphate-buffered saline continued to bind infected erythrocytes as well as unfixed cells. The mechanism of binding to fixed and unfixed cells appeared to be identical for the following reasons. First, erythrocytes infected by parasite strains that bound to unfixed cells also bound to fixed cells while those that did not bind to unfixed cells did not bind to fixed cells. Second, immune serum that inhibited binding to unfixed cells also inhibited binding to fixed cells. Third, electron microscopy showed that knobs were the points of attachment between infected erythrocytes and both fixed and unfixed melanoma cells. Fixed cells gave reproducible results over at least 2 months. Thus, we have developed a simplified, reproducible assay for measuring binding of P. falciparum-infected erythrocytes to target cells.     

Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein

Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P. falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.     

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.     

Monoclonal antibodies that react with human band 3 residues 542–555 recognize different conformations of this protein in uninfected andPlasmodium falciparum infected erythrocytes

A monoclonal antibody generated against synthetic peptides patterned on amino acids 542–555 of human band 3, designated 1F4, specifically immunostainedPlasmodium falciparum-infected erythrocytes and inhibited the cytoadherence ofP. falciparum-infected erythrocytes to C32 amelanotic melanoma cells. 1F4 did not recognize intact band 3 protein on immunoblots, however it was reactive towards proteolytic fragments of band 3.The binding region of another murine monoclonal antibody previously reported to recognize the membrane spanning domain of human band 3, designated B6, was found to also recognize residues 542–555, however its properties differed from 1F4. Mab B6 recognized both infected and uninfected red cells, and reacted only with intact band 3 on immunoblots. Mab B6 was without effect on cytoadherence.These results demonstrate that monoclonal antibodies reactive against a common peptide sequence may bind to different conformations of the peptide sequence and suggest that the adherent competency ofP. falciparum-infected erythrocytes may result from a change in the surface topography of human band 3 protein.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - KLH Keyhole Limpet Hemocyanin - PBS Phosphate Buffered Saline - Mab Monoclonal Antibody - PMSF Phenylmethyl sulfonyl fluoride - i.p. intraperitoneum - TBS Tris Buffered Saline - H₂DIDS dihydro 4,4-diisothocyanostilbene-2,2-disulfonic acid - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid     

Role of phenylalanine 150 in the receptor-binding domain of the K88 fibrillar subunit.

Recently, we reported the isolation of three peptides, Ile-83-Ala-Phe-85, Ser-148-Leu-Phe-150, and Ala-156-Ile-Phe-158, derived from the K88 fibrillar subunit and found to inhibit the binding of K88 fibrillae to cavia erythrocytes or pig intestinal epithelial cells (A. A. C. Jacobs, J. Venema, R. Leeven, H. van Pelt-Heerschap, and F. K. de Graaf, J. Bacteriol. 169:735-741, 1987). The gene encoding the K88 fibrillar adhesin was modified by oligonucleotide-directed site-specific mutagenesis such that each of the phenylalanine residues at positions 85, 150, and 158 were replaced by serine. Replacement of phenylalanine 85 or 158 had no apparent effect on the biosynthesis of the fibrillae or on their adhesive capacity. In contrast, substitution of phenylalanine 150 with serine resulted in a dramatic decrease in adhesive capacity of the K88 fibrillae. Apparently, phenylalanine 150 plays an essential role in the interaction of the adhesin with receptor molecules present on eucaryotic cells.     

Purification and characterization of a Bacteroides loeschei adhesin that interacts with procaryotic and eucaryotic cells.

The adhesin of Bacteroides loeschei PK1295 that mediates coaggregation with Streptococcus sanguis 34 and hemagglutination of erythrocytes was purified to electrophoretic homogeneity. The lectinlike protein has an estimated native Mr of 450,000 and consists of six subunits of identical molecular weight (Mr 75,000). The purified adhesin appears to be a basic protein with a pI between 7.4 and 8.0. Amino acid and N-terminal sequence analyses were carried out with the purified protein. These indicated that the protein contains a large number of Asx and Glx residues as well as basic amino acid residues. The binding site of the pure adhesin retained its native configuration during purification. When preincubated with streptococcal partner cells at pH 4.6, the adhesin prevented B. loeschei cells from coaggregating with the streptococci. An adhesin preparation adjusted to a pH of 6.8 rapidly agglutinated both streptococci and neuraminidase-treated erythrocytes. Galactosides inhibited the agglutination reactions.     

Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion

The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts.     

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.     

Plasmodium falciparum: effect of time in continuous culture on binding to human endothelial cells and amelanotic melanoma cells

An in vitro correlate of the binding in vivo of Plasmodium falciparum-infected erythrocytes to capillary and venular endothelium, using cultured human endothelial cells and amelanotic melanoma cells, was previously developed. The effects of different times in continuous culture on binding of erythrocytes infected with nine different isolates of P. falciparum is now reported. Four isolates, which bound at the time they were first tested, rapidly lost the ability to bind after 26-43 days in culture. One of these, the Cameroun isolate, tested 12 h after the blood was obtained from the patient, had the highest rate of binding of all isolates (680 infected erythrocytes per 100 melanoma cells). After 37 days in culture, only 18 infected erythrocytes per 100 melanoma cells bound. Three isolates first tested after 30-62 days in culture bound poorly. In contrast, two others, the Vietnam (VI) and Brazil (It), continued to bind during the period of study. The Brazil (It) isolate studied after 43 days in culture bound 505 infected erythrocytes per 100 melanoma cells; its clone ItG2G1 continued to bind equally well after 400 days in culture. The ultrastructural morphology of knobs on the binding and nonbinding infected erythrocytes were indistinguishable. Since evidence from other studies indicates that knobs are necessary for binding to endothelium, it is proposed that some parasites in continuous culture may not express the molecules responsible for binding, although the morphologic knobs are still present.     

An in Vitro Assay for Sequestration: Binding of Plasmodium falciparum-Infected Erythrocytes to Formalin-Fixed Endothelial Cells and Amelanotic Melanoma Cells1

Erythrocytes infected with Plasmodium falciparum bind specifically to cultured endothelial cells and to a line of amelanotic melanoma cells. We have fixed endothelial cells and amelanotic melanoma cells in various ways and determined whether the fixed cells were still able to bind infected erythrocytes. Only cells fixed with 1.0-2.5% formalin in phosphate-buffered saline continued to bind infected erythrocytes as well as unfixed cells. The mechanism of binding to fixed and unfixed cells appeared to be identical for the following reasons. First, erythrocytes infected by parasite strains that bound to unfixed cells also bound to fixed cells while those that did not bind to unfixed cells did not bind to fixed cells. Second, immune serum that inhibited binding to unfixed cells also inhibited binding to fixed cells. Third, electron microscopy showed that knobs were the points of attachment between infected erythrocytes and both fixed and unfixed melanoma cells. Fixed cells gave reproducible results over at least 2 months. Thus, we have developed a simplified, reproducible assay for measuring binding of P. falciparum-infected erythrocytes to target cells.     

Plasmodium falciparum: the effect of pH and Ca2+ concentration on the in vitro cytoadherence of infected erythrocytes to amelanotic melanoma cells

Cytoadherence of Plasmodium falciparum-infected erythrocytes to amelanotic melanoma cells was pH dependent; increased adherence was observed in the pH range of 6.1 to 6.8 and was greatest between pH 6.6 and 6.8 Ca2+ promoted cytoadherence, but at higher concentrations (40-50 mM) than is usually the case for cell-cell adhesion. The effects of pH and Ca2+ were interdependent--the pH optimum of cytoadherence was altered by the Ca2+ concentration in the medium. The adherent properties of several P. falciparum lines (including a knobless cytoadherent line) under varying pH and Ca2+ concentrations were similar.     

Production of a conserved adhesin by the human gastroduodenal pathogen Helicobacter pylori.

An adhesin protein with an approximate subunit molecular weight of 19,600 has been purified from the gastric pathogen Helicobacter pylori. The protein was loosely associated with the cell surface and was removed by gentle stirring or shearing. Released aggregates of the 19.6-kDa protein were removed from suspension by ultracentrifugation and separated from contaminating membranes by washing in 1.0% sodium dodecyl sulfate (SDS). The SDS-insoluble protein was purified further by Mono Q anion-exchange column chromatography. Electron microscopy of the purified adhesin demonstrated that it formed amorphous aggregates similar to the material attached to the bacterial cells and that the aggregates were morphologically distinct from typical fimbriae. Western blot (immunoblot) analysis with antiserum raised against the purified protein from one strain reacted with a protein with a similar subunit molecular weight present in all nine strains of H. pylori examined, but the protein was not present in other Helicobacter species examined. The N-terminal sequences of the 19.6-kDa protein purified from three different strains of H. pylori were identical for the first 28 amino acids, with the 10 amino-terminal residues showing limited sequence homology with the TcpA pilus protein of Vibrio cholerae. The H. pylori 19.6-kDa protein associated both with human and rabbit erythrocytes and with human buccal epithelial cells. Polystyrene microspheres coated with the protein agglutinated human, horse, and rabbit erythrocytes, suggesting that this protein species could mediate adhesion between H. pylori and eucaryotic cells. This ability to act as an adhesin may make this protein an important virulence factor for H. pylori and hence a potential target for a vaccine and therapy.     

Transient Expression of High Molecular Weight,Heat Sensitive,Trypsin-Resistant Form of Tyrosinase in B-16 Melanoma Cells

High molecular weight forms of tyrosinase have been found to be expressed during spontaneous remelanization of the amelanotic B-16 melanoma cells in culture as well as in melanotic tumors formed from amelanotic melanoma cells grown in C57BL/6J mice. Overnight extraction of the crude melanosomal fractions from such tumors and cultured melanoma cells reveal the presence of an additional DOPA-MBTH positive band well below the stacking gel. This band has been found to be α-PEP7 (antibody specific for tyrosinase) positive and α-PEPl (antibody specific for TRP-1) negative on Western blot analysis. Heat treatment at 60°C for 60 min results in the loss of this band and considerable loss of activity of the melanosomal extract. Trypsin treatment of these melanosomal extracts resulted in a minor change in the mobility of the high molecular weight band. SDS-PAGE under reduced conditions followed by Western blotting revealed that the high molecular weight band was lost and not detected by α-PEP7 or α-PEPl. These findings indicate that high molecular weight, heat sensitive and trypsin resistant forms of tyrosinase are transiently expressed in B-16 melanoma cells and tumors that are initiating remelanization following phenotypic drift towards the amelanotic state.     

IR spectroscopy of isotope-labeled helical peptides: probing the effect of N-acetylation on helix stability

The effect of N-acetylation on the conformation of alanine-rich helical peptides is examined using isotope-edited Fourier transform infrared (FTIR) spectroscopy. A series of peptides with sequence AA(AAKAA)(3)AAY has been prepared; each peptide incorporates four (13)C-labeled alanines. These peptides have two amide I' bands in their FTIR spectra: one corresponding to the (12)C amino acids, and one assigned to the (13)C amino acids. The intensity and frequency of the (13)C amide I' band varies systematically with the position of the labels in the sequence and the presence or absence of an N-acetyl capping group. The intensity of the (13)C amide I' band correlates with helix stability at the labeled residues as predicted by thermodynamic models of the helix-coil transition. These results suggest that FTIR spectroscopy combined with specific isotope labeling can be used to dissect the conformation of helical peptides at the residue level.     

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.     

Cloning and expression of cDNA encoding mouse tyrosinase.

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.