Epitope Of mAb Research Articles

The mouse natural killer T cell-associated antigen recognized by U5A2-13 monoclonal antibody is intercellular adhesion molecule-1

Natural Killer T (NKT) cells in mice are generally defined as NK1.1 + T cells, although NK1.1 antigen is expressed only in C57BL/6 and related strains. This has precluded investigations of other strains. To find a novel NKT cell surface marker, we generated a monoclonal antibody (mAb), U5A2-13, which recognizes phenotypically and functionally similar populations to NKT cells in naı̈ve mice irrespective of strain. Here, by using a COS-7 expressional cloning system, we molecularly cloned a cDNA encoding a protein reactive with the U5A2-13 mAb and then identified it as intercellular adhesion molecule-1 (ICAM-1). Importantly, the U5A2-13 mAb did not stain hepatic mononuclear cells from ICAM-1 gene disrupted mice. Furthermore, Pepscan method disclosed that the discontinuous epitope for U5A2-13 mAb is composed of three loops located in extracellular domain two of ICAM-1. Overall, U5A2-13, a mAb originally established for mouse NKT cells, recognizes a novel conformational epitope of ICAM-1.

A Novel CRM1-mediated Nuclear Export Signal Governs Nuclear Accumulation of Glyceraldehyde-3-phosphate Dehydrogenase following Genotoxic Stress

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

Identification of the immunodominant regions of the melanoma antigen tyrosinase by anti-tyrosinase monoclonal antibodies

Tyrosinase, the critical enzyme in melanin synthesis, is also found to be expressed in most malignant melanomas and can serve as a target for the immune response by both CD4+ and CD8+ T-cells. Therefore it could be used as a potential target for therapeutic intervention in tyrosinase-positive melanomas. In order to develop serological reagents for the immunodetection of human tyrosinase and to find the most immunogenic region of the protein, we have raised a panel of monoclonal antibodies (MAbs) against recombinant tyrosinase expressed and purified from bacteria. Epitope mapping revealed the 79 amino acid long stretch between 163 and 241 residues to be the most immunodominant region of the tyrosinase. This region could be further divided into three parts by binding different MAbs. These MAbs were very useful tools for the detection of tyrosinase expression from different constructs in tissue culture cells by immunocytochemistry and in melanocytes by immunohistochemistry. Some of the MAbs that recognized epitopes between 163 and 204 amino acids also recognized an additional distinct protein of about 70 kDa seen on Western blot analysis of transfected and non-transfected COS-7 cells. One of these, the MAb 4B1, was used in immunohistochemistry, and cross reaction with the basement membrane of the human tissue was observed. The analysis of the 4B1 MAb epitope showed that the C-terminal part of that region almost entirely overlaps with the sequence of the recently reported basement membrane protein beta-netrin.

Epitope mapping of pneumococcal surface protein A of strain Rx1 using monoclonal antibodies and molecular structure modelling

Pneumococcal surface protein A (PspA) is an antigenic variable vaccine candidate of Streptococcus pneumoniae. Epitope similarities between PspA from the American vaccine candidate strain Rx1 and Norwegian clinical isolates were studied using PspA specific monoclonal antibodies (mAbs) made against clinical Norwegian strains. Using recombinant PspA/Rx1 fragments and immunoblotting the epitopes for mAbs were mapped to two regions of amino acids, 1–67 and 67–236. The discovered epitopes were visualized by modelling of the PspA:Fab part of mAb in three dimensions. Flow cytometric analysis showed that the epitopes for majority of mAbs were accessible for antibody binding on live pneumococci. Also, the epitopes for majority of the mAbs are widely expressed among clinical Norwegian isolates.

Mapping of the low pH-sensitive conformational epitope of rabies virus glycoprotein recognized by a monoclonal antibody #1-30-44.

Monoclonal antibody (mAb) #1-30-44 recognized an acid-sensitive conformational epitope of rabies virus glycoprotein (G). The antigenicity of G protein exposed on the cell surface was lost when the infected cells were exposed to pH 5.8. By comparing the deduced amino acid sequence of G protein between the HEP-Flury strain and the epitope-negative CVS strain as well as the mAb-resistant escape mutants, two distant sites that contained Lys-202 and Asn-336 were shown to be involved in the epitope formation. Lys-202 is located in the so-called neurotoxin-like sequence, while Asn-336 is included in antigenic site III and is very near the amino acid at position 333, which is known to affect greatly the neuropathogenicity of rabies virus when changed. Consistent with this finding, antigenicity of a neurovirulent revertant of the HEP-Flury strain, in which Gln-333 of G protein was replaced by Arg, was also affected as shown by its greatly decreased reactivity with mAb #1-30-44 compared to that of the original avirulent HEP virus. Based on these results, we hypothesize that the neurotoxin-like domain and some amino acids in antigenic site III come into contact with each other to form a conformational epitope for mAb #1-30-44, and such a configuration would be lost when exposed to acidic conditions to perform a certain low pH-dependent function of G protein.

Conformational Changes in the Integrin औA Domain Provide a Mechanism for Signal Transduction via Hybrid Domain Movement

The ligand-binding head region of integrin beta subunits contains a von Willebrand factor type A domain (betaA). Ligand binding activity is regulated through conformational changes in betaA, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from betaA is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the beta(1) subunit hybrid domain, which is connected to the lower face of betaA. Changes in the expression of these epitopes are induced by conformational changes in betaA caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated alpha(5)beta(1) with a mutation L358A in the alpha7 helix of betaA has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the alpha subunit. Taken together, these data suggest that the transduction of conformational changes through betaA involves shape shifting in the alpha7 helix region, which is linked to a swing of the hybrid domain away from the alpha subunit.

High Affinity Ligand Binding by Integrins Does Not Involve Head Separation

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.

Characterization of Reduced Expression of Glycine N-Methyltransferase in Cancerous Hepatic Tissues Using Two Newly Developed Monoclonal Antibodies

Glycine N-methyltransferase (GNMT) is a protein with multiple functions. Recently, two Italian siblings who had hepatomegaly and chronic elevation of serum transaminases were diagnosed to have GNMT deficiency caused by inherited compound heterozygosity of the GNMT gene with missence mutations. To evaluate the expression of GNMT in cell lines and tissues from hepatocellular carcinoma (HCC) patients, we produced two monoclonal antibodies (mAbs) 4-17 and 14-1 using two recombinant GNMT fusion proteins. M13 phage peptide display showed that the reactive epitopes of mAbs 4-17 and 14-1 were amino acid residues 11–15 and 272–276 of human GNMT, respectively. The dissociation constants of the binding between GNMT and mAbs were 1.7 × 10^–8M for mAb 4-17 and 1.8 × 10^–9M for mAb 14-1. Both mAbs can identify GNMT present in normal human and mouse liver tissues using Western blotting (WB) and immunohistochemical staining assay (IHC). In addition, WB with both mAbs showed that none of 2 hepatoblastoma and 5 HCC cell lines expressed GNMT. IHC demonstrated that 50% (13/26) of nontumorous liver tissues and 96% (24/25) of HCC tissues did not express GNMT. Therefore, the expression of GNMT was downregulated in human HCC.

Discontinuous residues of factor IX constitute a surface for binding the anti-factor IX monoclonal antibody A-5

Anti-human factor IX monoclonal antibody, A-5 (Mab A-5), has been widely used in structure-function studies for factor IX. Mab A-5 recognizes the catalytic domain of human factor IX (FIX). Regions important for Mab A-5 binding have previously been localized to the amino terminus of the heavy chain of factor IX, encompassing amino acid residues 181–310 [Blood (74) 971]. We have selected 20 positions in this region for alanine-scanning mutagenesis. We found that Mab A-5 failed to react with the recombinant factor IX mutants with substitutions at positions 228 and 252. Mab A-5 also reacted to a lesser extent to FIXD276A (factor IX with alanine substitution for aspartic acid at residue 276) and FIXK201A/D203A (double alanine substitutions at residues 201 and 203). The apparent dissociation rate constants ( K D) in binding Mab A-5 were 6.0×10 −9, 1.4×10 −8 and 2.0×10 −8 M, for wild-type FIX, FIXK201A/D203A and FIXD276A, respectively. The increased K D values of the two FIX mutants are mainly owing to the increased dissociation rates. These affected residues constitute a surface opposite from the factor VIIIa binding surface. We conclude that the epitope for Mab A-5 is on a surface composed of residues 228, 252, 276, and 201 or 203. This surface, which may not be important for factor VIII binding, contains a strong antigenic region on factor IX.

Characterization of reduced expression of glycine N-methyltransferase in cancerous hepatic tissues using two newly developed monoclonal antibodies

Glycine N-methyltransferase (GNMT) is a protein with multiple functions. Recently, two Italian siblings who had hepatomegaly and chronic elevation of serum transaminases were diagnosed to have GNMT deficiency caused by inherited compound heterozygosity of the GNMT gene with missence mutations. To evaluate the expression of GNMT in cell lines and tissues from hepatocellular carcinoma (HCC) patients, we produced two monoclonal antibodies (mAbs) 4-17 and 14-1 using two recombinant GNMT fusion proteins. M13 phage peptide display showed that the reactive epitopes of mAbs 4-17 and 14-1 were amino acid residues 11-15 and 272-276 of human GNMT, respectively. The dissociation constants of the binding between GNMT and mAbs were 1.7 x 10(-8) M for mAb 4-17 and 1.8 x 10(-9) M for mAb 14-1. Both mAbs can identify GNMT present in normal human and mouse liver tissues using Western blotting (WB) and immunohistochemical staining assay (IHC). In addition, WB with both mAbs showed that none of 2 hepatoblastoma and 5 HCC cell lines expressed GNMT. IHC demonstrated that 50% (13/26) of nontumorous liver tissues and 96% (24/25) of HCC tissues did not express GNMT. Therefore, the expression of GNMT was downregulated in human HCC.

Discordant expression of selectin ligands and sialyl Lewis x–related epitopes on murine myeloid cells

Murine leukocytes are thought to express alpha2-3-sialylated and alpha1-3-fucosylated selectin ligands such as sialyl Lewis x (sLe(x)), although monoclonal antibodies (mAbs) to sLe(x) or Le(x) reportedly do not bind to murine leukocytes. We observed that P- and E-selectin bound to pronase-sensitive ligands on murine monocytic WEHI-3 cells and murine neutrophils, indicating that the ligands for both selectins are glycoproteins. CSLEX-1, HECA-452, and other widely used mAbs to sLe(x) and Le(x) did not bind to WEHI-3 cells and bound at very low levels to murine neutrophils. Only the anti-sLe(x) mAbs 2H5 and KM93, which also recognize nonfucosylated glycans, bound to WEHI-3 cells. 2H5 and KM93 bound to pronase-resistant structures, indicating that the mAbs did not identify selectin ligands. Treatment of WEHI-3 cells with glycosidases or chlorate demonstrated that sialic acid modifications, alpha1-3-galactosylation, or sulfation did not mask epitopes for mAbs to sLe(x) or Le(x). Compared to human promyelocytic HL-60 cells, WEHI-3 cells and murine neutrophils expressed low alpha1-3-fucosyltransferase activities. Consistent with very low endogenous fucosylation, forced fucosylation of intact WEHI-3 cells or murine neutrophils by exogenous alpha1-3-fucosyltransferase FTVI and GDP-fucose created many new epitopes for anti-sLe(x) mAbs such as HECA-452 and CSLEX-1. Nevertheless, forced fucosylation of intact cells did not significantly augment their ability to bind to fluid-phase P- or E-selectin or to roll on immobilized P- or E-selectin under flow. These data suggest that murine myeloid leukocytes fucosylate only a few specific glycans, which interact preferentially with P- and E-selectin.

Heterogeneity of the thyroglobulin epitopes associated with circulating thyroid hormone autoantibodies in hashimoto's thyroiditis and non-autoimmune thyroid diseases.

We previously implicated TG leakage from fine-needle aspiration biopsy (FNAB) as responsible for circulating thyroid hormone autoantibodies (THAb). However, THAb were not always associated with TGAb. In the literature these negative findings have been interpreted against a role of TG as the antigen for THAb. To evaluate the TGAb status more fully and to gain information on TG epitopes involved in THAb development, we measured: 1) TGAb with an independent hemagglutination assay (HA), and 2) epitope specificity in a competitive ELISA using 2 monoclonal Abs (mAb) against TG: mAb 42C3 and mAb 134C2. MAb 42C3 recognizes a cross-reactive iodinated epitope, whereas 134C2 is specific for human TG. We tested 12 Hashimoto's thyroiditis (HT) and 35 non-HT patients sampled prior to, 1 and 3 months after FNAB. We found that, irrespective of thyroid disease or post-FNAB THAb status, certain patients previously classified as TGAb negative by IRMA tested TGAb positive by HA or by competition ELISA and vice versa. A post FNAB positive response to the 42C3 iodinated epitope in only one THAb IgM-T4+ve HT and a few THAb negative non-HT patients was observed. Furthermore, we observed that the 3 non-HT patients who expressed IgM-T3 THAb failed to bind either TG-mAb epitope. We conclude that a single TGAb assay is not sufficient to define the TGAb status, which can be achieved reliably only by using multiple TGAb assays. In addition, the TG-iodinated epitope recognized by 42C3 is not a major epitope in post-FNAB THAb, and the T3-epitope involved in THAb remains distinct from the mAb epitopes. In light of recent data in the literature, we further suggest that the responsible epitopes are more likely to be expressed in leaked TG fragments, rather than leaked intact TG.

The requirement of varicella zoster virus glycoprotein E (gE) for viral replication and effects of glycoprotein I on gE in melanoma cells.

The glycoprotein E (gE) of varicella zoster virus (VZV), encoded by ORF68, is the most abundant viral glycoprotein. In the current experiments, we demonstrated that ORF68 deletion was incompatible with recovery of infectious virus from VZV cosmids. Replacing ORF68 at a nonnative AvrII site in the genome restored infectivity. Further, we found that VZV gE could be expressed under the control of the Tet-On promoter in stably transfected melanoma cell lines (Met-gE cells) without evidence of toxicity. In these Met-gE cells, gE colocalized with gamma-adaptin, a trans Golgi network marker, in perinuclear sites, but did not reach plasma membranes. In order to investigate how infection altered gE localization, we made a recombinant virus, vOka-MSPgE, with ORF68 from the VZV MSP strain. VZV MSP encodes a mutant gE protein (D150N) that lacks the mAb epitope, 3B3 (Santos et al., Virology 275, 306-317, 2000), whereas Met-gE protein binds mAb 3B3. Within 48 h after Met-gE cells were infected with vOka-MSPgE, the steady-state distribution of Met-gE protein extended beyond the perinuclear areas to other cytoplasmic sites and to plasma membranes. A second recombinant, vOka-MSPgE without gI (vOka-MSPgEdeltagI), was constructed to investigate Met-gE protein distribution in the absence of gI. The redistribution of Met-gE protein which was observed by 48 h after vOka-MSPgE infection did not occur until 5 days (140 h) within vOka-MSPgEdeltagI infected cells. After vOka-MSPgE infection of Met-gE cells, most Met-gE protein was in the final 94K mature form by 72 h. However, progression to predominance of mature gE was delayed in Met-gE cells infected with vOka-MSPgEdeltagI. These observations confirm our hypothesis that VZV gE is essential, based upon the demonstration of restored infectivity after replacing ORF68 in a nonnative site in the genome, and provide further evidence of the role of gI in facilitating the maturation and intracellular distribution of this critical VZV glycoprotein.

Mapping of two dominant sites of VP35 of Marburg virus.

Five types of anti-VP35 monoclonal antibodies (MAbs), four immune sera against Marburg virus (MBGV), and 11 overlapping recombinant VP35 fragments were used to map the epitopes for VP35 of MBGV. The purified full-size recombinant VP35 was highly immunogenic and retained the B-cell epitopes that were identical to those of the viral VP35. Two major sites on VP35 and a set of truncated VP35 fragments were found by use of an enzyme immunoassay and immunoblot. Site I was located in a region between amino acids 1 and 174 of the VP35 sequence, and only polyclonal antibodies (PAbs) against MBGV recognized epitopes at this site. Site II was mapped by use of anti-VP35 MAbs to the region between amino acid residues 167 and 278 of VP35. Amino acids 252-278 of VP35 might be involved in the formation of the epitopes for MAbs. B-cell epitopes were not found on the C-terminus of VP35 by use of PAbs or MAbs.

Structural analysis of chick ephrin-A2 by function-blocking and non-blocking monoclonal antibodies

Ephrins, ligands for the Eph family of receptor tyrosine kinases, play key roles in diverse biological processes. In this study, we determined the epitopes and kinetic parameters of function-blocking (B3) and non-blocking (IV) monoclonal antibodies (mAbs) recognizing chick ephrin-A2. We show that the epitope for the non-blocking mAb is the residue Asp 105 of chick ephrin-A2. However, the binding of the function-blocking mAb depends mostly on residue Ser 108 and its epitope may reside within residues 105–132, which appear crucial for the receptor interaction site. Kinetic studies suggest a possible mechanism why mAb IV, despite recognizing a region very close to the mAb B3 epitope, fails to block the ligand–receptor interaction.

Mapping of B-cell epitopes in the nucleocapsid protein of Puumala hantavirus.

Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/Hällnäs, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/Hällnäs and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined.

The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model.

Streptococcus parasanguis is a primary colonizer of the tooth surface and plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis are important in mediating adhesion to saliva-coated hydroxylapatite (SHA), an in vitro tooth adhesion model. The Fap1 adhesin has been identified as the major fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated the presence of rhamnose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide was similar to that of the intact molecule. The functionality of the glycan moiety was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein. These antibodies were grouped into two categories based on their ability to block adhesion of S. parasanguis to SHA and their corresponding specificity for either protein or glycan epitopes of the Fap1 protein. 'Non-blocking' MAb epitopes were mapped to unique protein sequences in the N-terminus of the Fap1 protein using non-glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed in Escherichia coli. In contrast, the 'blocking' antibodies did not bind to the recombinant Fap1 proteins, and were effectively competed by the binding to the purified glycopeptide. These data suggest that the 'blocking' antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.

Epitope mapping of the O-chain polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide by saturation-transfer-difference NMR spectroscopy.

Two modifications of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5-N-acetimidoyl-7-N-acetyllegionaminic acid) in the O-chain polysaccharide (OPS) of the Legionella pneumophila serogroup 1 lipopolysaccharide (LPS) concern N-methylation of the 5-N-acetimidoyl group in legionaminic acid. Both N-methylated substituents, the (N,N-dimethylacetimidoyl) amino and acetimidoyl(N-methyl)amino group, could be allocated to one single legionaminic acid residue in the long- and middle-chain OPS, respectively. Using mutants devoid of N-methylated legionaminic acid derivatives, it could be shown that N-methylation of legionaminic acid correlated with the expression of the mAb 2625 epitope. In the present study we investigated the binding of the LPS-specific monoclonal antibody mAb 2625 to isolated OPS with surface-plasmon-resonance biomolecular interaction analysis and saturation-transfer-difference (STD) NMR spectroscopy in order to map the mAb 2625 epitope on a molecular level. It could be demonstrated that the binding affinity of the N-methylated legionaminic acid derivatives was independent from the size of the isolated OPS molecular species. In addition, STD NMR spectroscopic studies with polysaccharide ligands with an average molecular mass of up to 14 kDa revealed that binding was mainly mediated via the N-methylated acetimidoylamino group and via the closely located 7-N-acetyl group of the respective legionaminic acid residue, thus indicating these derivatives to represent the major epitope of mAb 2625.

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.

Hemagglutinin-neuraminidase-independent fusion activity of simian virus 5 fusion (F) protein: difference in conformation between fusogenic and nonfusogenic F proteins on the cell surface.

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47 degrees C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.