Erythrocyte-binding Activity Research Articles

Four molecules of the 33 kDa haemagglutinin component of the Clostridium botulinum serotype C and D toxin complexes are required to aggregate erythrocytes

Normally, large-sized botulinum toxin complexes (L-TC) of serotype C and D are composed of a single neurotoxin, a single non-toxic non-haemagglutinin, two HA-70 molecules, four HA-33 molecules and four HA-17 molecules that assemble to form a 650 kDa L-TC. The 540 and 610 kDa TC species (designated here as L-TC2 and L-TC3, respectively) were purified in addition to the 650 kDa L-TC from the culture supernatants of serotype D strains (D-4947 and D-CB16) and serotype C strains (C-6814 and C-Yoichi). The 650 kDa L-TC from D-4947, D-CB16 and C-6814 showed haemagglutination and erythrocyte-binding activity, but their L-TC2 and L-TC3 species had only binding activity. In contrast, every TC species from C-Yoichi having the C-terminally truncated variant of HA-33 exhibited neither haemagglutination activity nor erythrocyte-binding activity. Four strain-specific HA-33/HA-17 complexes were isolated from the 650 kDa L-TC of each strain. The 650 kDa HA-hybrid L-TCs were reconstituted by various combinations of isolated HA-33/HA-17 complexes and haemagglutination-negative L-TC2 or L-TC3 from each strain. HA-hybrid 650 kDa L-TC, including at least one HA-33/HA-17 complex derived from C-Yoichi, lost haemagglutination activity, leading to the conclusion that the binding of four HA-33 molecules is required for haemagglutination activity of botulinum L-TC. The results of the modelling approach indicated that the structure of a variant C-Yoichi HA-33 molecule reveals clear deformation of the beta-trefoil domain responsible for the carbohydrate recognition site.

Targeted disruption of maebl in Plasmodium falciparum

Malaria parasites have a complex life cycle that requires invasion of several different cell types in both vertebrate and mosquito hosts. In Plasmodium falciparum, the merozoite must attach to and invade a new erythrocyte in order to continue parasite development in the blood of an infected host. Merozoite entry into erythrocytes is a multi-step process requiring merozoite adhesion, re-orientation, junction formation, and invasion [1]. Each step is thought to be mediated by the coordinated interactions of numerous specific merozoite ligands and erythrocyte surface receptors [2]. A number of mediators involved in the invasion process are positioned on the merozoite surface and in the organelles of the apical complex. However, the functions of molecules involved in this interaction are still poorly characterized. Understanding the complex process of P. falciparum merozoite invasion requires identification and characterization of numerous potential parasite ligands and their potential interactions. MAEBL is a paralogue of the DBL-EBP family (Duffy binding like-erythrocyte binding protein), but has a chimerical structure and shares similarity with AMA1 [3]. M1 and M2 are tandem cysteine-rich regions with similarity to AMA1 and are present in the N-terminal portion of the MAEBL ectodomain. MAEBL was identified in P. yoelii and P. falciparum blood stage parasites as a minor membrane protein with erythrocyte binding activity expressed in the apical organelles and on the surface of invasive merozoites [3–6]. Expressed abundantly in sporozoites, MAEBL appears to be important for sporozoite invasion into the mosquito salivary glands and in establishing exoerythrocytic schizonts [6–12]. It has been reported that sera from infected individuals living in a malaria endemic region of western Kenya recognized M2 recombinant antigen and had the ability to inhibit M2-erythrocyte binding [6]. However, whether MAEBL is essential or even has a significant role for erythrocytic stage growth is unclear.

Conserved residues in the Plasmodium vivax Duffy-binding protein ligand domain are critical for erythrocyte receptor recognition

Malaria merozoite invasion of human erythrocytes depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium vivax merozoite invasion is totally dependent on the recognition of the Duffy blood group antigen by the parasite ligand Duffy-binding protein (DBP). Receptor recognition by P. vivax relies on a cysteine-rich domain, the DBL domain or region II, at the N terminus of the extracellular portion of DBP. The minimal region of the DBP implicated for receptor recognition lies between cysteines 4 and 8 of the DBL domain, which is a region that also has the highest rate of allelic polymorphisms among parasite isolates. We previously found that allelic polymorphisms in this region altered the P. vivax DBL domain antigenic character, which contrasts with changes in receptor specificity attributed to polymorphisms in some homologous ligands of Plasmodium falciparum. To further investigate the relative importance of conserved and polymorphic residues within this DBL central region, we identified residues critical for receptor recognition by site-directed mutagenesis. Seventy-seven surface-predicted residues of the Sal-1 DBL domain were substituted with alanine and assayed for erythrocyte binding activity by expression of the mutant proteins on the surface of transiently transfected COS cells. The functional effect of alanine substitution varied from nil to complete loss of DBL erythrocyte-binding activity. Mutations that caused loss of ligand function mostly occurred in discontinuous clusters of conserved residues, whereas nearly all mutations in polymorphic residues did not affect erythrocyte binding. These data delineate DBL domain residues essential for receptor recognition.

Antigenic drift in the ligand domain of Plasmodium vivax duffy binding protein confers resistance to inhibitory antibodies.

Interaction of the Duffy binding protein (DBP) with its erythrocyte receptor is critical for maintaining Plasmodium vivax blood-stage infections, making DBP an appealing vaccine candidate. The cysteine-rich region II is the ligand domain of DBP and a target of vaccine development. Interestingly, most of the allelic diversity observed in DBP is due to the high rate of nonsynonymous polymorphisms in this critical domain for receptor recognition. Similar to the hypervariability in influenza hemagglutinin, this pattern of polymorphisms in the DBP ligand domain suggests that this variation is a mechanism to evade antibody neutralization. To evaluate the role that dbp allelic diversity plays in strain-specific immunity, we examined the ability of an anti-Sal1 DBP serum to inhibit the erythrocyte-binding function of variant dbp alleles expressed on COS cells. We observed that the PNG-7.18 allele was significantly less sensitive to immune inhibition of its erythrocyte-binding activity than were the Sal1 and PNG-27.16 alleles. This result suggested that the unique polymorphisms of resistant PNG-7.18 were part of a protective epitope on the DBP ligand. To confirm this, Sal1 was converted to the refractory phenotype by introduction of 3 polymorphisms unique to PNG-7.18, via site-directed mutagenesis. The results of the present study indicate that linked polymorphisms have an additive, synergistic effect on DBP antigenic character.

Epidermal growth factor-like motifs 1 and 2 of Plasmodium vivax merozoite surface protein 1 are critical domains in erythrocyte invasion

Plasmodium vivax merozoite surface protein 1 (PvMSP1) is believed to be important in erythrocyte invasion. However, the detailed mechanism of PvMSP1-mediated invasion has been unclear. We demonstrate that the C-terminal 19 kDa domain (PvMSP1 19) of PvMSP1, the 42-kDa fragment of PvMSP1 is further cleaved to a 33 kDa N-terminal polypeptide and a 19 kDa C-terminal fragment in a secondary processing step, is a critical domain in the binding between parasite ligand and erythrocyte receptor. Also, its cytoadherence was successfully blocked by naturally acquired immunity, was partially sensitive to neuraminidase and trypsin. When expressed separately epidermal growth factor (EGF)-like motifs 1 and 2, subunits of the PvMSP1 19, mediated 64% and 66% of the erythrocyte-binding activity, respectively, relative to their expression together as a single intact ligand domain. These results suggest that the EGF-like motifs 1 and 2 of PvMSP1 19 function as a core-binding portion in the attachment of PvMSP1 to erythrocytes.

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ( 21 INGKIIKNSEKDEIIKSNLRY 40 ), 20637 ( 157 KEKLQGQQSDSEQERRAY 173 ), 20638 ( 174 KEKLQEQQSDLEQERLAY 190 ) and 20639 ( 191 KEKLQEQQSDLEQERRAY 207 ) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48 kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ( 21 INGKIIKNSEKDEIIKSNLRY 40 ) and 20633 ( 81 DKELTMSNVKNVSQTNFKSLY 100 ) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 “High Activity Binding Peptides” could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.

Cellular localization of Babesia bovis merozoite rhoptry-associated protein 1 and its erythrocyte-binding activity.

The cellular localization of Babesia bovis rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca(2+) accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in B. bovis merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.

Immune complex-like moieties in immunoglobulin for intravenous use (i.v.Ig) bind complement and enhance phagocytosis of human erythrocytes.

Treatment with i.v.Ig can, on rare occasions, lead to detrimental effects such as enhanced erythrocyte sequestration and an increase in serum immune complexes with inflammatory sequellae such as exacerbation of glomerular nephritis. In this study, i.v.Ig (Sandoglobin) was examined for complement binding moieties which resemble immune complexes and can mediate the binding of IgG and C'3b to human erythrocytes via CR1 and enhance erythrocyte susceptibility to sequestration. Sephacryl S-200 HR separated i.v.Ig into two fractions: monomeric IgG (74%) and larger complexes of the molecular weight of an IgG dimer or greater (> or = 300 kD) (26%). In the presence of complement, the 'dimers' bound to human erythrocytes, rendering them susceptible to phagocytosis in vitro. Removal of erythrocyte-specific isoantibodies from the i.v.Ig had no effect on 'dimer' binding to the erythrocytes. Monomeric IgG contained virtually no complement-activating, erythrocyte-binding activity. Erythrocyte binding of complement-bearing IgG 'dimers' and subsequent phagocytosis resembles the binding of complement-bearing immune complexes to erythrocyte CR1. Exposure to Factor I leads to the release of complement-bearing IgG 'dimers' from erythrocyte CR1 and to the abrogation of erythrophagocytosis. Binding of complement-bearing IgG 'dimers' to the erythrocyte is blocked by To5, a CR1-specific monoclonal antibody.

Erythrocyte-binding activity of Plasmodium yoelii apical membrane antigen-1 expressed on the surface of transfected COS-7 cells

Malaria merozoite surface and apical organellar molecules facilitate invasion into the host erythrocyte. The underlying molecular mechanisms of invasion are poorly understood, and there are few data to delineate roles for individual merozoite proteins. Apical membrane antigen-1 (AMA-1) is a conserved apicomplexan protein present in the apical organelle complex and at times on the surface of Plasmodium and Toxoplasma zoites. AMA-1 domains 1/2 are conserved between Plasmodium and Toxoplasma and have similarity to the defined ligand domains of MAEBL, an erythrocyte-binding protein identified from Plasmodium yoelii. We expressed selected portions of the AMA-1 extracellular domain on the surface of COS-7 cells to assay for erythrocyte-binding activity. The P. yoelii AMA-1 domains 1/2 mediated adhesion to mouse and rat erythrocytes, but not to human erythrocytes. Adhesion to rodent erythrocytes was sensitive to trypsin and chymotrypsin, but not to neuraminidase. Other parts of the AMA-1 ectodomain, including the full-length extracellular domain, mediated significantly less erythrocyte adhesion activity than the contiguous domains 1/2. The results support the role of AMA-1 as an adhesion molecule during merozoite invasion of erythrocytes and identify highly conserved domains 1/2 as the principal ligand of the Plasmodium AMA-1 and possibly the Toxoplasma AMA-1. Identification of the AMA-1 ligand domains involved in interaction between the parasite and host cell should help target the development of new therapies to block growth of the blood-stage malaria parasites.

Multivalent binding of K99 fimbriae to the N-glycolyl-GM3 ganglioside receptor

The ganglioside N-glycolyl-GM3 binds laterally at numerous positions to K99 fimbriae, as shown by electron microscopic detection and erythrocyte-binding activity. The data demonstrate the multivalent nature of K99 fimbriae with respect to their receptor-binding sites.

Identification of erythrocyte-binding antigens in Helicobacter pylori.

The surface antigens of Helicobacter pylori conferring erythrocyte-binding activity were obtained by adsorption onto formaldehyde-treated dog and goat erythrocytes from supernatant fractions of sonicated bacteria and elution using a high concentration of NaCl. The desorbed material was analysed by SDS-PAGE and immunoblotting with anti-whole-cell serum to agar-grown bacteria which had been absorbed with broth-grown, non-haemagglutinating cells (haemagglutination-associated antiserum). Two polypeptides with molecular masses of 25 and 59 kDa were revealed as erythrocyte-binding antigens. Strains which agglutinated both dog and goat erythrocytes possessed both these erythrocyte-binding antigens, whereas an antigenically cross-reactive 24 kDa polypeptide was present in a strain which only agglutinated goat erythrocytes. Haemagglutinin material was extracted from H. pylori using n-octylglucopyranoside and purified by Sepharose chromatography and sucrose density gradient ultracentrifugation. The purified extract directly agglutinated erythrocytes in a neuraminyl-lactose-sensitive and neuraminidase-sensitive manner. The 59 kDa polypeptide was not present in the purified haemagglutinin preparation. The haemagglutination-associated antiserum reacted strongly with the 25 kDa polypeptide band which was the most prominent polypeptide band on analysis of the purified haemagglutinin preparation by SDS-PAGE and silver staining. Thus, H. pylori possesses at least two adhesins, one of which recognises a N-acetylneuraminic acid (alpha 2-3) moiety of receptors, the other being of unknown receptor specificity. Differences in the antigenicity and molecular masses of these adhesins in individual strains may underlie differences in receptor-binding specificities and haemagglutination profiles.