Macrophage recognition of saccharide chains on the erythrocytes damaged by iron-catalyzed oxidation

Mouse erythrocytes oxidized with an iron catalyst ADP/Fe3+ chelate attached to the monolayers of mouse resident and thioglycollate-induced peritoneal macrophages in the absence of serum, indicating that the macrophages recognized the oxidized erythrocytes. The recognition was partially prevented when the oxidized cells were treated with dithiothreitol, suggesting that disulfide formation is involved, in part, in the generation of the membrane sites recognized by macrophages. Phosphatidylserine is unlikely to be the determinant on the oxidized cells because it was not detected on the outer surface of the oxidized cells. The recognition by resident macrophages was effectively inhibited by N-acetylneuramin lactose, N-acetylneuraminic acid, glycophorin A, and disialoganglioside GD1a, but poorly by lactose, asialoglycophorin A, and monosialoganglioside GM1. In addition, the recognition was partially inhibited by L-fucose and human lactoferrin. The recognition by thioglycollate-induced macrophages was not inhibited by glycophorin A but was partially inhibited by L-fucose, lactoferrin, and oligosaccharides from band 3 glycoprotein. Enzymatic cleavage of the poly-N-acetyllactosaminyl saccharide chains of band 3 and lactoferrin resulted in a loss of the inhibitory activity. These results suggest that sialosaccharide chains of ADP/Fe(3+)-oxidized erythrocytes, possibly those on glycophorin A, are mainly involved in the recognition by resident macrophages, and poly-N-acetyllactosaminyl saccharide chains, possibly those on band 3, are partly involved in the recognition both by resident and thioglycollate-induced macrophages. Oxidation of erythrocytes may induce change in these membrane glycoproteins, like aggregation, which renders their saccharide chains susceptible to the macrophage recognition.     

Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.     

Determination of antibody affinity using threshold values of serum activity in the passive hemagglutination test

Agglutination of erythrocytes of the antigenic diagnostic agents with the IgG antibodies is attributed to the fact that the reactive centres of the same molecule of the antibody attached to the antigenic determinants located on different erythrocytes. A possibility of agglutination of erythrocytes under conditions of the maximal dilution of the immune serum depended on the number of molecules located on a single erythrocyte. When the erythrocytes are sensitized with the optimal dose of the antigen those serum antibodies are involved in the passive hemagglutination test (PHAT) which are characterized by the mean integrated association constant. When the antibodies participate in the PHAT with several batches of the erythrocytic diagnostic agent characterized by an average number of the antigen molecules on a single erythrocyte there appears a possibility of determining the extent of heterogeneity of the antibodies. Not only the amount of antibodies, but also their affinity can be ascertained by the PHAT, and also the heterogeneity of antibodies can be evaluated.     

Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes

We and others previously showed that immunization of rabbits with different forms of oxidized low density lipoprotein (LDL) significantly reduced atherogenesis. We now investigated the effect of continued immunization on atherosclerosis in LDL receptor-deficient (LDLR-/-) mice to determine whether a similar reduction of atherosclerosis occurred in murine models and whether this was due to humoral immune responses, ie, formation of high titers of antibodies to oxidation-specific epitopes. Three groups of LDLR-/- mice were repeatedly immunized with homologous malondialdehyde-modified LDL (MDA-LDL), native LDL, or phosphate-buffered saline (PBS) for 7 weeks. Extensive hypercholesterolemia and accelerated atherogenesis were then induced by feeding a cholesterol-rich diet for 17 weeks, during which immunizations were continued. Binding of immunoglobulin (Ig) M and IgG antibodies, as well as IgG1 and IgG2a isotypes, to several epitopes of oxidized LDL were followed throughout the study. After 24 weeks of intervention, atherosclerosis in the aortic origin was significantly reduced by 46.3% and 36.9% in mice immunized with MDA-LDL and native LDL, respectively, compared with PBS (133 558 and 157 141 versus 248 867 microm2 per section, respectively). However, the humoral immune response to oxidative neoepitopes in the MDA-LDL group was very different from that of the LDL or PBS group. IgG antibody binding to MDA-LDL and other epitopes of oxidized LDL, such as oxidized phospholipid (cardiolipin), oxidized cholesterol, or oxidized cholesteryl linoleate, but not native LDL, increased markedly in mice immunized with MDA-LDL, but not in mice immunized with native LDL or PBS. In the MDA-LDL group, both T helper cell (Th)2-dependent IgG1 antibody and Th1-dependent IgG2a antibody binding to oxidative neoepitopes increased significantly over time. The fact that mice immunized with both MDA-LDL and native LDL had a significant reduction in atherosclerosis, whereas only the MDA-LDL group developed very high titers of antibodies to oxidation-specific epitopes, suggests that the antiatherogenic effect of immunization is not primarily dependent on very high titers of antibodies to oxidation-specific epitopes but is more likely to result from the activation of cellular immune responses.     

Evaluation of biochemical changes during in vivo erythrocyte senescence in the dog

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, approximately 115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age >/=110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs >/=112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.     

Growth and body composition of preterm infants: influence of nutrient fortification of mother''s milk in hospital and breastfeeding post-hospital discharge

Enhanced oxidative stress is a feature of inflammatory and infectious conditions. Proteins may be important targets of oxidation and this may alter their function. We evaluated whether metal-catalyzed oxidation of IgG could alter its ability to bind to Fc receptors on macrophages. Human IgG incubated with an FeCl3/EDTA/ascorbate metal-catalyzed oxidation system resulted in a significant increase in carbonyl content, a measure of protein oxidation, compared to IgG treated with EDTA alone (control). Western blot analysis using an antibody to oxidized protein revealed an increase in antibody binding to both the heavy (Fc portion-containing) and light chains of IgG treated with the oxidizing system. Western blot analysis of papain-digested IgG confirmed oxidative modification of the Fc portion. Binding studies carried out with J774.16 macrophages demonstrated significantly diminished ability of the oxidized IgG to bind to macrophage Fc receptors compared to control IgG. These data demonstrate that IgG is susceptible to metal-catalyzed oxidation and that this impairs its ability to bind to macrophage Fc receptors. Oxidation of IgG might play a role in modulating immune function in infection and disorders associated with immune complex formation by diminishing IgG binding to phagocytic cells.     

Treatment of carbon monoxide poisoning with hyperbaric oxygen

This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.     

Effect of taurine on angiotensin II-induced hypertrophy of neonatal rat cardiac cells

T Lymphocytes (T cells) recognize short antigenic peptides bound to either MHC I or II molecules, in contrast to antibodies which can bind to native antigen. The mechanism by which antigens are processed into peptides, and the nature of the interactions of antigenic peptides with MHC molecules and with the T cell receptor have now been defined in some detail. Of significance to glycobiologists is the recent appreciation that the carbohydrate of glycoprotein antigens can contribute to the T cell recognition of epitopes presented by MHC molecules. Experiments using model T cell epitopes have demonstrated that carbohydrate can modulate T cell responses in a variety of ways; for example, there are a number of cases where glycopeptide-specific T cell responses have been identified. Many of these glycopeptide-specific T cell responses involve a peptide bearing a single glycosyl residue, thus it appears very likely that both glycan and peptide make contact with the T cell receptor binding site. Significantly, glycopeptide-specific T cell responses have also been detected to native glycoproteins. The ability of carbohydrate to influence T cell recognition of antigen has important consequences for a wide range of immune responses as well as the current strategies for mapping T cell determinants.     

Erythrocyte thiol status regulates band 3 phosphotyrosine level via oxidation/reduction of band 3-associated phosphotyrosine phosphatase

Oxidative stress-induced tyrosine phosphorylation has been ascribed to activation of phosphotyrosine kinase or to inhibition of phosphotyrosine phosphatase (PTP). We have previously identified a PTP associated with band 3 in the human erythrocyte membrane, a PTP that is normally highly active and prevents the appearance of band 3 phosphotyrosine. Here we show that treatment of erythrocytes with the thiol-oxidizing agent diamide leads to the formation of PTP disulfides (PTP-band 3 mixed disulfides) and inhibition of dephosphorylation, allowing the accumulation of band 3 phosphotyrosine. Upon reduction of the disulfides, the band 3 phosphotyrosine is dephosphorylated. Erythrocyte thiol alkylation by N-ethylmaleimide results in irreversible PTP inhibition and irreversible phosphorylation. The results are consistent with the notion that alterations in cellular thiol status affect the cell phosphotyrosine status and that oxidative stress-induced tyrosine phosphorylation involves inhibition of PTP.     

Human preformed IgG combining with membrane-bound porcine serotransferrin lyse porcine endothelial cells through antibody-dependent cellular cytotoxicity

Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100% homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab')2gamma specifically bound to psTf. Neutralization by psTf abolished that binding. Although alpha1,3-linked galactose residues (Gal(alpha)1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Gal(alpha)1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.     

Urease-specific monoclonal antibodies prevent Helicobacter felis infection in mice

Experiments were performed to determine the antigenic specificity of a monoclonal antibody (immunoglobulin A [IgA] 71) previously demonstrated to neutralize the ability of Helicobacter felis to colonize mice. Immunoprecipitation of radiolabeled H. felis outer membrane proteins with IgA 71 revealed specificity for a 62-kDa protein. Another of our monoclonal antibodies, IgG 40, precipitated a protein of similar molecular weight. IgA 71 but not IgG 40 also precipitated purified recombinant H. pylori urease. The antigenic specificity of both antibodies was confirmed to be urease by the ability of each to select Escherichia coli clones expressing the H. felis urease genes. The two antibodies were shown to bind nonoverlapping epitopes in a competition enzyme-linked immunosorbent assay. Both IgA 71 and IgG 40 could effectively neutralize H. felis infectivity by incubating the bacteria with the antibodies prior to oral administration to naive mice. The mechanism of protection does not appear to be inhibition of urease activity, as IgA 71 does not inhibit the conversion of urea to ammonia by H. pylori urease in vitro. These results support a protective role for the secretory humoral immune response in Helicobacter immunity and provide further evidence that the urease enzyme can serve as a protective antigen.     

Binding of human monoclonal IgG rheumatoid factors to Fc is influenced by carbohydrate

OBJECTIVE: The precise nature of the epitope on the Fc portion of the IgG molecule to which IgG rheumatoid factors (RF) bind has not been identified. As patients with rheumatoid arthritis (RA) have abnormal glycosylation of the Fc portion of IgG, we investigated the impact of the sugar present in the Fc on the binding of IgG RF. METHODS: Binding of monoclonal IgG RF to Fc fragments with varying oligosaccharide chains was detected using an immunoblot assay. RESULTS: We demonstrated that the binding of human hybridoma derived monoclonal IgG RF was strongly influenced by the presence of carbohydrate and was maximal when the carbohydrate molecule was intact. The RF did not bind directly to the carbohydrate moiety itself. CONCLUSION: This suggests that conformational changes in the polypeptide chain induced by the carbohydrate are responsible for the observed binding patterns.     

Osteogenesis imperfecta: the distinction from child abuse and the recognition of a variant form

Inactivation of glutathione peroxidase correlates with the rate of hemoglobin chain oxidation. The enzyme inactivation is mainly present in those conditions where the autoxidation of the oxygenated chains is followed by transformation of the oxidized molecule into a hemichrome. Free hemoglobin chains have been encapsulated in human red blood cells by a dialysis technique that involves transient hypotonic hemolysis followed by isotonic resealing. Chain-loaded erythrocytes represent a good in vitro model of thalassemia. The presence of free human chains in the cell alters the intraerythrocytic glutathione peroxidase activity (alpha chains are more effective in the inactivation of the enzyme with respect to the beta chains).     

Antigenic determinants of Staphylococcus aureus type 5 and type 8 capsular polysaccharide vaccines

Bacterial capsular polysaccharides (CP) are carbohydrate polymers comprised of repeating saccharide units. Several of these CP have side chains attached to their backbone structures. The side chains may include O-acetyl, phosphate, sialic acid, and other moieties. Those moieties represent the immunodominant epitopes and the most functional ones. The clinically significant Staphylococcus aureus type 5 CP (CP 5) and type 8 CP (CP 8) are comprised of a trisaccharide repeat unit with one O-acetyl group attached to each repeat unit. The immunogenicity of these CP and the functionality of antibodies to the backbone and the O-acetyl moieties were investigated. Immunization with the native CP conjugates (CP with 75% O-acetylation) elicited a high proportion of antibodies directed against the O-acetyl moiety. Nonetheless, all of the vaccinees produced antibodies to the backbone moieties as well. Conjugate vaccines made of de-O-acetylated CP elicited backbone antibodies only. Antibodies to both backbone and O-acetyl groups were found to be opsonic against S. aureus strains which varied in their O-acetyl content. Absorption studies with O-acetylated and de-O-acetylated CP showed that (i) native CP conjugates generated antibodies to both backbone and O-acetyl groups and (ii) O-acetylated isolates were opsonized by both populations of antibodies while the non-O-acetylated strains were predominantly opsonized by the backbone antibodies. These results suggest that S. aureus CP conjugate vaccines elicit multiple populations of antibodies with diverse specificities. Moreover, the antibodies of different specificities (backbone or O-acetyl) are all functional and efficient against the variations in bacterial CP that may occur among clinically significant S. aureus pathogenic isolates.     

Histamine metabolism of the guinea-pig gastric mucosa

(1) Preparative dodecyl sulfate gel electrophoresis of human erythrocyte membrane proteins has been used to isolate dodecylsulfate band 3 containing the M,N-glycoprotein and the major "intrinsic" membrane protein (Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617; Bretscher, M.S. (1971) J. Mol. Biol. 59,351-357; Bretscher, M.S. (1971) Nat. New Biol. 231, 229-232 and Marchesi, V.T. and Andrews, E.P. (1972) Science 174, 1247-1248). Subsequent isoelectric focusing in polyacrylamide gels containing Triton X-100 separates these two entities and allows their simultaneous purification. (2) The proteins thus obtained retain their antigenic properties. They are pure according to electrophoretic and immunoelectrophoretic criteria. However, crossed immunoelectrophoresis yields evidence for molecular microheterogeneity of the major "intrinsic" protein. (3) Analyses utilizing crossed immunoelectrophoresis with antibodies absorbed with intact erythrocytes show that the major "intrinsic" protein possesses antigenic determinants on both membrane surfaces and therefore spans the erythrocyte membrane. All determinants of the M,N-glycoprotein detectable with our antibodies were found solely on the exterior membrane surface. (4) Neither the major "intrinsic" membrane protein nor the major M,N-glycoprotein bound significantly to concanavalin A in crossed immunoaffinoelectrophoresis.     

Indices of membrane alterations in beta-thalassemic erythrocytes

Analytical protocols for the study of thalassemic erythrocyte membrane alterations are described. Denatured hemoglobin derivatives and aggregated band 3 are separated from detergent membrane extracts by gel-filtration as high-molecular-weight aggregates and quantitated spectrophotometrically. Membrane-bound, low-molecular-weight iron is measured on SDS-solubilized ghosts by a ferrozine-based colorimetric test. We adapted these methods for microscale preparation and analysis of erythrocyte ghosts in order to have suitable tools to estimate oxidative membrane damage in human samples. Data from 11 beta-thalassemia intermedia patients and from 10 normal controls are reported as an example of the application of these methods.     

Efficacy of ELISA compared to conventional tests (RBPT and CFT) for the diagnosis of Brucella melitensis infection in sheep

The ability of monoclonal antibodies (MAb) to bind or not simultaneously to the antigen (Ag) is used to establish antigenic maps considering that two different MAb do not bind to the Ag when the corresponding epitopes are overlapped (steric effect). Nevertheless, MAb inducing negative allosteric effect on the Ag could prevent the binding of the second MAb even if it is directed to a separate epitope. We report here that a knowledge-based expert module included in our previously described antigenic model-builder program (MAPAG) was able to differentiate between steric and negative allosteric effects between some MAb.     

The effects of macrolides on the expression of bacterial virulence mechanisms

Human erythrocytes in the circulation undergo dynamic oxidative damage involving membrane lipid peroxidation and protein aggregation during aging. The present study was undertaken to determine the effect of n-3 fatty acid supplementation on lipid peroxidation and protein aggregation in the circulation and also the in vitro susceptibility of rat erythrocyte membranes to oxidative damage. Wistar male rats were fed a diet containing n-6 fatty acid-rich safflower oil or n-3 fatty acid-rich fish oil with an equal amount of vitamin E for 6 wk. n-3 Fatty acid content in erythrocyte membranes of rats fed fish oil was significantly higher than that of rats fed safflower oil. The degree of membrane lipid peroxidation and protein aggregation of rats fed fish oil was not significantly higher than that of rats fed safflower oil when the amounts of phospholipid hydroperoxides, thiobarbituric acid-reactive substances, and detergent-insoluble protein aggregates were measured. When isolated erythrocytes were oxidized under aerobic conditions in the presence of Fe(III), the degree of membrane lipid peroxidation of erythrocytes from rats fed fish oil was increased to a greater extent than that of rats fed safflower oil, whereas the degree of membrane protein aggregation of both groups was increased in a similar extent. Hence, n-3 fatty acid supplementation did not affect lipid peroxidation and protein aggregation in membranes of circulating rat erythrocytes, and the supplementation increased the susceptibility of isolated erythrocytes to lipid peroxidation, but not to protein aggregation, under the aerobic conditions. If a sufficient amount of vitamin E is supplied, n-3 fatty acid supplementation may give no undesirable oxidative effects on rat erythrocytes in the circulation.     

Violence against women: the physician''s role

We have measured by indirect ELISA the binding of neutralizing and non-neutralizing anti-lactate dehydrogenase-elevating virus (LDV) polyclonal and monoclonal antibodies to synthetic peptides representing unmodified hydrophilic segments of LDV proteins. Using this method a single neutralization epitope has been shown to be located in the very short (about 30 amino acid long) ectodomain of the primary envelope glycoprotein, VP-3P, encoded by ORF 5. Although the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs differ slightly in amino acid sequences, the neutralizing antibodies bind strongly to the epitopes of both groups of viruses. However, the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs are associated with different numbers of polylactosaminoglycan chains (1 and 3, respectively) which may affect the binding of neutralizing antibodies to the virions of these LDVs. The ELISA using synthetic peptides containing the neutralization epitope provides a novel, rapid, sensitive, and inexpensive method for quantitating LDV neutralizing antibodies in infected mice.     

The mast cell function-associated antigen exhibits saccharide binding capacity

The mast cell function-associated antigen (MAFA) is a membrane glycoprotein first identified on rat mucosal type mast cells (line RBL-2H3) and known to inhibit the Fc epsilon RI-mediated secretory response. In its extracellular domain, an amino acid stretch homologous to the carbohydrate binding domain of calcium-dependent animal lectins has been found. To investigate its carbohydrate binding capacity, the MAFA has been expressed in the Spodoptera frugiperda insect cell line (Sf9) using the baculovirus expression system. Analysis by flow cytometry and surface labeling with 125I showed that the recombinant MAFA (rMAFA) was expressed as a monomeric and disulfide-linked homodimeric glycoprotein in the membrane of the insect cells, and both forms exhibited the same epitopes as the protein isolated from RBL-2H3 cells. Immunoaffinity-purified rMAFA was then employed for studies of its saccharide binding capacity by using different neoglycans and glycoproteins. The rMAFA was found to bind specifically terminal mannose residues in a Ca(2+)-dependent manner. These results support the notion that the extracellular domain of the MAFA is indeed able to bind ligands, which may be modulatory for the mast cell response.