Bivalent binding of IgA1 to FcalphaRI suggests a mechanism for cytokine activation of IgA phagocytosis

FcalphaRI, the receptor specific for the Fc region of immunoglobulin A (IgA), is responsible for IgA-mediated phagocytosis, oxidative burst, and antibody-dependent cellular cytotoxicity. Using the techniques of analytical ultracentrifugation and equilibrium gel-filtration, we show that two FcalphaRI molecules bind to a single Fcalpha homodimer. Surface plasmon resonance studies confirm the 2:1 stoichiometry of binding, with equilibrium dissociation constants of 176 nM and 431 nM for the first and second binding events, respectively. The binding affinity decreases at acidic pH in a manner consistent with protonation of a single histidine residue in the binding site. A thermodynamic analysis indicates that the histidine residue does not participate in a salt-bridge in the complex; in fact, less than 10% of the free energy of binding was contributed by electrostatic interactions. The bivalent, pH-dependent interaction between FcalphaRI and IgA has important implications for cytokine-dependent phagocytosis of IgA and the FcalphaRI-mediated degradation or recycling of IgA.     

The interaction of Fc alpha RI with IgA and its implications for ligand binding by immunoreceptors of the leukocyte receptor cluster

This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.     

Episodes of natural selection shaped the interactions of IgA-Fc with FcalphaRI and bacterial decoy proteins

FcalphaRI, a receptor for IgA-Fc, recruits myeloid cells to attack IgA-coated pathogens. By competing with FcalphaRI for IgA, bacterial decoys, like SSL7 of Staphylococcus aureus, subvert this defense. We examined how pathogen selection has driven the diversification and coevolution of IgA and FcalphaRI. In higher primates, the IgA binding site of FcalphaRI diversified under positive selection, a strong episode occurring in hominoid ancestors about the time of the IgA gene duplication. The differential binding of SSL7 to IgA-Fc of different species correlates with substitution at seven positions in IgA-Fc, two of which were positively selected in higher primates. Two others, which reduce SSL7 binding, emerged during episodes of positive selection in the rabbit and rodent lineages. The FcalphaRI-IgA interaction evolves episodically under two types of positive selection: pressure from pathogen decoys selects for IgA escape variants which, in turn, selects for FcalphaRI variants to keep up with the novel IgA. When FcalphaRI cannot keep up, its function is lost and the gene becomes susceptible to elimination, as occurred in the mouse genome, either by chance or selection on one of the many linked, variable immune system genes. A cluster of positively selected residues presents a putative binding site for unknown IgA-binding factors.     

The staphylococcal superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding and serum killing of bacteria

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (K(D), 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcalphaRI (CD89) and to a soluble form of the FcalphaRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (K(D), 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.     

Structural requirements for the interaction of human IgA with the human polymeric Ig receptor

Transport of polymeric IgA onto mucosal surfaces to become secretory IgA is mediated by the polymeric Ig receptor (pIgR). To study the interaction of human dimeric IgA (dIgA) (the predominant form of IgA polymer) with the human pIgR (hpIgR), we generated recombinant wild-type dIgA1 and dIgA2m(1) and various mutant dIgA1 and analyzed their interaction with a recombinant human secretory component and membrane-expressed hpIgR. We found that wild-type dIgA1 and dIgA2m(1) bound to recombinant human secretory component with similar affinity and were transcytosed by the hpIgR to the same extent. Mutation of the IgA Calpha2 domain residue Cys311 to Ser reduced binding to hpIgR, possibly through disruption of noncovalent interactions between the Calpha2 domain and domain 5 of the receptor. Within the Calpha3 domain of IgA1, we found that combined mutation of residues Phe411, Val413, and Thr414, which lie close to residues previously implicated in hpIgR binding, abolished interaction with the receptor. Mutation of residue Lys377, located very close to this same region, perturbed receptor interaction. In addition, 4 aa (Pro440-Phe443), which lie on a loop at the domain interface and form part of the binding site for human FcalphaRI, appear to contribute to hpIgR binding. Lastly, use of a monomeric IgA1 mutant lacking the tailpiece revealed that the tailpiece does not occlude hpIgR-binding residues in IgA1 monomers. This directed mutagenesis approach has thus identified motifs lying principally across the upper surface of the Calpha3 domain (i.e., that closest to Calpha2) critical for human pIgR binding and transcytosis.     

Inside-out regulation of Fc alpha RI (CD89) depends on PP2A

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcalphaRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcalphaRI tail is important for FcalphaRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcalphaRI activation. PP2A binds to the intracellular tail of FcalphaRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcalphaRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcalphaRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcalphaRI ligand binding regulation, a key step in initiating an immune response.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.     

Porcine pancreatic alpha-amylase shows binding activity toward N-linked oligosaccharides of glycoproteins.

Porcine pancreatic alpha-amylase was shown by interaction analyses using a resonance mirror detector and alpha-amylase-immobilized Sepharose to bind with glycoproteins possessing N-glycans but not O-linked mucin-type glycans. Direct binding of three types of N-glycans to the alpha-amylase was demonstrated by surface plasmon resonance. Binding with biotin-polymer sugar probes revealed that the alpha-amylase has affinity to alpha-mannose, alpha-N-acetylneuraminic acid, and beta-N-acetyllactosamine, which are components of N-glycans. The binding of glycoproteins or carbohydrates enhanced the enzyme activity, indicating that the recognition site for N-glycans is different from its catalytic site. The binding activity was unique to porcine pancreatic alpha-amylase and was not observed for alpha-amylase from saliva, wheat, and fungus.     

Implications of the near-planar solution structure of human myeloma dimeric IgA1 for mucosal immunity and IgA nephropathy

IgA is unique in being able to form a diverse range of polymeric structures. Increases in the levels of dimeric IgA1 (dIgA1) in serum have been implicated in diseases such as IgA nephropathy. We have determined the solution structure for dIgA1 by synchrotron x-ray and neutron scattering and analytical ultracentrifugation. The Guinier radius of gyration (RG) of 7.60-8.65 nm indicated that the two monomers within dIgA1 are arranged in an extended conformation. The distance distribution curve P(r) gave an overall length (L) of 22-26 nm. These results were confirmed by the sedimentation coefficient and frictional ratio of dIgA1. Constrained scattering modeling starting from the IgA1 monomer solution structure revealed a near-planar dimer structure for dIgA1. The two Fc regions form a slightly bent arrangement in which they form end-to-end contacts, and the J chain was located at this interface. This structure was refined by optimizing the position of the four Fab regions. From this, the best-fit solution structures show that the four Fab Ag-binding sites are independent of one another, and the two Fc regions are accessible to receptor binding. This arrangement allows dIgA1 to initiate specific immune responses by binding to FcalphaRI receptors, while still retaining Ag-binding ability, and to be selectively transported to mucosal surfaces by binding to the polymeric Ig receptor to form secretory IgA. A mechanism for the involvement of dIgA1 oligomers in the pathology of IgA nephropathy is discussed in the light of this near-planar structure.     

FcalphaRI-positive liver Kupffer cells: reappraisal of the function of immunoglobulin A in immunity

Despite the well-recognized involvement of immunoglobulin (Ig) A in mucosal immunity, the function of its receptor, FcalphaRI (CD89), is poorly understood. The ability of FcalphaRI to activate leukocytes seems to conflict with the proposed anti-inflammatory activity of secretory IgA. We show here that in a transgenic mouse model, inflammatory mediators induced expression of FcalphaRI on Kupffer cells, which enabled efficient phagocytosis in vivo of bacteria coated with serum IgA. Secretory IgA did not initiate phagocytosis. Therefore, interactions between serum IgA and FcalphaRI on Kupffer cells may provide a 'second line of defense' in mucosal immunity, by eliminating invasive bacteria entering through the portal circulation and thus preventing disease.     

Do rodent and human brains have different N-glycosylation patterns?

A large number of studies on the structure of N-glycosidically linked oligosaccharides from glycoproteins of different organs and/or different species have been carried out in the past using various combinations of techniques such as monosaccharide analysis, permethylation, peracteylation, exoglycosidase sequencing, normal and reversed phase HPLC, mass spectrometry and nuclear magnetic resonance spectroscopy. Although it is widely accepted that the processing of N-glycans in the ER and Golgi of mammalian cells follows the same principal metabolic rules, analyses have revealed that the glycosylation pattern of a particular protein may differ depending on the cell type in which it is expressed. N-glycans from brain glycoproteins have been shown to include a variety of hybrid- and complex-type structures with structural features that are not so commonly found on glycoproteins from other organs and which have, therefore, been classified as 'brain-specific'. Comparison of the N-glycans of glycoproteins from homogenates of rat, mouse and human brains confirm that, in general, glycoproteins from human brain show a similar profile of brain-specific N-glycans as glycoproteins from mouse and rat brain.     

Signaling through mutants of the IgA receptor CD89 and consequences for Fc receptor gamma-chain interaction

The prototypic receptor for IgA (FcalphaRI, CD89) is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. The functions of FcalphaRI and activating receptors for IgG (FcgammaRI and FcgammaRIII) are dependent on the FcR gamma-chain dimer. This study increases our understanding of the molecular basis of the FcalphaRI-FcR gamma-chain transmembrane interaction, which is distinct from that of other activatory FcRs. FcalphaRI is unique in its interaction with the common FcR gamma-chain, because it is based on a positively charged residue at position 209, which associates with a negatively charged amino acid of FcR gamma-chain. We explored the importance of the position of this positive charge within human FcalphaRI for FcR gamma-chain association and FcalphaRI functioning with the use of site-directed mutagenesis. In an FcalphaRI R209L/A213H mutant, which represents a vertical relocation of the positive charge, proximal and distal FcR gamma-chain-dependent functions, such as calcium flux, MAPK phosphorylation, and IL-2 release, were similar to those of wild-type FcalphaRI. A lateral transfer of the positive charge, however, completely abrogated FcR gamma-chain-dependent functions in an FcalphaRI R209L/M210R mutant. By coimmunoprecipitation, we have demonstrated the loss of a physical interaction between FcR gamma-chain and FcalphaRI M210R mutant, thus explaining the loss of FcR gamma-chain-dependent functions. In conclusion, not only the presence of a basic residue in the transmembrane region of FcalphaRI, but also the orientation of FcalphaRI toward the FcR gamma-chain dimer is essential for FcR gamma-chain association. This suggests the involvement of additional amino acids in the FcalphaRI-FcR gamma-chain interaction.     

Crystal structure of the ectodomain of human FcalphaRI

Human FcalphaRI (CD89) is the receptor specific for IgA, an immunoglobulin that is abundant in mucosa and is also found in high concentrations in serum. Although FcalphaRI is an immunoglobulin Fc receptor (FcR), it differs in many ways from FcRs for other immunoglobulin classes. The genes of most FcRs are located on chromosome 1 at 1q21-23, whereas FcalphaRI is on chromosome 19, at 19q13.4, a region called the leukocyte receptor complex, because it is clustered with several leukocyte receptor families including killer cell inhibitory receptors (KIRs) and leukocyte Ig-like receptors (LIRs). The amino acid sequence of FcalphaRI shares only 20% homology with other FcRs but it has around 35% homology with its neighboring LIRs and KIRs. In this work, we analyzed the crystal structure of the ectodomain of FcalphaRI and examined structure similarities between FcalphaRI and KIR2DL1, KIR2DL2 and LIR-1. Our data show that FcalphaRI, KIRs, and LIRs share a common hydrophobic core in their interdomain interface, and FcalphaRI is evolutionally closer to LIR than KIR.     

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.     

Heterogeneity of expression of IgA receptors by human, mouse, and rat eosinophils

IgA is the most abundant class of Abs at mucosal surfaces where eosinophils carry out many of their effector functions. Most of the known IgA-mediated functions require interactions with IgA receptors, six of which have been identified in humans. These include the IgA FcR FcalphaRI/CD89 and the receptor for the secretory component, already identified on human eosinophils, the polymeric IgR, the Fcalpha/muR, asialoglycoprotein (ASGP)-R, and transferrin (Tf)R/CD71. In rodents, the existence of IgA receptors on mouse and rat eosinophils remains unclear. We have compared the expression and function of IgA receptors by human, rat, and mouse eosinophils. Our results show that human eosinophils express functional polymeric IgR, ASGP-R, and TfR, in addition to CD89 and the receptor for the secretory component, and that IgA receptors are expressed by rodent eosinophils. Indeed, mouse eosinophils expressed only TfR, whereas rat eosinophils expressed ASGP-R and CD89 mRNA. These results provide a molecular basis for the differences observed between human, rat, and mouse regarding IgA-mediated immunity.     

Phage-based molecular directed evolution yields multiple tandem human IgA affibodies with intramolecular binding avidity

Affibodies are a group of affinity proteins that are based on a 58-amino-acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. A single human IgA affibody with high IgA affinity has been generated by directed evolution. It remains interesting whether tandem IgA affibody proteins could increase binding capacity. Here, we report the generation of multiple tandem IgA affibodies by directed evolution using a combinatorial phage library displaying the IgA affibody A1 and/or A2 linked with three random amino acids. These affibodies exhibited markedly increased IgA binding capacity, as shown by enzyme linked immunosorbent assay, immunoblotting and surface plasmon resonance assays. We further showed that these tandem IgA affibodies displayed preferential binding to intact IgA molecules compared to individual IgA chain, suggesting intramolecular binding avidity. Our data demonstrates that artificial multiple tandem human IgA affibodies with relevant biological binding avidity were successfully yielded by phage-based molecular evolution. These results have broad implications for the design and development of binding proteins that target important biological molecules.     

Xenopus galectin-VIIa binds N-glycans of members of the cortical granule lectin family (xCGL and xCGL2)

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.     

Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors

Antitumor Abs are promising therapeutics for cancer. Currently, most Ab-based therapies focus on IgG Ab, which interact with IgG FcR (FcgammaR) on effector cells. In this study, we examined human and mouse neutrophil-mediated tumor cell lysis via targeting the IgA FcR, FcalphaRI (CD89), in more detail. FcalphaRI was the most effective FcR in triggering tumor cell killing, and initiated enhanced migration of neutrophils into tumor colonies. Importantly, immature neutrophils that are mobilized from the bone marrow upon G-CSF treatment efficiently triggered tumor cell lysis via FcalphaRI, but proved incapable of initiating tumor cell killing via FcgammaR. This may provide a rationale for the disappointing results observed in some earlier clinical trials in which patients were treated with G-CSF and antitumor Ab-targeting FcgammaR.     

Species-specific distribution of alpha-galactosyl epitopes on the gastric H/K ATPase beta-subunit: relevance to the binding of human anti-parietal cell autoantibodies.

The gastric H/K ATPase beta-subunit, an abundant glycoprotein of the secretory membranes of gastric parietal cells, is the major autoantigen recognized by human parietal cell autoantibodies in gastric autoimmunity. Our previous studies demonstrated that the human autoantibodies recognize the H/K ATPase beta-subunit from a number of species and that glycosylation of the beta-subunit with complex N-glycans is required for autoantibody binding. The N-glycans of the beta-subunit contain polylactosamine chains. The lactosamine chains of the rabbit beta-subunit are terminated with alpha-linked galactosyl residues (alpha-galactosyl epitope) (Tyagarajan et al., Biochemistry, 1996, 35, 3238-3246). Here we have investigated the expression of alpha-galactosyl epitopes on the H/K ATPase beta-subunit from a number of species. Using the alpha-galactosyl binding lectin, BS1-IB4, and naturally occurring anti-alpha-galactosyl antibodies, we have demonstrated that the rat H/K ATPase beta-subunit also contains terminal alpha-galactosyl residues, but not the beta-subunit from pig, dog, and mouse, indicating species-specific differences in the terminal saccharide sequences of the beta-subunit. We also investigated the potential contribution of the alpha-galactosyl epitopes to the binding by human sera. The reactivity of human pernicious anemia serum with gastric parietal cells could not be inhibited with saccharide inhibitors and, in addition, no binding was observed with normal human sera. We conclude that the H/K ATPase beta-subunit oligosaccharides from rabbit and rat are terminated with alpha-galactosyl epitopes, and although the presence of this epitope does not contribute to binding by human parietal cell autoantibodies at the concentrations routinely used, it is recommended that neither rat or rabbit stomachs be used for screening human sera.     

Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii

The primary structure of a lectin, designated Oscillatoria agardhii agglutinin (OAA), isolated from the freshwater cyanobacterium O. agardhii NIES-204 was determined by the combination of Edman degradation and electron spray ionization-mass spectrometry. OAA is a polypeptide (Mr 13,925) consisting of two tandem repeats. Interestingly, each repeat sequence of OAA showed a high degree of similarity to those of a myxobacterium, Myxococcus xanthus hemagglutinin, and a marine red alga Eucheuma serra lectin. A systematic binding assay with pyridylaminated oligosaccharides revealed that OAA exclusively binds to high mannose (HM)-type N-glycans but not to other N-glycans, including complex types, hybrid types, and the pentasaccharide core or oligosaccharides from glycolipids. OAA did not interact with any of free mono- and oligomannoses that are constituents of the branched oligomannosides. These results suggest that the core disaccharide, GlcNAc-GlcNAc, is also essential for binding to OAA. The binding activity of OAA to HM type N-glycans was dramatically decreased when alpha1-2 Man was attached to alpha1-3 Man branched from the alpha1-6 Man of the pentasaccharide core. This specificity of OAA for HM-type oligosaccharides is distinct from other HM-binding lectins. Kinetic analysis with an HM heptasaccharide revealed that OAA possesses two carbohydrate binding sites per molecule, with an association constant of 2.41x10(8) m-1. Furthermore, OAA potently inhibits human immunodeficiency virus replication in MT-4 cells (EC50=44.5 nm). Thus, we have found a novel lectin family sharing similar structure and carbohydrate binding specificity among bacteria, cyanobacteria, and marine algae.