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Key Points The pattern and degree of sulfation influenced FHR5–GAG interactions. Surface heparan sulphate enhanced the ability of FHR5 to limit FH binding to C3b., Complement activation is an important mediator of kidney injury in glomerulonephritis. Complement factor H (FH) and FH-related protein 5 (FHR-5) influence complement activation in C3 glomerulopathy and IgA nephropathy by differentially regulating glomerular complement. FH is a negative regulator of complement C3 activation. Conversely, FHR-5 in vitro promotes C3 activation either directly or by competing with FH for binding to complement C3b. The FH–C3b interaction is enhanced by surface glycosaminoglycans (GAGs) and the FH–GAG interaction is well-characterized. In contrast, the contributions of carbohydrates to the interaction of FHR-5 and C3b are unknown. Using plate-based and microarray technologies we demonstrate that FHR-5 interacts with sulfated GAGs and that this interaction is influenced by the pattern and degree of GAG sulfation. The FHR-5–GAG interaction that we identified has functional relevance as we could show that the ability of FHR-5 to prevent binding of FH to surface C3b is enhanced by surface kidney heparan sulfate. Our findings are important in understanding the molecular basis of the binding of FHR-5 to glomerular complement and the role of FHR-5 in complement-mediated glomerular disease.

R-10G antigen of human iPS and ES cells has been assigned as a unique mono-sulfated glycan. We highlight its relationship to a known bioactive glycan sequence, the ligand on high endothelial venules for the lymphocyte homing receptor, L-selectin. Details of the sequences of four other glycan antigens on the podocalyxin are re-evaluated and ambiguities resolved. Regulation of biosynthesis and possible involvements of these glycans in podocalyxin-signaling in stem cells are discussed., Graphical Abstract Highlights R-10G is a mono-sulfated glycan antigen of human iPS and ES cells on podocalyxin. R-10G and nonsulfated i are reciprocal antigens on type 2 glycan backbones. TRA-1–60 and -81 and FC10.2 are antigens expressed on unsulfated type 1 backbones. These assignments open the way to probing their roles in podocalyxin-signaling., Glycan antigens recognized by monoclonal antibodies have served as stem cell markers. To understand regulation of their biosynthesis and their roles in stem cell behavior precise assignments are required. We have applied state-of-the-art glycan array technologies to compare the glycans bound by five antibodies that recognize carbohydrates on human stem cells. These are: FC10.2, TRA-1–60, TRA-1–81, anti-i and R-10G. Microarray analyses with a panel of sequence-defined glycans corroborate that FC10.2, TRA-1–60, TRA-1–81 recognize the type 1-(Galβ-3GlcNAc)-terminating backbone sequence, Galβ-3GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, Galβ-4GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By Beam Search approach, starting with an antigen-positive keratan sulfate polysaccharide, followed by targeted iterative microarray analyses of glycan populations released with keratanases and mass spectrometric monitoring, R-10G is assigned as a mono-sulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Galβ-4GlcNAc(6S)β-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc. Microarray analyses using newly synthesized glycans corroborate the assignment of this unique determinant raising questions regarding involvement as a ligand in the stem cell niche.

Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step., Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection. IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

FCT Investigator IF/00033/2012. Wellcome Trust (086827, 075470, 099215, 099197 and 101873) Wellcome Trust ISSF award (105625), MRC CiC (MC_PC_14114) and MRC Centre for Medical Mycology and University of Aberdeen for funding and a Wellcome Trust Strategic Award (097377). Wellcome Trust grant 099197MA . The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis. publishersversion published

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-β. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-β secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.

Amino acid replacement mutations in certain chronic lymphocytic leukemia (CLL) stereotyped B cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously, and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found that IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bound naïve B cells. However, only subset 4 IGs reacted with memory B cells. Furthermore, subset 4 IGs did not bind DNA nor i or I carbohydrate antigens that are common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis., Late diagnosis and ineffective treatment of fungal infections lead to high mortality. Here, Rudkin et al. generate anti-Candida human monoclonal antibodies with diagnostic and therapeutic potential, by expressing recombinant antibodies from genes cloned from B cells of patients suffering candidiasis.

Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012–2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

Human polyomavirus 9 (HPyV9) is a closely related homologue of simian B-lymphotropic polyomavirus (LPyV). In order to define the architecture and receptor binding properties of HPyV9, we solved high-resolution crystal structures of its major capsid protein, VP1, in complex with three putative oligosaccharide receptors identified by glycan microarray screening. Comparison of the properties of HPyV9 VP1 with the known structure and glycan-binding properties of LPyV VP1 revealed that both viruses engage short sialylated oligosaccharides, but small yet important differences in specificity were detected. Surprisingly, HPyV9 VP1 preferentially binds sialyllactosamine compounds terminating in 5- N -glycolyl neuraminic acid (Neu5Gc) over those terminating in 5- N -acetyl neuraminic acid (Neu5Ac), whereas LPyV does not exhibit such a preference. The structural analysis demonstrated that HPyV9 makes specific contacts, via hydrogen bonds, with the extra hydroxyl group present in Neu5Gc. An equivalent hydrogen bond cannot be formed by LPyV VP1. IMPORTANCE The most common sialic acid in humans is 5- N -acetyl neuraminic acid (Neu5Ac), but various modifications give rise to more than 50 different sialic acid variants that decorate the cell surface. Unlike most mammals, humans cannot synthesize the sialic acid variant 5- N -glycolyl neuraminic acid (Neu5Gc) due to a gene defect. Humans can, however, still acquire this compound from dietary sources. The role of Neu5Gc in receptor engagement and in defining viral tropism is only beginning to emerge, and structural analyses defining the differences in specificity for Neu5Ac and Neu5Gc are still rare. Using glycan microarray screening and high-resolution protein crystallography, we have examined the receptor specificity of a recently discovered human polyomavirus, HPyV9, and compared it to that of the closely related simian polyomavirus LPyV. Our study highlights critical differences in the specificities of both viruses, contributing to an enhanced understanding of the principles that underlie pathogen selectivity for modified sialic acids.

SummaryBroadly neutralizing HIV antibodies are much sought after (a) to guide vaccine design, both as templates and as indicators of the authenticity of vaccine candidates, (b) to assist in structural studies, and (c) to serve as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies has been limited. Here, we describe a set of human monoclonal antibodies that define what is, to the best of our knowledge, a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process., Highlights • Changes in receptor binding of HA during H5N1 human infection were identified. • Single A134V and L151F substitutions caused reduced affinity for avian receptors. • Glycan array analyses were used to identify changes in receptor binding specificity. • Structural basis for altered receptor binding was examined by X-ray crystallography.

11 pags, 7 figs, CLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., This work was funded by the Wellcome Trust, the National Research Foundation, the Deutscher Akademischer Austauschdienst, the University of Cape Town, the UK Research Council Basic Technology Initiative “Glycoar-rays” (GRS/79268), and the UK Medical Research Council. A. S. P is a fellowof the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal) and M. A. C. of the Consejo Superior de Investigaciones Cientificas, Programe “Junta para la Ampliación de Estudios” (JaeDoc/098/2011) cofinanced by the Fondo Social Europeo.

11 pags, 6 figs, It is well established that murine T-lymphocyte activation is accompanied by major changes in cell-surface sialylation, potentially influencing interactions with sialic acid-binding immunoglobulin-like lectins (siglecs). In the present study, we analyzed early activation of murine CD4+ and CD8+ T-lymphocytes at 24 h. We observed a striking and selective up-regulation in the binding of a recombinant soluble form of siglec-E, an inhibitory siglec, which is expressed on several myeloid cell types including antigen-presenting dendritic cells. In contrast, much lower levels of T cell binding were observed with other siglecs, including sialoadhesin, CD22, and siglec-F and the plant lectins Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin. By mass spectrometry, the sialic acid content of 24-h-activated CD4+ and CD8+ T-lymphocytes exhibited an increased proportion of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) in N-glycans. Reduced levels of NeuGc on the surface of activated T cells were demonstrated using an antibody specific for NeuGc and the expression levels of the gene encoding NeuAc- to NeuGc-converting enzyme, CMP-NeuAc hydroxylase, were also reduced. Siglec-E bound a wide range of sialylated structures in glycan arrays, had a preference for NeuAc versus NeuGc-terminated sequences and could recognize a set of sialoglycoproteins that included CD45, in lysates from activated T-lymphocytes. Collectively, these results show that early in T cell activation, glycan remodelling involves a switch from NeuGc- to NeuAc-terminating oligosaccharides on cell surface glycoproteins. This is associated with a strong up-regulation of siglec-E ligands, which may be important in promoting cellular interactions between early activated T-lymphocytes and myeloid cells expressing this inhibitory receptor. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc., This work was funded by the Biotechnology and Biological Sciences Research Council and the Engineering and Physical Sciences Research Council through the Radical Solutions for Researching the Proteome (RASOR) Project BB/C511572/1, and the Wellcome Trust (WT081882MA).This work was also supported in part by funding from the Analytical Gly-cotechnology Core (Core C) of the Consortium for Functional Glycomics (GM 62116) and the Biotechnology and Biological Sciences Research Council (BBSRC) Grant number BBF0083091 (to A. D. and S. M. H.), the MRCand the RCUK Basic Technology Initiative Glycoarrays (GRS/79268) andEPSRC Translational Grant (EP/G037604/1) (EP/G037604/1) (to T. F.)

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-A resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000 kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray in analyses of one of the monoclonal antibodies, AE3, that has been regarded the ‘most carcinoma specific’ in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewisa/b, Lewisx/y and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galβ1-3GalNAcβ1-4(3-O-sulfate)Galβ1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

Immunologically, "self" carbohydrates protect the HIV-1 surface glycoprotein, gp120, from antibody recognition. However, one broadly neutralizing antibody, 2G12, neutralizes primary viral isolates by direct recognition of Manalpha1-->2Man motifs formed by the host-derived oligomannose glycans of the viral envelope. Immunogens, capable of eliciting antibodies of similar specificity to 2G12, are therefore candidates for HIV/AIDS vaccine development. In this context, it is known that the yeast mannan polysaccharides exhibit significant antigenic mimicry with the glycans of HIV-1. Here, we report that modulation of yeast polysaccharide biosynthesis directly controls the molecular specificity of cross-reactive antibodies to self oligomannose glycans. Saccharomyces cerevisiae mannans are typically terminated by alpha1-->3-linked mannoses that cap a Manalpha1-->2Man motif that otherwise closely resembles the part of the oligomannose epitope recognized by 2G12. Immunization with S. cerevisiae deficient for the alpha1-->3 mannosyltransferase gene (DeltaMnn1), but not with wild-type S. cerevisiae, reproducibly elicited antibodies to the self oligomannose glycans. Carbohydrate microarray analysis of DeltaMnn1 immune sera revealed fine carbohydrate specificity to Manalpha1-->2Man units, closely matching that of 2G12. These specificities were further corroborated by enzyme-linked immunosorbent assay with chemically defined glycoforms of gp120. These antibodies exhibited remarkable similarity in the carbohydrate specificity to 2G12 and displayed statistically significant, albeit extremely weak, neutralization of HIV-1 compared to control immune sera. These data confirm the Manalpha1-->2Man motif as the primary carbohydrate neutralization determinant of HIV-1 and show that the genetic modulation of microbial polysaccharides is a route towards immunogens capable of eliciting antibody responses to the glycans of HIV-1.

Total syntheses of two natural sulphoglycolipids, disulphated glycosphingolipid SB1a and the structurally related monosulphated SM1a, are described. They have common glycan sequences and ceramide moieties and are associated with human epithelial carcinomas. The syntheses featured efficient glycan assembly and the glucosyl ceramide cassette as a versatile building block. The binding of the synthetic sulphoglycolipids by the carcinoma-specific monoclonal antibody AE3 was investigated using carbohydrate microarray technology.

Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy., Author Summary HAdVs are common pathogens in humans, causing disease mainly in eyes, airways and gastrointestinal tract. Most HAdVs are equipped with twelve protruding fiber proteins that mediate attachment to host cell receptor molecules. Recently, a new human gastroenteritis-associated adenovirus (HAdV-52) was identified and classified as the first member of a novel species (HAdV-G). Unlike most other HAdVs, this virus contains two different fiber proteins, a long and a short one, a feature shared only with the two members of species HAdV-F (HAdV-40 and -41). To gain further insights into the mechanisms of HAdV-52 infection of human cells, we set out to identify the host cell receptors used by the long and short fibers. We find that the long fiber binds to a protein-based receptor known as the coxsackievirus and adenovirus receptor (CAR), and that the short fiber binds to glycoproteins that contain sialic acid-capped glycans. The crystal structure determination of a complex of the short fiber knob bound to sialic acid demonstrates that this interaction is unique among HAdVs, and bioinformatic analysis indicates that simian AdVs may also engage sialic acids in the manner seen in HAdV-52. The results presented here provide insights into the plasticity of adenovirus-host cell interactions.

The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N-glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5-positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N-glycans containing a 3-fucosyl N-acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N-acetyllactosamine sequence [Lewisx (Le(x)) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15.

Carbohydrates are thought to function as tags that mark circulatory glycoproteins for rapid clearance. To examine the role of the mannose receptor (MR) in glycoprotein clearance, we generated mice genetically deficient in MR. MR−/−mice were defective in clearing proteins bearing accessible mannose andN-acetylglucosamine residues and had elevated levels of eight different lysosomal hydrolases. Proteomic analysis of MR−/−and control mouse sera showed that an additional 4 out of 52 proteins identified were elevated in MR−/−serum. Each of these is up-regulated during inflammation and wound healing. Thus, MR appears to operate as an essential regulator of serum glycoprotein homeostasis.

Monoclonal antibodies (MAbs) directed to Lewis(x) (Le(x)) and related carbohydrate sequences have been invaluable in anticipating biological roles for these oligosaccharides by detecting the remarkable changes that occur in their expression from the earliest stages of embryogenesis, through development and sequential stages of cell differentiation and maturation. A notable impact has been in the molecular dissection of ligand-receptor interactions in key cell adhesion events at the initial stages of leukocyte recruitment in inflammation, and almost certainly in the metastasis of epithelial tumours. Antibodies that recognise Le(x) and the 3'-sialyl forms were observed to identify leukocyte subsets; these were subsequently found to match those recognized by the leukocyte-endothelium adhesion molecules, the E- and P-selectins. We now describe a MAb (rat hybridoma MIN/3/60) raised to 3'-sulpho-Le(x), a carbohydrate sequence which, in vitro, is bound not only by the E-, L-, and P-selectins, but also by the cysteine-rich domain of the macrophage endocytosis receptor. We observe that MIN/3/60 is bispecific, however; it binds 3'-sulpho-Le(a) as well as 3'-sulpho-Le(x). Nevertheless, our exploratory studies reveal that it may be a useful histochemical reagent when used in conjunction with a monospecific antibody to 3'-sulpho-Le(a). The MIN/3/60 antibody reveals a sub-population of epithelial glycans in the crypts of Lieberkühn in normal human colon.

The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrP(Sc), even in the earliest detectable brain lesions of scrapie-infected mice. Determining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis. Toward this aim, we have examined preparations of heparan sulfate, with differing sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenicity.

NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NK-like T cells and is an activator of cytotoxicity. Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coli and folded in vitro. The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure. A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure. The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry. These are characteristic of a long form CRD. The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the (15)N-labeled CRD are those of a folded protein. Chemical shifts of H(alpha) and NH protons indicate a considerable amount of beta-strand structure. Successful folding in the absence of Ca(2+), coupled with the lack of chemical shift changes upon addition of Ca(2+), suggests that the NKR-P1A-CRD may not be a Ca(2+)-binding protein.

There is growing interest in carbohydrate-recognizing receptors of the innate immune system. Among them are members of the C-type lectin family, which include the collectins and the selectins and which operate by ligating exogenous (microbial) or endogenous carbohydrates. De novo assignments of the sequences of ligands for carbohydrate-recognizing receptors are among the most challenging topics in cell biology. This is because of the heterogeneity of oligosaccharides on proteins and lipids, and their availability only in limited amounts. To address the need for a microprocedure for direct binding studies with oligosaccharides derived from glycoproteins, we introduced the neoglycolipid technology for generating solid phase oligosaccharide probes for binding experiments. The technology has enabled assignments of unsuspected oligosaccharide ligands for the selectins and given valuable insights into those for the collectins. The ligands so far identified appear not to be unique for a given receptor system; there are considerable cross-reactions. Specificity can be created, however, through different modes of oligosaccharide presentation on macromolecular carriers, or the expression of a particular oligosaccharide sequence on a selected cell type in a given body compartment, and the regulated expression of the receptor protein at the desired location. The existence of unique ligand structures is not ruled out, however. Co-ligation of a receptor may also occur to a second carbohydrate or even to a non-carbohydrate ligand to create a unique assembly. A further group of C-type lectin-like proteins occurs on natural killer (NK) cells and NK T cells, and is associated with activation or inhibition of the cell effector functions. An important challenge is to determine whether carbohydrates are among physiological ligands for this important group of receptors.

The closing years of the second millennium have been uplifting for carbohydrate biology. Optimism that oligosaccharide sequences are bearers of crucial biological information has been borne out by the constellation of efforts of carbohydrate chemists, biochemists, immunochemists, and cell- and molecular biologists. The direct involvement of specific oligosaccharide sequences in protein targeting and folding, and in mechanisms of infection, inflammation and immunity is now unquestioned. With the emergence of families of proteins with carbohydrate-binding activities, assignments of information content for defined oligosaccharide sequences will become more common, but the pinpointing and elucidation of the bioactive domains on oligosaccharides will continue to pose challenges even to the most experienced carbohydrate biologists. The neoglycolipid technology incorporates some of the key requirements for this challenge: namely the resolution of complex glycan mixtures, and ligand binding coupled with sequence determination by mass spectrometry.

The occurrence of mannitol-terminating oligosaccharides (2-substituted or 2,6-disubstituted) among the O-glycans released by alkaline borohydride treatment from glycoproteins of the nervous system has prompted the development of a microscale method to analyze the core-branching pattern and sequence by the neoglycolipid (NGL) technology, analogous to a method previously described for GalNAcol-terminating oligosaccharides (M. S. Stoll, E. F. Hounsell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 1990). The approach involves the selective cleavage at the core mannitol by mild periodate treatment and analysis of the reaction products as NGLs by in situ TLC/liquid secondary ion mass spectrometry. Oxidation conditions have been optimized using as reference compounds 2-, 3-, 4-, or 6-monosubstituted mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylated oligosaccharides isolated from brain glycopeptides. When a 2:1 molar ratio of periodate to alditol is used, the core mannitol is cleaved at the C3-C4 threo-diol bond and in the absence of a threo-diol cleavage occurs to a lesser extent at erythro-diols. Saccharide ring diols are not cleaved under these conditions, and it is also shown that the side chain of sialic acid on the oligosaccharide is largely unaffected. Substituents at 2- and 6-positions of the core mannitol can be identified, and the method is applicable to neutral and sialylated oligosaccharide alditols. Typically, the starting material is 5 nmol of oligosaccharide and 0.5-1 nmol of derivatives is applied for analysis. By this strategy, the core-branching pattern and position of sialic acid of two branched monosialylated mannitol-terminating oligosaccharide isomers have been determined.

The interaction between glycosphingolipids and recombinant human GM2-activator was studied in a microwell binding assay. A-series gangliosides like GM3, GM2 and GM1 were strongly bound by the recombinant human GM2 activator. A weak binding was observed to GD1b and sulfatide, while neutral glycolipids were not bound. Optimal binding occurred at pH 4.2 and was inhibited by increasing concentrations of citrate buffer and NaCl. In contrast with these in vitro results the recombinant human GM2-activator is able to restore the degradation of GA2 in fibroblasts from patients with the AB variant of GM2 gangliosidosis in vivo.

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells also binds to selected regions in nervous tissue. The antigen is a carbohydrate that has attracted much interest as its expression is developmentally regulated in nervous tissue, and it is found, and proposed to be a ligand, on several of the adhesive glycoproteins of the nervous system. It is also expressed on glycolipids and proteoglycans, and is the target of monoclonal auto-antibodies that give rise to a demyelinating disease. The epitope, as characterized on glycolipids isolated from the nervous system, is expressed on 3-sulfated glucuronic acid joined by β1-3-linkage to a neolacto backbone. Here we exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides. The immunoreactive oligosaccharides detected are tetra- to octasaccharides that are very minor components among a heterogeneous population, each representing less than 0.1% of the starting material. Their peripheral and backbone sequences resemble those of the HNK-1-positive glycolipids. An unexpected finding is that they terminate not with N-acetylgalactosaminitol but with hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as 2-substituted mannitol.

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV-16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV-16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell-binding receptors for HPV-16, heparin-preincubated virus bound to the extracellular matrix (ECM) via laminin-332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S-domains of heparan sulfate (HS) chains of HSPGs, allowed HPV-16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope-specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV-16 virion occur during infection by interaction with'heparin-like' domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV-16 infection.

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types., Author Summary Viruses need to bind to receptors on host cells for viral entry and infection, and the type of receptor bound determines the range of hosts and tissues the virus can infect. Viruses within a family often vary in their tissue distribution and pathogenicity because changes in receptor specificity can produce a virus with different spread and infectivity. In fact, many transmissions between species are based on a virus acquiring binding capability for a new receptor. The structural changes that underlie such receptor switching are not well understood. We have analyzed the structural requirements for receptor binding and switching of the human BK polyomavirus (BKPyV), the causative agent of polyomavirus-associated nephropathy. We show that BKPyV uses specific gangliosides that all contain a common α2,8-disialic acid motif to infect cells, and have characterized the interaction in atomic detail. Our data explains the requirement for this disialic acid motif and in particular highlights a single amino acid that is central to determining specificity. Mutation of this residue switches the receptor specificity, enabling BKPyV to infect cells bearing a different class of gangliosides. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram binding experiments and in situ liquid secondary ion mass spectrometry (LSIMS) with neoglycolipids derived from their endoglycoceramidase-released oligosaccharides, in conjunction with compositional and linkage analyses, one of the glycolipid ligands in kidney was identified as the Le x -active extended globo-glycolipid: Galβ1-4GlcNAcβ1-6GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-Cer;|||Fucα1,3||Ga1β1,3|. Neoglycolipids enriched for the ligand structures were obtained from oligosaccharides released by endo-β-galactosidase from the 32D c13 cells. By TLC-LSIMS and antibody binding, the main E-selectin binding determinant on these was identified as sialyl-Le a .

Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of α2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.

Unnatural, NMR- and MRI-active fluorinated sugar probes, designed and synthesised to bind to the pathogenic protein TgMIC1 from Toxoplasma gondii, were found to display binding potency equal to and above that of the natural ligand. Dissection of the binding mechanism and modes, including the first X-ray crystal structures of a fluoro-oligosaccharide bound to a lectin, demonstrate that it is possible to create effective fluorinated probe ligands for the study of, and perhaps intervention in, sugar-protein binding events.

There is evidence to suggest that carbohydrate antigens restricted to neutrophils and monocytes among human blood cells, but distributed widely in epithelial tissues and secretions, are ligands for cell-cell adhesion molecules that are transiently expressed on endothelia and platelets at sites of inflammation. If these combining specificities are confirmed, it means that biological specificities may arise through regulated focal expression of proteins that recognize oligosaccharides that are not necessarily unique.

The soluble beta-galactoside-specific bovine lectin of subunit 14 kDa has been expressed in vitro by transcription and then translation in a rabbit reticulocyte lysate. The protein thus expressed shows the predicted binding to lactose coupled to Sepharose. Several mutants of the 134 amino acid protein have been expressed and insight gained into (a) the polypeptide length required to form the carbohydrate recognition domain and (b) the functional importance of some of the highly conserved amino acids. The following amino acids have been deleted: 1-9, 1-23, 88-122, 88-134, 107-134, or 124-134. In addition, a frame-shift mutant has been made in which the 23 amino acids at the C-terminal end were completely changed. Among these seven mutants only mutant 1-9 shows carbohydrate binding but with congruent to 30% of the activity of the wild-type protein (as assessed by the percentage of the protein bound to lactose-Sepharose). On the other hand, carbohydrate binding is relatively well preserved (75-90%) in mutant proteins where the C-terminal octapeptide sequence of the bovine lectin has been changed to sequences that resemble those in the chick 14-kDa lectin. When the single tryptophan at position 68 is changed by point mutagenesis to phenylalanine or to a leucine residue, a weak binding activity (congruent to 20%) is retained only with the former. When either of the cysteines 2 or 60 is changed to serine, binding activity is reduced to congruent to 60%, and when both are changed, to congruent to 20% of that for the wild-type protein. The susceptibility of the lectin to oxidative inactivation is unaffected when these 2 cysteines and cysteine 130 are changed to serine individually or in tandem (cysteines 2 and 60). In a second approach we show that the natural protein isolated from bovine heart is protected from proteolysis by trypsin and V8-protease in the presence of saccharide ligand. Although further work is required to identify residues which come into contact with the carbohydrate ligand, these results indicate that almost the complete polypeptide chain is necessary for the integrity of the carbohydrate recognition domain.

SUMMARYEvidence is presented that monoclonal antibody BA-1, directed against a marker (CD24) of human lymphocytes of B cell lineage, recognizes a sialic aeul-dependent cpitopc. This conclusion is based on a scries of experiments exploiting the reaction of this antibody with bovine and ovine submaxillary mucins. Expression of the cpitopc was enhanced following alkaline saponification of bovine submaxillary mucin, which converts 0-acctylatcd ncuraminic acid residues to N-acelylncuraminic acid. The epitope was destroyed following ncuraminidase or mild acid treatment of the mucins, and its expression was diminished following neuraminidasc treatment of B lymphobtastoid cells. Glycopcptidcs obtained by digestion of the bovine mucin with papain, trypsin or pronasc were lacking in antigenicity. However, anligcnic activity could be regenerated after conjugation of pronase glycopepiides to poly-L-lysinc. These results indicate that multivalenl display of sialo-oligosacchar-ide on peptide rather than a protease-susceptible polypcplide domain is required for BA-1 antibody binding.

B-cell chronic lymphocytic leukemia (CLL) clones from different patients can express stereotyped BCR immunoglobulins (IGs) that share very similar IGHV-D-J rearrangements and HCDR3s, often with similar IGLV gene usage, suggesting that they were selected by common antigenic epitope(s). Patients bearing IGHV4-34/D5-18/JH6/VK2-30/JK2 rearrangements belong to one of such stereotyped subsets, subset 4. In addition, subset 4 BCRs are always IgG switched and exhibit IGHVsomatic mutations. The germline IGHV4-34 gene is inherently autoreactive and is often found in antibodies that bind to the surfaces of erythrocytes and B lymphocytes through the linear i blood group antigen, poly-N-acetyllactosamine (NAL). To test the reactivity of subset 4 monoclonal antibodies (mAbs) to auto and foreign antigens, we expressed a panel of CLL BCR IGs carrying IGHV4-34, 1-69, 3-21, and 4-39 as recombinant human IgGs and tested the binding of these CLL mAbs to HEp-2 cells by intracellular immunostaining. Three subset 4 mAbs (183, 240 and 342) showed only weak binding to permeabilized HEp-2 cells compared to other CLL mAbs which often showed strong binding to cytoplasmic structures. Furthermore, because apoptotic cells are a source of autoantigens in autoimmune conditions, the reactivity of CLL mAbs to non-permeabilized, irradiated Ramos B and Jurkat T cell line cells was also tested by flow cytometry. Surprisingly, the average binding percentage to apoptotic Ramos B cells of subset 4 mAbs 183, 240, and 342 was only 6, 5, and 4%, respectively. Moreover, all three subset 4 mAbs exhibited minimum binding to live or apoptotic Jurkat T cells (1-10% and 4-6% to each population, respectively). We next tested the reactivity of subset 4 mAbs to i/I carbohydrate epitopes, the known targets of many non-stereotyped IGHV4-34 mAbs, using an i/I focused microarray containing 60 i/I related oligosaccharides. Notably, the subset 4 mAbs showed no binding at a concentration of 10 μg/mL. Together, the above results suggest that CLL subset 4 mAbs have weak or no interaction to the known autoantigens recognized by other non-stereotyped IGVH4-34 IGs. Hence we hypothesized that the restricted IGHV-D-Jrearrangement, somatic mutations, and IgG switch of subset 4 were acquired by selection for other auto or foreign antigens. To test this possibility, we screened CLL mAbs against a library of over 8,000 full length human proteins, including an influenza A virus and some human viral gene encoded proteins. Each subset 4 mAb showed selective and strong binding to influenza A virus H3N2 Texas 1/77 at concentration of 50 μg/mL; this binding was 6-15 times higher than that of other non-subset 4 mAbs, which included two IGHV4-34, non-stereotyped CLL mAbs. Because the binding to virus is usually mediated via its glycoprotein hemagglutinin (HA), we tested reactivity of subset 4 mAbs to the HA of influenza A H3N2 Texas 1/77. Subset 4 mAbs had on average 2 fold (1.8-2.2) higher binding signals than the two non-stereotyped IGHV4-34mAbs. To explore if subset 4 mAbs recognize other influenza A virus strains, we tested the binding of a representative subset 4 mAb 183 to HAs from 11 strains that belong to 3 subtypes: H1 (4 strains), H2 (1 strain) and H3 (6 strains). Clear preferential binding to all 6 H3s and to some of H1s was found. mAb 183 did not bind to H2 (A/Japan/305/1957 H2N2). The average binding signal ratio to H3s, H1s and H2 was 2.4:1.6:1. Testing to another hemagglutinin subtype H7 (A/Anhui/1/13 H7N9) showed that mAb 183 did not interact with H7. Intrigued by the observation that CLL subset 4 BCR IGs are always the IgG isotype, we tested if isotype switching contributed to antigen recognition by expressing CLL 183 IGHV-D-Jas divalent human IgM (mAb183M) and checked its reactivity to virus A H3N2 Texas 1/77. Switching isotype from IgG to IgM led to a 48% loss in reactivity to intact virus and 74% loss to hemagglutinin H3 (A/Philippines/2/1982 H3N2). Taken together, our results indicate that unlike other IGHV4-34 bearing IGs, CLL subset 4 mAbs have only limited reactivity to autoantigens, including linear NAL epitopes. Instead, this subset has acquired specific recognition of subtypes of an infectious agent, influenza A (H3 and H1), apparently via its envelope protein hemagglutinin. The shift in antigen recognition appears to be dependent upon both the stereotyped IGVH-D-Jrearrangement and isotype switching to IgG. Disclosures No relevant conflicts of interest to declare.

Much interest has been stimulated in carbohydrate biology as a result of selectin research. The three selectins, E-, L- and P-selectins are leukocyte-endothelium adhesion molecules (Brandley et al. 1990; Harlan et al. 1992; Bevilacqua et al. 1993). As discussed by Dr Klaus Ley (this volume), the three molecules are key players in mechanisms of innate immunity, for they have important roles at the initial stages of recruitment and emigration of leukocytes from the blood vascular compartment into sites of infection and injury. L-selectin, which is also involved in lymphocyte recirculation in and out of peripheral lymphoid tissues, is expressed constitutively on most types of leukocytes. E-selectin is expressed on activated endothelium, and P-selectin, which is in storage granules of platelets and endothelium, reaches the surface following cell activation. The selectins are long molecules that project from the cell surface. The special feature of these molecules is that they capture and mediate the rapid, but transient, attachment of leukocytes in the flowing blood stream to desired sites on the endothelium or to other leukocytes, and they tether and cause them to roll along the endothelial cell surface. This in turn allows cascades of other much tighter cell adhesive events to take place, mediated by other cell adhesion molecules, before the leukocytes can migrate through the endothelium and the underlying basement membrane. If the selectin-mediated rolling and tethering is inhibited, the subsequent steps are severely compromised. This knowledge has stimulated research into defining the ligands for the selectins in the hope of being able to design analogues that could serve as inhibitory, therapeutic substances for the treatment of disorders of inflammation.