Removal Of Terminal Sialic Acid Research Articles

Host Cell Response to Rotavirus Infection with Emphasis on Virus-Glycan Interactions, Cholesterol Metabolism, and Innate Immunity.

Although rotavirus A (RVA) is the primary cause of acute viral gastroenteritis in children and young animals, mechanisms of its replication and pathogenesis remain poorly understood. We previously demonstrated that the neuraminidase-mediated removal of terminal sialic acids (SAs) significantly enhanced RVA-G9P[13] replication, while inhibiting RVA-G5P[7] replication. In this study, we compared the transcriptome responses of porcine ileal enteroids (PIEs) to G5P[7] vs. G9P[13] infections, with emphasis on the genes associated with immune response, cholesterol metabolism, and host cell attachment. The analysis demonstrated that G9P[13] infection led to a robust modulation of gene expression (4093 significantly modulated genes vs. 488 genes modulated by G5P[7]) and a significant modulation of glycosyltransferase-encoding genes. The two strains differentially affected signaling pathways related to immune response, with G9P[13] mostly upregulating and G5P[7] inhibiting them. Both RVAs modulated the expression of genes encoding for cholesterol transporters. G9P[13], but not G5P[7], significantly affected the ceramide synthesis pathway known to affect both cholesterol and glycan metabolism. Thus, our results highlight the unique mechanisms regulating cellular response to infection caused by emerging/re-emerging and historical RVA strains relevant to RVA-receptor interactions, metabolic pathways, and immune signaling pathways that are critical in the design of effective control strategies.

Increased α-2,6 sialic acid on microglia in amyloid pathology is resistant to oseltamivir.

Terminal sialic acid residues are present on most glycoproteins and glycolipids, but levels of sialylation are known to change in the brain throughout the lifespan as well as during disease. Sialic acids are important for numerous cellular processes including cell adhesion, neurodevelopment, and immune regulation as well as pathogen invasion into host cells. Neuraminidase enzymes, also known as sialidases, are responsible for removal of terminal sialic acids in a process known as desialylation. Neuraminidase 1 (Neu1) cleaves the α-2,6 bond of terminal sialic acids. Aging individuals with dementia are often treated with the antiviral medication oseltamivir, which is associated with induction of adverse neuropsychiatric side effects; this drug inhibits both viral and mammalian Neu1. The present study tested whether a clinically relevant antiviral dosing regimen of oseltamivir would disrupt behavior in the 5XFAD mouse model of Alzheimer's disease amyloid pathology or wild-type littermates. While oseltamivir treatment did not impact mouse behavior or modify amyloid plaque size or morphology, a novel spatial distribution of α-2,6 sialic acid residues was discovered in 5XFAD mice that was not present in wild-type littermates. Further analyses revealed that α-2,6 sialic acid residues were not localized the amyloid plaques but instead localized to plaque-associated microglia. Notably, treatment with oseltamivir did not alter α-2,6 sialic acid distribution on plaque-associated microglia in 5XFAD mice which may be due to downregulation of Neu1 transcript levels in 5XFAD mice. Overall, this study suggests that plaque-associated microglia are highly sialylated and are resistant to change with oseltamivir, thus interfering with microglia immune recognition of and response to amyloid pathology.

IgA Complexes Induce Neutrophil Extracellular Trap Formation More Potently Than IgG Complexes.

Neutrophil extracellular trap (NET) formation is a powerful instrument to fight pathogens, but may induce collateral damage in the affected tissues. Besides pathogen-derived factors, immune complexes are potent inducers of NET formation. Neutrophils express IgA and IgG specific Fc receptors (FcRs) and therefore respond to complexed IgA and IgG. Especially in the context of autoimmune diseases, IgA and IgG immune complexes have been shown to trigger NET formation, a process that putatively contributes to disease severity. However, it is of question if both antibody classes stimulate neutrophils to the same extent. In this study, we compared the capability of IgA and IgG complexes formed by heat aggregation to induce NET formation. While stimulation of neutrophils with IgA complexes robustly induced NET formation, complexed IgG only marginally increased the amount of NETs compared to the unstimulated control. Mixing IgA with IgG before heat aggregation did not increase the effect of complexed IgA on neutrophils. By contrast, the presence of IgG complexes seemed to disturb neutrophil stimulation by IgA complexes. The capacity of complexed IgG to induce NET formation could not be increased by the addition of autologous serum or the removal of terminal sialic acid in the Fc glycan. Together, our data show that IgA is a much more potent inducer of NET formation than IgG. IgA may thus be the main driving force in (auto)immune complex-mediated NET formation.

Genetic Removal of Terminal Sialic Acid Residues from the O-Linked Glycans Adjacent to the HPA-9b Polymorphism of Platelet Membrane Glycoprotein IIb Improves the Binding and Detection of HPA-9b Patient Alloantibodies

Sialic acids occupy the terminal position of glycan chains, and have the potential to influence the antigenicity of glycoproteins. Antibody binding sites on a glycoprotein can be solely protein in nature, or can include or be affected by nearby glycan chains, which may either mask the epitope, or conversely comprise part of the antibody binding site. The polymorphisms responsible for formation of the Human Platelet Alloantigens (HPA)-3 (Ile843Ser) and HPA-9 (Val837Met) are next to each other near the C-terminus of the extracellular domain of platelet membrane glycoprotein (GP)IIb, and are adjacent to sialyl-Core 1 O-glycans that emanate from serines 845 and 847. Previous studies have shown that these O-linked glycans are required to support the binding of some of HPA-3a alloantibodies. Loss of these glycans, especially terminal sialic acid residues, during platelet storage or preparation, can present major difficulties in detecting clinically important anti-HPA-3a alloantibodies in suspected cases of fetal/neonatal alloimmune thrombocytopenia (FNAIT). Similarly, detection and identification of anti-HPA-9b alloantibodies from FNAIT patient sera can also be extremely challenging, resulting in the inability to resolve clinical cases of this bleeding disorder. Whether the nearby O-glycans on serines 845 and 847 of GPIIb affect the antigenicity of HPA-9b, and/or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of FNAIT is unknown.We previously reported the generation of bioengineered, HLA class I-negative, HPA-9a or -9b allele-specific megakaryocytes (MKs) from human induced pluripotent stem cells (iPSCs) that are suitable for whole-cell flow cytometric detection of anti-HPA-9b alloantibodies (Blood 2019;134(22):e1-e8). Unexpectedly, treatment of these allele-specific MKs with neuraminidase actually enhanced the binding of anti-HPA-9b alloantibodies, suggesting that terminal sialic acids on GPIIb partially mask the HPA-9b epitope. To test the hypothesis that removal of terminal sialic acids on nearby O-glycans, or removal of the entire O-glycan chains emanating from Ser845/847 of GPIIb, might enhance the detection of anti-HPA-9b patient alloantibodies, we created a series of deletion mutants in two major sialidases, ST3GAL1 and ST3GAL2, known to be responsible for transferring terminal sialic acid residues to Core 1 O-glycans, in our HPA-9a and -9b allele-specific iPSCs. Immunoprecipitation/western blot analysis confirmed the complete removal of terminal sialic acids on the O-glycan chains of GPIIb in ST3GAL1/2 knockout (KO) iPSC-derived MKs, as reported by the binding of the lectin PNA to the exposed Core 1 structure on GPIIb. These sialylation-deficient ST3GAL1/2 KO HPA-9b MKs exhibited dramatically increased anti-HPA-9b alloantibody binding, further confirming the notion that HPA-9b epitopes are partially masked by terminal sialic acids on nearby GPIIb O-glycan chains. Finally, allele-specific iPSCs lacking the complete O-glycan chains attached to serines 845 and 847 of GPIIb were generated by mutating those residues to alanines using a similar CRISPR/Cas9 gene editing approach. Interestingly, O-glycan chain-deficient Ala845/847 mutant MKs carrying the HPA-9b polymorphism exhibited slightly to moderately reduced binding of anti-HPA-9b alloantibodies, indicating that the presence of the Core 1 O-glycan chains attached to GPIIb serine residues 845 and 847 contribute to the presentation of the HPA-9b epitope - perhaps by stabilizing the conformation of the glycoprotein in this region. Taken together, these data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of iPSC-derived HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues, but retain the glycan chains to which they are attached. DisclosuresCurtis: Rallybio: Consultancy. Newman: Rallybio: Consultancy, Research Funding.

Infection of porcine small intestinal enteroids with human and pig rotavirus A strains reveals contrasting roles for histo-blood group antigens and terminal sialic acids.

Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Growing evidence suggests that host cellular glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established a porcine crypt-derived 3D intestinal enteroids (PIEs) culture system which contains all intestinal epithelial cells identified in vivo and represents a unique physiologically functional model to study RV-glycan interactions in vitro. PIEs expressing different HBGAs (A+, H+, and A+/H+) were established and isolation, propagation, differentiation and RV infection conditions were optimized. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] or PRV OSU G5P[7] virulent and attenuated strains and virus replication was measured by qRT-PCR. Our results indicated that virulent HRV G1P[8] Wa replicated to the highest titers in A+ PIEs, while a distinct trend was observed for PRV G9P[13] or G5P[7] with highest titers in H+ PIEs. Attenuated Wa and Gottfried strains replicated poorly in PIEs while the replication of attenuated G9P[13] and OSU strains in PIEs was relatively efficient. However, the replication of all 4 attenuate strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for G1P[8] Wa replication; however, they may only serve as a cofactor for PRVs G9P[13] and OSU G5P[7]. Interestingly, contrasting outcomes were observed following sialidase treatment which significantly enhanced G9P[13] replication, but inhibited the growth of G5P[7]. These observations suggest that some additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. Overall, our results confirm that differential HBGAs-RV and SA-RV interactions determine replication efficacy of virulent group A RVs in PIEs. Consequently, targeting individual glycans for development of therapeutics may not yield uniform results for various RV strains.

Characterization of glycoconjugates and sialic acid modification in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis).

Diverse glycoconjugates are expressed in the vertebrate olfactory bulb and serve as guidance cues for axons of nasal receptor neurons. Although the involvement of glycoconjugates in the segregation of the olfactory pathway has been suggested, it is poorly understood in salamanders. In this study, lectin histochemistry was used to determine glycoconjugate distribution in the olfactory bulb of the Chinese fire-bellied newt (Cynops orientalis). Succinylated wheat germ agglutinin (sWGA), Ricinus communis agglutinin-I and Lens culinaris agglutinin showed different bindings in the nerve fibre layer or glomerular layer, or both, between the main and accessory olfactory bulbs. We then investigated the lectin-binding pattern after the removal of terminal sialic acids using neuraminidase. Desialylation resulted in a change in the binding reactivities with seven lectins. Wheat germ agglutinin, sWGA, soybean agglutinin (SBA) and peanut agglutinin showed different degrees of binding between the main and accessory olfactory bulbs. In addition, SBA showed a heterogeneous labelling of glomeruli in the rostral region of the main olfactory bulb. Our results suggest that terminal sialic acids mask the heterogeneity of glycoconjugates in the olfactory bulb of C.orientalis.

Bifunctional properties and characterization of a novel sialidase with esterase activity from Bifidobacterium bifidum.

Sialidases catalyze the removal of terminal sialic acid from various complex carbohydrates. In the gastrointestinal tract, sialic acid is commonly found in the sugar chain of mucin, and many enteric commensals use mucin as a nutrient source. We previously identified two different sialidase genes in Bifidobacterium bifidum, and one was cloned and expressed as an extracellular protein designated as exo-α-sialidase SiaBb2. The other exo-α-sialidase gene (siabb1) from the same bifidobacterium encodes an extracellular protein (SiaBb1) consisting of 1795 amino acids with a molecular mass of 189kDa. SiaBb1 possesses a catalytic domain that classifies this enzyme as a glycoside hydrolase family 33 member. SiaBb1 preferentially hydrolyzes α2,3-linked sialic acid over α2,6-linked sialic acid from sialoglycan, which is the same as SiaBb2. However, SiaBb1 has an SGNH hydrolase domain with sialate-O-acetylesterase activity and an N-terminal signal sequence and C-terminal transmembrane region. SiaBb1 is the first bifunctional sialidase identified with esterase activity. Abbreviations: GalNAc: N-acetyl-D-galactosamine; Fuc: L-fucose; Gal: D-galactose.

Low-glycosylated forms of both FSH and LH play major roles in the natural ovarian stimulation

Background: The natural ovarian stimulation is mediated by four gonadotrophin glycoforms: FSHtri with three, FSHtetra with four, LHdi with two, and LHtri with three N-glycans. The aim of the study was to determine the serum concentrations of the four glycoforms and their contents of anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU) residues throughout the menstrual cycle.Methods: Serum samples were collected from 78 healthy women with regular menstrual cycles. The serum glycoform molecules were identified by their distributions at electrophoreses. Analyses were also performed after removal of terminal SA. The hormones were measured with time-resolved sandwich fluoroimmunoassays.Results: The concentration profiles of the four glycoforms were markedly different. FSHtri, which had a 3-fold higher biopotency than FSHtetra, had peak levels on cycle day 5 and at midcycle and nadirs on cycle days 9 and 21–23. FSHtetra had a raised level on cycle days 5–12, followed by a decrease. LHdi and LHtri had similar patterns, but the peak/nadir ratio was much more pronounced for LHdi than for LHtri, 18 versus 4. The numbers of SA residues per molecule were at a maximum around midcycle when the corresponding numbers of SU were at a minimum. The SU/SA ratio was at a minimum on cycle day 12.Conclusion: The results indicate that the LHdi and the FSHtri molecules play major roles in the natural ovarian stimulation. The SU/SA ratios per molecule favoured a prolonged circulatory half-life of all glycoforms at the midcycle phase. The observations may lead to more successful inductions of ovulation in anovulatory women.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed β-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to β-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind β-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors.

New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability.

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

Effects of Desialylation on Human α1-Acid Glycoprotein–Ligand Interactions

Human α1-acid glycoprotein (AGP), an acute-phase glycoprotein, exists predominantly in blood. With its ability to bind basic, lipophilic, and acidic drugs, AGP has served as a drug carrier. It has been shown that the carbohydrate composition of AGP changes in response to tissue injury, inflammation, or infection and can have a great impact on AGP's drug binding activities. The molecular-level details of the effects of desialylation on the AGP conformation and AGP-ligand interactions, however, are unknown. Here we report the use of hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to reveal the changes in AGP conformational dynamics induced by the removal of terminal sialic acid. HDX-MS also reveals the changes in the conformational dynamics of sialylated and unsialylated AGP upon formation of complexes of holo-AGP with progesterone or propranolol. Our HDX-MS results demonstrate that desialylation stabilizes two loop regions that are exterior to the β-sheet barrel in AGP, and this stabilization minimizes the conformational changes of AGP upon binding with progesterone or propranolol.

A surface‐exposed neuraminidase affects complement resistance and virulence of the oral spirochaete Treponema denticola

Neuraminidases (sialidases) catalyse the removal of terminal sialic acid from glycoconjugates. Bacterial pathogens often utilize neuraminidases to scavenge host sialic acid, which can be utilized either as a nutrient or as a decorating molecule to disguise themselves from host immune attacks. Herein, a putative neuraminidase (TDE0471) was identified in Treponema denticola, an oral spirochaete associated with human periodontitis. TDE0471 is a cell surface-exposed exo-neuraminidase that removes sialic acid from human serum proteins; it is required for T.denticola to grow in a medium that mimics gingival crevice fluid, suggesting that the spirochaete may use sialic acid as a nutrient in vivo. TDE0471 protects T.denticola from serum killing by preventing the deposition of membrane attack complexes on the bacterial cell surface. Animal studies revealed that a TDE0471-deficient mutant is less virulent than its parental wild-type strain in BALB/C mice. However, it causes a level of tissue damage similar to the wild type in complement-deficient B6.129S4-C3(tm1) (Crr) /J mice albeit the damage caused by both bacterial strains is more severe in these transgenic mice. Based on these results, we propose that T.denticola has evolved a strategy to scavenge host sialic acid using its neuraminidase, which allows the spirochaete to acquire nutrients and evade complement killing.

B-cell maturation antigen is modified by a single N -glycan chain that modulates ligand binding and surface retention

Glycosylation, an important posttranslational modification process, can modulate the structure and function of proteins, but its effect on the properties of plasma cells is largely unknown. In this study, we identified a panel of glycoproteins by click reaction with alkynyl sugar analogs in plasma cells coupled with mass spectrometry analysis. The B-cell maturation antigen (BCMA), an essential membrane protein for maintaining the survival of plasma cells, was identified as a glycoprotein exhibiting complex-type N-glycans at a single N-glycosylation site, asparagine 42. We then investigated the effect of N-glycosylation on the function of BCMA and found that the dexamethasone-induced apoptosis in malignant plasma cells can be rescued by treatment with BCMA ligands, such as a proliferation-inducing ligand (APRIL) and B-cell-activating factor (BAFF), whereas removal of terminal sialic acid on plasma cells further potentiated the ligand-mediated protection. This effect is associated with the increased surface retention of BCMA, leading to its elevated level on cell surface. In addition, the α1-3,-4 fucosylation, but not the terminal sialylation, assists the binding of BCMA with ligands in an in vitro binding assay. Together, our results highlight the importance of N-glycosylation on BCMA in the regulation of ligand binding and functions of plasma cells.

Specific N- and O-Linked Carbohydrate Structures Mediate Von Willebrand Factor Interaction with Galectins -1 and -3

Abstract 2234Von Willebrand Factor (VWF) is extensively glycosylated with both N- and O-linked carbohydrates. Moreover, these complex glycan structures influence VWF functional properties, including susceptibility to ADAMTS13 proteolysis, and plasma clearance. The molecular mechanisms through which VWF glycosylation (including ABO blood group antigens) act to influence VWF physiology remains unexplained. However, recent data suggest that VWF circulates in normal plasma bound to various carbohydrate-binding proteins, including specific members of the galectin family. In addition, galectin-3 binding has been reported to influence VWF cleavage by ADAMTS13. In this context, we sought to elucidate the role of specific VWF glycan determinants in modulating galectin interaction.VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. VWF glycosylation was then modified using exoglycosidases and quantified by specific lectin ELISAs. Blood group specific VWF was also purified from pooled group AB, O, or Bombay plasmas. Galectins-1 and -3 were transiently expressed in competent E-coli cells with an N-terminal histidine tag, and purified by nickel chromatography. Finally, binding interactions were characterized via modified immunosorbant assay.In keeping with the previous report of Lenting et al, human pdVWF bound to both galectin-1 and galectin-3 in a dose-dependent manner. Enzymatic desialylation of pdVWF with α2-3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced binding to galectin-1 (231±6%, p<0.0001). Similarly, removal of terminal sialic acid also increased binding to galectin-3, albeit to a lesser extent (136±6%, p<0.05). To further define the role of VWF glycans in regulating galectin binding, pdVWF was exposed to sequential neuraminidase and galactosidase digestions to remove terminal sialic acid and sub-terminal galactose residues (NeuGal-VWF). In contrast to the enhanced binding of Neu-VWF, binding of NeuGal-VWF to both galectin -1 and -3 was significantly reduced (51±5% and 52±6% compared to pdVWF; p<0.005). Cumulatively these findings suggest that loss of capping sialic acid and exposure of sub-terminal galactose critically regulates VWF-galectin binding. Treatment with PNGase F to completely remove N-linked carbohydrate structures (PNG-VWF) markedly decreased binding to galectin -1 and -3 (13±1% and 57±2%, p<0.001). Moreover, combined PNGase F and O-glycosidase digestions further attenuated galectin-3 binding (21±1%, p<0.001), suggesting that both the N- and O-linked glycans are involved in mediating the VWF-galectin interaction.ABO(H) blood group antigens are expressed on both the N-linked and O-linked glycans of human VWF. Moreover, ABO(H) determinants influence VWF susceptibility to ADAMTS13 proteolysis and plasma VWF half-life, through unknown mechanisms. Purified VWF from normal group AB individuals bound to both galectin-1 and galectin-3 significantly better than group O VWF (146±8% and 483±19%; p<0.01). Conversely, no significant difference in binding was observed between Group O and Bombay VWF. Consequently, although terminal A (GalNAc) and B (Gal) sugar moieties promote galectin binding, expression of terminal α1–2 fucose residues is not important. The glycosylation profile of platelet-VWF differs from that of pdVWF. In particular, platelet-VWF expresses reduced levels of both capping sialic acid and sub-terminal galactose residues (∼50%), and lacks AB blood group antigens. To characterize the effects of this differential sugar expression on galectin binding, platelet-derived VWF was isolated and purified (platelet freeze-thawing followed by immuno-affinity chromatography with monoclonal CLB-Rag20). In keeping with the reduction in Gal and AB blood group antigen expression, platelet VWF bound less well to galectin-1 and galectin-3 (72±6% and 67±7% versus pdVWF; p<0.05).These novel data demonstrate that both the N- and O-linked oligosaccharide structures of VWF are involved in mediating galectin binding. In particular, expression of terminal AB blood group antigens, and expression of sub-terminal galactose moieties following loss of capping sialic acid, both markedly enhance galectin binding affinity. Further studies will be required to define how galectin binding is involved in mediating the functional consequences of variation in VWF glycans. Disclosures:No relevant conflicts of interest to declare.

Involvement of sialic acid in the regulation of -aminobutyric acid uptake activity of -aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.

Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, fromClostridium perfringens

Sialidases catalyse the removal of terminal sialic acids from a range of glycoproteins, glycolipids and oligosaccharides. They have been found in bacteria, viruses and parasites, where they play important roles in pathogenesis and/or microbial nutrition, and in mammalian cells, where they modulate cell-surface glycosylation associated with a range of cellular activities. Clostridium perfringens, a causative agent of gas gangrene and peritonitis in humans, possesses three sialidases: nanH, nanI and nanJ, with molecular weights of 42, 77 and 129 kDa, respectively. The two larger enzymes are secreted by the bacterium and are involved in the pathogenesis and nutrition of Clostridium. As part of a study to examine the structures of all three enzymes, crystallization of the 77 kDa nanI isoenzyme was attempted. The expressed full-length protein was found to degrade easily; a stable 50 kDa catalytic domain was therefore subcloned. This domain was overexpressed in Escherichia coli and produced crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.98, b = 69.41, c = 72.69 A and one monomer per asymmetric unit. The crystals diffract to at least 0.92 A. A molecular-replacement solution was obtained using the catalytic domain of the sialidase from the leech Macrobdella decora.

Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe

Human neutrophils are activated by the β-galactoside-binding lectin galectin-3, provided that the cells are primed by in vivo extravasation or by in vitro preactivation with, for example, LPS. Removal of terminal sialic acid can change neutrophil functionality and responsiveness due to exposure of underlying glycoconjugate receptors or change in surface charge. Here, we investigated whether such alteration of the cell surface carbohydrate composition can alter the responsiveness of the cells to galectin-3. Neutrophils were treated with neuraminidases (NA) of different origins: Clostridium perfringens (CP), Salmonella typhimurium, Vibrio cholerae, and Newcastle disease virus (NDV). In the presence of NDV-NA, but no other NA, the otherwise non-responding neutrophils responded readily to galectin-3 by activation of the NADPH-oxidase. The galectin-3 priming effect was inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy- N-acetyl-neuraminic acid. Earlier studies have shown that priming of the neutrophil response to galectin-3 with, for example, LPS is paralleled by degranulation of intracellular vesicles and granules and upregulation of potential galectin-3 receptors. Also, NDV-NA (but not CP-NA) treatment induced degranulation, shown as an upregulation of complement receptor 3. Since not only the galectin response but also the response to the chemoattractant fMLF was primed, NDV-NA appears to induce a general priming phenomenon, possibly due to receptor upregulation by degranulation.

Sialic acid derivatives and their distribution in rat sublingual gland acini during pre- and post-natal development.

Sialoglycoconjugates in rat sublingual gland acinar cells, at different stages of pre- and post-natal development, were investigated in situ with specific lectins and by the selective removal of terminal sialic acids. Cleavage of acetyl substituents sited in the pyranose ring and/or polyhydroxyl side chain was used as an additional means of characterising the glycoconjugates. The first expression of terminal sialic acid linked to beta-galactose was found at gestational day 17 and progressive different derivatives were observed. The terminal disaccharide sialic acid-N-acetylgalactosamine was constantly visualized in the sublingual gland from gestational day 18. In both terminal disaccharides, sialic acids were characterized by variable degrees of acetylation and were found to be highly packaged and responsible for the hydration coat. The complex data obtained indicated that the sublingual gland is characterized by a marked fluctuation of complex sialoglycoconjugates that differ from those in the submandibular gland of the same species.

The Glycosylation of the Influenza A Virus Hemagglutinin by Mammalian Cells: A SITE-SPECIFIC STUDY

We have characterized the glycans at individual sites on the hemagglutinin of three influenza A variants to obtain information on the role of cell-specific glycosylation in determining the receptor binding properties of this virus. The variants differ in whether they have a glycosylation site at residue 129 on the tip of the hemagglutinin and whether amino acid 184 (near to the receptor binding site) is His or Asn. We found that all sites on each variant are glycosylated in Madin-Darby bovine kidney cells, that the glycosylation is site-specific, and that the glycans at the same site in each variant are highly similar. One site that is buried in the hemagglutinin trimer contains only oligomannose glycans. The remaining sites carry complex glycans of increasing size as the distance of the site from the viral membrane decreases. Most of these complex glycans are terminated with alpha-galactose residues, a consequence in bovine cells of the removal of terminal sialic acids by the viral neuraminidase. Although the glycans at residue 129 are among the smallest on the molecule, they are large enough to reach the receptor binding pocket on their own and adjacent monomers. The results suggest that the reduction in receptor binding observed with Madin-Darby bovine kidney cell-grown virus is due to the combined effect of large complex glycans at the tip of the hemagglutinin and a His to Asn substitution close to the receptor binding pocket.

Altered glycosylation in Madin-Darby canine kidney (MDCK) cells after transformation by murine sarcoma virus

The changes in glycosylation of an immortalized epithelial cell line (MDCK) before and after progression towards a more malignant phenotype have been studied. The parental MDCK-3 cells were immortalized after long-term passage in vitro and have shown no tendency for spontaneous acquisition of malignancy-related phenotypes such as tumorigenicity, invasion and metastasis. They conserved morphological and functional characteristics of the epithelial tissue of origin. The ras-MDCK cells acquired the fully malignant phenotype after transformation with a Harvey murine sarcoma virus; they were immortalized, invasive in vitro and produced invasive and also metastatic tumors after subcutaneous injection into nude mice. Using immobilized lectins and gel chromatography, before and after liberation of O-linked glycans from the peptide moieties and also after removal of terminal sialic acid, we have found differences in the glycosylpeptides of both whole cells and cell surface trypsinates from ras-MDCK cultures as compared to the parental MDCK-3 cultures: (i) more sialic acid in the N-linked tri- and tetra-antennary structures; (ii) more fucosylation in the N-glycosylpeptides; (iii) more bi-antennary N-glycosylpeptides and less O-linked glycans; and (iv) a lower molecular weight of the O-linked glycans probably due to a decreased sialylation. It is concluded that alterations in sialylation and fucosylation of the cell surface exposed glycans accompanied progression of MDCK-3 cells towards a more malignant phenotype.