Showing papers on "Sialic acid published in 1997"

Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity.

Abstract: The design, synthesis, and in vitro evaluation of the novel carbocycles as transition-state-based inhibitors of influenza neuraminidase (NA) are described. The double bond position in the carbocyclic analogues plays an important role in NA inhibition as demonstrated by the antiviral activity of 8 (IC50 = 6.3 μM) vs 9 (IC50 > 200 μM). Structure−activity studies of a series of carbocyclic analogues 6a−i identified the 3-pentyloxy moiety as an apparent optimal group at the C3 position with an IC50 value of 1 nM for NA inhibition. The X-ray crystallographic structure of 6h bound to NA revealed the presence of a large hydrophobic pocket in the region corresponding to the glycerol subsite of sialic acid. The high antiviral potency observed for 6h appears to be attributed to a highly favorable hydrophobic interaction in this pocket. The practical synthesis of 6 starting from (−)-quinic acid is also described.

Perspectives on the significance of altered glycosylation of glycoproteins in cancer

Abstract: No abstract Abbreviations:Sia, sialic acid, type unspecified; Tn antigen, GalNAcα 1-O-Ser/Thr; T antigen, Galβ1-3GalNAcα-O-Ser/Thr; Sialyl LewisX, Siaα2-3Galβ1-4(Fucα1-3)GlcNAc; Sialyl Lewisa, Siaα2-3Galβ1-3(Fucα1-4)GlcNAc; Sialyl-Tn antigen, Siaα2-6GalNAcα1-O-Ser/Thr; FucT, fucosyltransferase; ST, sialyltransferase.

Sialic acids as ligands in recognition phenomena.

Abstract: The sialic acids are acidic monosaccharides typically found at the outermost ends of the sugar chains of animal glycoconjugates. They potentially can inhibit intermolecular and intercellular interactions by virtue of their negative charge. However, they can also act as critical components of ligands recognized by a variety of proteins of animal, plant, and microbial origin (sialic acid binding lectins). Recognition can be affected by specific structural variations and modifications of sialic acids, their linkage to the underlying sugar chain, the structure of these chains, and the nature of the glycoconjugate to which they are attached. Presented here is a summary of the various proteins that can recognize and bind to this family of monosaccharides, comparing and contrasting the structural requirements and mechanisms involved in binding. Particular attention is focused on the recently evolving information about sialic acid recognition by certain C-type lectins (the selectins), I-type lectins (e.g., CD22 a...

Contribution of novel choline‐binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae

Abstract: The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.

Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides.

Abstract: Helicobacterpylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3'-sialyllactose (NeuAc alpha2-3Gal beta1-4Glc; 3'SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC50). Its alpha2-6 isomer (6'SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3'SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (P. G. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. H. Trust, J. Bacteriol. 177:6049-6057, 1995). Pretreatment of the epithelial monolayer with neuraminidase reduced the extent of binding by those bacteria that are sensitive to inhibition by 3'SL. Other potent inhibitors of bacterial binding are the glycoproteins alpha1-acid glycoprotein, fetuin, porcine gastric and bovine submaxillary mucins, and the glycolipid sulfatide, all of which present multivalent sialylated and/or sulfated galactosyl residues under the conditions of the binding assay. Consistent with this pattern, a multivalent neoglycoconjugate containing 20 mol of 3'SL per mol of human serum albumin inhibited bacterial binding with micromolar IC50. The H. pylori isolate most sensitive to inhibition by 3'SL was least sensitive to inhibition by sulfatide, gastric mucin, and other sulfated oligosaccharides. Bacteria that have been allowed to bind epithelial cells are also effectively detached by 3'SL. These results describe a heterogeneous adherence repertoire for these bacteria, but they also confirm the critical role of the 3'SL structure on human gastric epithelial cells as an adherence ligand for recent isolates of H. pylori.

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.

Abstract: Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.

Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.

Abstract: Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.

Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis.

Abstract: Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells1–3, and mobilizes bacterial nutrients4. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described5,7. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus8,9, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly10–12. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Contribution of Sialic Acid to the Voltage Dependence of Sodium Channel Gating : A Possible Electrostatic Mechanism

Abstract: A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium chan- nels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line ( lec2 ). The steady-state half-activation voltages (V a ) of channels under each condition of reduced sialylation were z 10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for chan- nels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements.

Synthesis of New α-Thiosialodendrimers and Their Binding Properties to the Sialic Acid Specific Lectin from Limax flavus

Abstract: Carbohydrate−protein binding interactions can be greatly amplified using the cluster or multivalent effect. In previous studies, sialylated multibranched l-lysine dendrimers were found to be potent inhibitors of the hemagglutination of human erythrocytes by Influenza viruses. In order to further the understanding of multivalency and its role in carbohydrate−protein interactions, glycoconjugates with differing carbohydrate densities, conformations, and interglycosidic spacings must be prepared. The synthesis and biological testing of structurally similar divergent and tethered α-sialodendrimers are presented herein. α-Thiosialoside-containing dendrimers scaffolded on an orthogonally protected 3,3‘-iminobis(propylamine) core were efficiently prepared via Cbz-protecting group and HOBt/DIC coupling strategies. The potential of these sialodendrimers to cross-link and precipitate Limax flavus lectin (LFA) was confirmed by turbidimetric analysis. When tested in enzyme-linked lectin inhibition assays using human ...

Myelin-associated Glycoprotein Interacts with Neurons via a Sialic Acid Binding Site at ARG118 and a Distinct Neurite Inhibition Site

Abstract: Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid–dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid– dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid–dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

Abstract: The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Complement factor C3 deposition and serum resistance in isogenic capsule and lipooligosaccharide sialic acid mutants of serogroup B Neisseria meningitidis

Abstract: Serogroup B meningococci express sialic acids on their surfaces as a modification of the lipooligosaccharide (LOS) and as capsular material consisting of alpha2,8-linked sialic acid homopolymers. The aim of this study was to elucidate the impact of each sialic acid component on the deposition of complement factor C3 and serum resistance. For this purpose, we used isogenic mutants deficient in capsule expression (a polysialyltransferase mutant) or sialylation of the LOS (a galE mutant) or both (a mutant with a deletion of the cps gene locus). Bactericidal assays using 40% normal human serum (NHS) demonstrated that both the capsule and LOS sialic acid are indispensable for serum resistance. By immunoblotting with monoclonal antibody MAb755 that is specific for the C3 alpha-chain, we were able to demonstrate that C3 from 40% NHS was covalently linked to the surface structures of meningococci as C3b and iC3b, irrespective of the surface sialic acid compounds. However, C3b linkage was more pronounced and occurred on a larger number of target molecules in galE mutants with nonsialylated LOS than in meningococci with wild-type LOS, irrespective of the capsule phenotype. C3b deposition was caused by both the classical pathway (CP) and the alternative pathway of complement activation. Use of 10% NHS revealed that at low serum concentrations, C3 deposition occurred via the CP and was detected primarily on nonsialylated-LOS galE mutants, irrespective of the capsular phenotype. Accordingly, immunoglobulin M (IgM) binding to meningococci from heat-inactivated NHS was demonstrated only in both encapsulated and unencapsulated galE mutants. In contrast, inhibition of IgA binding required both encapsulation and LOS sialylation. We conclude that serum resistance in wild-type serogroup B meningococci can only be partly explained by an alteration of the C3b linkage pattern, which seems to depend primarily on the presence of wild-type LOS, since a serum-resistant phenotype also requires capsule expression.

New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones

Abstract: Sialic acids are a family of 9-carbon carboxylated sugars, where different substitutions of the backbone define over 30 members. Biological roles of these substitutions have been missed until recently because of their low abundance and lability to conventional isolation/purification methods. This new approach characterizes sialic acids using electrospray ionization-mass spectrometry (ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene) derivatives (quinoxalinones). A combination of retention times and spectra characteristics allows definition of the type and position of the various substituents. This approach requires no previous purification, involving a simple derivatization reaction followed by direct injection on the microbore HPLC column. A complete spectrum, including molecular ions and CAD fragments of a sialic acid quinoxalinone, is obtained by injecting 10-20 pmol of the compound. Individual quinoxalinones can be purified by regular RP-HPLC and analyzed by direct-injection ESI-MS or LSIMS. Using this approach, we identified 28 different sialic acids, including the following new species: Neu5Gc9Lt (BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro (horse serum hydrolyzates), KDN5(7)Ac and KDN5(7),9Ac2 (amphibian Pleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydro derivatives of Neu5Gc8Me (glycolipids of the starfish Pisaster brevispinus), and Neu5Ac8S (in addition to Neu5Gc8S, in the glycolipids of the sea urchin Lovenia cordiformis). Results show the usefulness of LC-ESI-MS to study sialic acid diversity, and identification of small amounts of unexpected sialic acids or new members of their family.

A transfected sialyltransferase that is elevated in breast cancer and localizes to the medial/trans-Golgi apparatus inhibits the development of core-2-based O-glycans.

Abstract: The alpha2,3 sialyltransferase, alpha2,3 SAT (O), catalyzes the transfer of sialic acid to Galbeta1,3 N-acetyl-D-galactosamine (GalNAc) (core-1) in mucin type O-glycosylation, and thus terminates chain extension. A Core-2 branch can also be formed from core-1 by the core-2 beta1,6 N-acetyl-d-glucosamine transferase (beta1,6 GlcNAc T) that leads to chain extension. Increased levels of the alpha2,3 SAT (O) and decreased levels of the core-2 beta1,6 GlcNAc T are seen in breast cancer cells and correlate with differences in the structure of the O-glycans synthesized (Brockhausen et al., 1995; Lloyd et al., 1996). Since in mucin type O-glycosylation sugars are added individually and sequentially in the Golgi apparatus, the position of the transferases, as well as their activity, can determine the final structure of the O-glycans synthesized. A cDNA coding for the human alpha2,3 SAT (O) tagged with an immunoreactive epitope from the myc gene has been used to map the position of the glycosyltransferase in nontumorigenic (MTSV1-7) and malignant (T47D) breast epithelial cell lines. Transfectants were analyzed for expression of the enzyme at the level of message and protein, as well as for enzymic activity. In T47D cells, which do not express core-2 beta1,6 GlcNAc T, the increased activity of the sialyltransferase correlated with increased sialylation of core-1 O-glycans on the epithelial mucin MUC1. Furthermore, in MTSV1-7 cells, which do express core-2 beta1,6 GlcNAc T, an increase in sialylated core-1 structures is accompanied by a reduction in the ratio of GlcNAc: GalNAc in the O-glycans attached to MUC1, implying a decrease in branching. Using quantitative immunoelectron microscopy, the sialyltransferase was mapped to the medial- and trans-Golgi cisternae, with some being present in the TGN. The data represent the first fine mapping of a sialyltransferase specifically active in O-glycosylation and demonstrate that the structure of O-glycans synthesized by a cell can be manipulated by transfecting with recombinant glycosyltransferases.

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases

Abstract: The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4°C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18°C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1

Abstract: Selectins interact with glycoconjugate ligands in important normal and pathological situations. While high affinity recognition of natural ligands is associated with alpha 1-3(4)fucosylated, alpha 2-3sialylated (and/or sulfated) lactosamine sequences, small oligosaccharides that potently inhibit the selectins have not been found. One possibility suggested by other investigators is that high affinity may require unusual sequences not yet tested, for example, the "major capping group" (6'-sulfo-sialyl Le(x)) of the L-selectin ligand GlyCAM-1. To explore this possibility, we synthesized a spectrum of novel synthetic and semisynthetic oligosaccharides related to those on natural ligands. In studying these molecules, we noted that binding of recombinant soluble selectins to immobilized sialyl Le(a) or 3'-sulfo-Le(x) is markedly inhibited by concentrations of chloride above the physiological range. This indicates the ionic nature of the interactions, and shows that buffers typically used in screening assays for inhibitors are not optimal. Using parameters that more closely approximate physiological conditions, we confirmed that alpha 2-3-linked sialic acids, and alpha 1-3(4)fucosylation are important for recognition. Similar results obtained with both types of immobilized targets for the three selectins indicated that the binding sites for sialic acid and sulfate are very close, or identical. While O-sulfate esters mostly improved L- and P-selectin recognition, effects depended upon their position and number. Furthermore, sulfation can also impart some "negative" specificity: the major capping group does not interact with E-selectin. The branched Core 2 sequence seemed to enhance L- and P-selectin binding, however, the best inhibitors still appeared to be sialyl Le(a) and 3'-sulfo-Le(x), with the aglycone group of the latter affecting binding. Of particular note, the "major capping group" of GlyCAM-1 was not an unusually potent nor highly selective inhibitor of L-selectin, even when studying the interaction of L-selectin with native GlyCAM-1 itself.

Carbohydrate composition and immunomodulatory activity of different glycoforms of alpha1-acid glycoprotein.

Abstract: The acute phase protein, alpha1-acid glycoprotein (AGP), is a normal constituent of human blood (0.2-1 mg ml(-1)) and its glycosylation and concentration in the blood change during inflammation. In this review of our recent work, we discuss the immunomodulatory properties of AGP in connection with the structure of its carbohydrate chains. AGP samples prepared from normal donor serum (nAGP), serum obtained during abortion (fAGP), serum of cancer patients (cAGP), and ascitic fluid of patients with stomach cancer (sAGP) were subjected to analysis. All the samples except for fAGP had five N-linked chains of the 'complex' type, however, the numbers of bi-, tri-, and tetra-antennary chains, as well as glycan structures terminating these chains, were different. fAGP had three N-linked chains of the lactosamine and polylactosamine type and three O-chains which were not present in AGP isolated from the other sources. The glycoforms of nAGP and sAGP that were isolated using a ConA affinity column were similar in respect to their branching, but differed in their terminal oligosaccharides. sAGP was enriched in units ending in Le(x) and asialoagalacto (GlcNAc-terminating) forms. Immunomodulatory activity of different AGP preparations was tested in vitro by measuring their effect on the proliferative response of human lymphocytes stimulated by PHA, and by determining their influence on the production of IL-1, IL-2, IL-6, and TNF in the stimulated cells. nAGP was less active compared to cancer or fetal AGP in the proliferation test, but more active in affecting cytokine production. Some AGP glycoforms had opposite immunomodulatory effects. A new approach was developed in order to clarify the role of carbohydrate chains in the biological activity of AGP. A pool of N-linked oligosaccharide chains were attached to a soluble polyacrylamide matrix. This 'pseudoglycoprotein' was similar to AGP in its molecular weight; in its relative amounts of tetra-, tri-, and bi-antennary chains; and in the content of mono-, di-, tri-, and tetra-sialylated-oligosaccharides. This pseudo-AGP displayed a similar activity to its parent AGP in the biological tests. Analytical flow cytometry of leukocyte subpopulation from human peripheral blood showed that monocytes and granulocytes but not lymphocytes were the main targets for the binding of AGP and pseudo-AGP. This binding was inhibited by synthetic glycoconjugates containing mannose or sialic acid. The binding curve data suggested that there are two monocyte and granulocyte populations. These may have different carbohydrate specificities. All the evidence provided by these studies indicate that it is the carbohydrate chains on AGP that are important in modulating the immune system and not the AGP molecule itself.

Sialylation of Neisseria meningitidis lipooligosaccharide inhibits serum bactericidal activity by masking lacto-N-neotetraose.

Abstract: Exogenous sialylation of gonococcal lipooligosaccharide causes resistance to serum bactericidal activity. The aim of this study was to determine how lipooligosaccharide sialylation affects the serum sensitivities of group C Neisseria meningitidis strains. The relationship between the degree of sialylation or expression of the lipooligosaccharide sialic acid acceptor, lacto-N-neotetraose (LNnT), of nine meningococcal strains and their sensitivities to a pool of normal human sera was assessed. All strains expressed LNnT that was variously endogenously sialylated. Susceptibility to serum bactericidal activity ranged from extremely sensitive to resistant in 50% serum. For endogenously sialylated strains, the amount of killing correlated with the amount of free LNnT above a threshold of expression; strains that expressed less than the threshold survived in 25% serum. All strains added more sialic acid when they were grown in medium that contained cytidine monophospho-N-acetylneuraminic acid. Exogenous sialylation reduced the expression of free LNnT and significantly increased serum resistance. Exogenous sialylation affected killing through both classical and alternative complement pathways. The killing of exogenously sialylated strains also correlated with the amount of free LNnT. The amounts of endogenous, exogenous, and total sialic acid bound to LNnT did not correlate with the resistance of strains to serum bactericidal activity; rather, the loss of free LNnT expression by sialylation was associated with resistance. In conclusion, the expression of free LNnT by group C meningococcal strains is directly associated with the amount of killing of organisms in pooled human sera. Both endogenous and exogenous lipooligosaccharide sialylation are associated with increased serum resistance by masking LNnT.

Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase.

Abstract: Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid. This polysaccharide is an essential virulence factor of these neuropathogenic bacteria. The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster. Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo. Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone. NeuNAc synthetase was purified to homogeneity from extracts of cells harboring pNEUB. The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene. The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene. NeuNAc synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction. The enzyme condenses manNAc and PEP with the release of phosphate. The E. coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4. This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity. These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E. coli K1.