Sialyl Lactose Research Articles

Role of neuraminidase in influenza virus-induced apoptosis.

The virulent influenza virus clone 7a produced a greater level of apoptosis in MDCK cells compared with the attenuated strain A/Fiji. In both cases, apoptosis could be partially blocked by treatment with three anti-neuraminidase compounds [4-amino-(GR121158A) and 4-guanidino- (GG167; Zanamivir) 2,3-dehydro-N-acetylneuraminic acid and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA)] when they were given to cells during the virus attachment/entry phase, but not subsequent to this phase. In contrast, GG167, which does not enter cells, did not affect the numbers of infected cells and, in addition, acted late in the infection cycle to inhibit virus yields. Clone 7a neuraminidase was more active than A/Fiji neuraminidase when fetuin was used as the substrate. Similar differences in activity between the two viruses were seen when alpha-2,6 sialyl lactose was used as a substrate, but not with alpha-2,3 sialyl lactose. No sequence differences in the enzyme active site of the two neuraminidases were observed, indicating that differences in neuraminidase specificity and activity may be dictated by other residues. These results suggest that neuraminidase plays some role in the induction of apoptosis and that it acts prior to or during virus entry. However, apoptosis was considerably reduced when UV-irradiated virus, which retains >75% of its neuraminidase activity, was used. In addition, ammonium chloride, used to prevent virus entry, reduced virus-induced apoptosis. Amantadine, which inhibits virus uncoating, also inhibited apoptosis induced by the amantadine-sensitive strain A/Udorn/307/72 (H3N2), but not the amantadine-resistant clone 7a. Hence, one or more intracellular processes are also involved in influenza virus-induced apoptosis.

Regioselective α(2→3)-Sialylation of LeX and Lea by Sialidase-Catalyzed Transglycosylation1

Considerable effort has been devoted to the development of new methods for α-selective sialylation due to the growing importance of the synthetic sialoglycoconjugates in glycobiology3. The synthesis of α-sialoside has been establised by chemical routes,4 which often involve many steps and are complicated. The promising chemoenzymatic procedure through the use of sialyltransferases has already become a preparative technique.5 However, laborious isolation and the pronounced acceptor specificity of the transferases limit their synthetic potential. Recently, a novel procedure for α-sialylation has been reported, which uses sialosides of synthetic substrate as donors and is catalyzed by sialidase in place of sialyltransferase. Thiem et a1.6 have reported the enzymatic synthesis of α(2→6)-linked sialyl galactose, glucose, lactose and lactosamine in preference to the corresponding α(2→3)-linked derivatives employing sialidase from vibrio cholerae, while Ajisaka et al.7 have synthesized α(2→3)-linked sialyl lactose and lactosamine with sialidase from new castle disease virus. 1. Synthetic studies on sialoglycoconjugates, Part 101. For Part 100, see H. Ito, H. Ishida, M. Kiso and A. Hasegawa, Carbohydr. Res., 306, 1 (1998).

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.

Inhibition of acid sialidase by inorganic sulfate

Sulfated glycosaminoglycans are known to inhibit mammalian acid-active sialidase. Although the inhibition depends clearly on the presence of sulfate groups on these macromolecules, there was no information on the intrinsic inhibitory potency of inorganic sulfate. In this study, we demonstrate that inorganic sulfates inhibit acid-active Mu-Neu5Ac sialidase of U937 cells. This inhibition was found to be reversible and it appeared to be of the mixed competitive type. Sulfate-induced inhibition was also observed in other cells as well as with other substrates such as sialyl lactose and bovine mixed brain gangliosides. We conclude that the intrinsic inhibitory potency of sulfate groups may be significantly involved in the inhibition of acid-active sialidase by sulfated glycosaminoglycans. In addition, inorganic sulfate by its apparent potency to selectively inhibit acid sialidases might constitute an interesting tool for the characterisation of the minor forms of sialidases occurring in mammalian cells.

Screening of bacteriophages producing endo- N-acetylneuraminidase

Seven different bacteriophages were isolated from sewage with Escherichia coli K1 strains as host bacteria. These phages showed specific degrading activity for the host capsular polysaccharide, α2,8-linked polysialic acid. The bacteriophage-associated endo- N-acetylneuraminidases degraded only a homopolymer of α2,8-linked sialic acid and were not able to degrade other sialylated carbohydrates such as sialyl lactose. With respect to the minimum substrate size, each enzyme had its own specificity. Among the seven phages isolated, three enzymes exhibited novel endo- N-acetylneuraminidase activity in that they produced sialyl dimer as a main product and two of them degraded sialyl trimer or tetramer.

Removal of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite-host cell interaction

The 35/50 kDa mucin-like surface glycoprotein (gp35/50) of Trypanosoma cruzi metacyclic trypomastigotes has beenimplicated in mammalian cell invasion. In this study we investigated whether the sialyl residues of gp35/50 are required for interaction of parasites with target cells. After treatment with bacterial neuraminidase, the metacyclic forms (G strain) remained reactive with the monoclonal antibody (mAb) 10138 but lost their reactivity with mAb 3C9, that recognizes sialic acid-containing epitopes on gp35/50, and entered HeLa cells in significantly higher numbers as compared to untreated controls. Resialylation of gp35/50, by incubation of parasites with T. cruzi trans-sialidase and sialyl lactose, restored the reactivity with mAb 3C9 as well as the affinity for sialic acid specific lectin. Accordingly, the rate of invasion of resialylated parasites was reduced to levels similar to those observed before desialylation. Purified G strain gp35/50, desialylated by neuraminidase treatment, bound to HeLa cells more than its sialylated counterpart. The Ca 2+ signaling activity, which has been associated with cell invasion, was also determined by measuring the cytosolic Ca 2+ concentration ([Ca 2+] i), in HeLa cells upon interaction with sonicated extracts from untreated or neuraminidase-treated parasites, or with purified gp35/50 in its sialylated or desialylated form. Consistent with the results of cell invasion assay, the desialylated parasite preparations, as well as the sialic acid free gp35/50, induced an average elevation in [Ca 2+] i significantly higher than that triggered by untreated controls. None of these effects, namely the increase in infectivity and Ca 2+ signaling activity, was observed with neuraminidase-treated CL strain metacyclic trypomastigotes, which express a variant form of sialic acid gp35/50 molecule that is not recognized by mAb 10138 and apparently is not involved in target cell invasion.

Towards a better semiquantitative estimation of binding constants: molecular dynamics exploration of the conformational behavior of isolated sialyllactose and sialyllactose complexed with influenza A hemagglutinin

The binding of influenza A virus is mediated by the attachment of the viral hemagglutinin (HA) to sialic acids (Sia) which are components of virus receptors. Molecular dynamics simulations were applied to study the conformational behavior of isolated sialyllactose (SL) and SL complexed with HA in order to achieve a deeper understanding of the process of binding. If a flexible molecule binds to a protein the binding process must select one conformation from the conformational ensemble. The process of freezing the degrees of freedom of the ligand implies a decrease in entropy of the oligosaccharide on binding. The comparison of the conformational behavior of isolated SL and the SL-HA complex showed that the conformational freedom of SL in the bound state is considerably reduced. The specific conformation which has to be frozen out to form the complex with HA, however, is populated in the free state for all functional groups of SL, which is essential for the process of binding. Molecular dynamics (MD) simulations indicate that for the HA-SL complex the strongest decrease in entropy should occur when freezing out the desired conformation of the carboxyl group, both glycosidic linkages and the pendant groups of galactose and glucose.

Towards a better semiquantitative estimation of binding constants: molecular dynamics exploration of the conformational behavior of isolated sialyllactose and sialyllactose complexed with influenza A hemagglutinin

The binding of influenza A virus is mediated by the attachment of the viral hemagglutinin (HA) to sialic acids (Sia) which are components of virus receptors. Molecular dynamics simulations were applied to study the conformational behavior of isolated sialyllactose (SL) and SL complexed with HA in order to achieve a deeper understanding of the process of binding. If a flexible molecule binds to a protein the binding process must select one conformation from the conformational ensemble. The process of freezing the degrees of freedom of the ligand implies a decrease in entropy of the oligosaccharide on binding. The comparison of the conformational behavior of isolated SL and the SL-HA complex showed that the conformational freedom of SL in the bound state is considerably reduced. The specific conformation which has to be frozen out to form the complex with HA, however, is populated in the free state for all functional groups of SL, which is essential for the process of binding. Molecular dynamics (MD) simulations indicate that for the HA-SL complex the strongest decrease in entropy should occur when freezing out the desired conformation of the carboxyl group, both glycosidic linkages and the pendant groups of galactose and glucose.

Sialyllactose-mediated Cell Interaction during Granulosa Cell Differentiation: IDENTIFICATION OF ITS BINDING PROTEINS

The present study was designed to prove the carbohydrate-binding proteins interacting with cell surface sialyllactosylceramide (GM3, NeuAc alpha 2-->3Gal beta 1-->4Glc beta 1-->1'Cer), which is highly expressed during differentiation of rat ovarian granulosa cells. As a specific ligand for the sialyllactose (SL)-binding proteins on granulosa cells, we used a radioiodinated multivalent SL-linked albumin (Alb-(SL)17). The specific association of the ligand to the putative proteins on the intact cells was competitively inhibited by GM3 more effectively than other gangliosides, sialyllactotetraosylceramide, sialylneolactotetraosylceramide, and several glycoproteins with N-linked oligosaccharides. However, the proteins had no specificity for the side chain (N-acetyl or N-glycolyl forms) of sialic acid in GM3. Scatchard analysis of Alb-(SL)17 binding showed high (Kd = 6.4 x 10(-10)M) and low (Kd = 3.1 x 10(-8)M) affinity population of binding sites. By direct binding of 125I-Alb-(SL)17 to SL-binding proteins on Western blots, the putative proteins with molecular masses of 35, 18, and 14 kDa were detected. The interaction of the multivalent derivative with these binding proteins was differently modulated by Ca2+ and Mn2+. The SL-binding proteins occurred in immature granulosa cells and progressively decreased during differentiation, whereas their endogenous ligand GM3 increased. These results indicate that relatively low molecular weight SL-binding proteins exist on the surface of immature granulosa cells and that they may serve as receptor sites for newly synthesized GM3 during differentiation.

Mechanism of macrophage recognition of SH-oxidized erythrocytes: recognition of glycophorin A on erythrocytes by a macrophage receptor for sialosaccharides

Mouse erythrocytes treated with diamide, an SH-oxidizing agent, attach to mouse resident peritoneal macrophages in the absence of serum. The mechanism by which macrophages recognize the SH-oxidized erythrocytes was investigated. Although phosphatidylserine-liposomes inhibited the macrophage recognition, there was no detectable phosphatidylserine on the outer surface of diamide-treated erythrocytes. It is unlikely that phosphatidylserine, that has been proposed to be a determinant in the recognition of some pathologic erythrocytes by macrophages, is involved in the recognition of diamide-treated erythrocytes. Sialyl lactose and glycophorin A effectively inhibited the macrophage recognition, while lactose and neuraminidase-treated glycophorin A did not. Disialoganglioside G D1a, but not monosialoganglioside G M1, partially inhibited the recognition. Trypsinized erythrocytes, in which majority of glycophorin A glycopeptides were expected to be removed from the cell surface, poorly attached to macrophages after diamide treatment. Therefore, it is likely that an interaction between glycophorin A on diamide-treated erythrocytes and a macrophage receptor for sialosaccharides is involved in the recognition. Similar inhibition specificity was observed in the macrophage recognition of erythrocytes treated with periodate, an oxidant that induces disulfide-dependent erythrocyte changes causing macrophage recognition, and of erythrocytes treated with SH-blocking agents, N-ethylmaleimide and p-chloromercuribenzoic acid, that were also found to be susceptible to macrophage recognition in the absence of serum. These results suggest that the macrophages recognize sialosaccharide chains of glycophorin A molecules on SH-oxidized or SH-blocked erythrocytes through a receptor for sialosaccharides.

Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins.

Pseudomonas aeruginosa infection is a leading cause of deterioration of pulmonary function in patients with cystic fibrosis (CF). The interaction of the bacterium with CF and non-CF tracheobronchial mucins was examined to understand the biochemical basis for the high susceptibility of the lungs of CF patients to infection by P.aeruginosa. The binding of radiolabelled bacteria to pure mucins in solid-phase assays was not significantly above non-specific binding to various blocking agents, such as bovine serum albumin, Tween 20, milk powder and polyvinyl pyrrolidine. Further, there was a tendency for the bacteria to be excluded from plastic wells and membranes coated with mucin. Therefore, an indirect approach involving the binding of radiolabelled P.aeruginosa to asialo GM1 ganglioside, the putative receptor for the bacteria on tracheal cells, was used to compare the interaction of CF and non-CF mucins with the bacteria. Highly purified preparations of CF mucin were consistently better inhibitors of the binding of the bacteria to asialo GM1 ganglioside than non-CF mucin preparations. In the case of the binding of a stable mucoid strain, the difference was statistically significant (P < 0.001) at all concentrations of mucin tested. For the non-mucoid strain, the difference was significant only at the higher concentrations. Of the saccharides tested similarly, sialyl lactose and the oligosaccharide portion of asialo GM1 were found to be good inhibitors. The increased binding of the bacteria to CF mucin was further confirmed by a solution binding assay in which the binding of 125I-labelled mucin to unlabelled bacteria was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Galactose terminated oligosaccharides activate macrophage respiratory burst

Respiratory burst is a primary event exhibited by macrophages in response to specific stimuli. We present results on activation of respiratory burst by lactose and sialyl lactose which were comparable to that of known activators like phorbol ester, lipopolysaccharide and Ca 2+-ionophore. These oligosaccharides were used as models of erythrocyte membrane glycoproteins whose terminal sugars are eigher galactose or sialic acid. Maximum activation at as low as 3 pM or 6 pM of sialyl lactose or lactose was obtained while desialylated erythrocyte membranes (5 ng) with exposed ß-galactosyl residues containing small amounts of sialic acid proved to be effective activators of respiratory burst. We could establish from kinetics of activation that maximum respiratory burst occurs between 5–9 min with either oligosaccharides or membranes as activators. The probable mechanism of activation by oligosaccharides and membranes was found to be operating via protein kinase C or calmodulin signal mediation pathways and inhibition by intracellular cAMP suggests the role for dephosphorylation and inactivation of respiratory burst. Taken together, our results suggests that exposed ß-galactosyl residues are capable of triggering the generation of reactive O 2 species which are implicated in killing and digestion of the engulfed organisms by phagocytes.

Trypanosoma rangeli sialidase lacks trans-sialidase activity

Extracts and tissue culture supernatants of axenic forms of T. rangeli were assayed for the presence of sialidase and transsialidase activities. Using sialyl(α2–3)lactose, sialyl(a2–6)lactose, poly( α2−8) N-acetylneuraminic acid, fetuin and 4-methylumbelliferyl- N-acetylneuraminic acid as sialic acid donors, and lactose as a sialic acid acceptor, no trans-sialidase activity was detected. Nevertheless, T. rangeli lysates and culture supernatants contain a sialidase that hydrolyzes sialyl(α2–3)lactose, and much less efficiently sialyl(α2–6)lactose, but not poly( α2−8) N-acetylneuraminic acid. T. cruzi trans-sialidase hydrolyzed only sialyl(α2–3)lactose under the same conditions. The T. rangeli and the T. cruzi enzymes differ antigenically and in their pH optimum for hydrolase activity.

The porcine paramyxovirus LPM specifically recognizes sialyl (alpha 2,3) lactose-containing structures.

The porcine paramyxovirus LPM recognizes alpha, but not beta, anomers of sialic acid containing structures, specifically sialyl (alpha 2,3) lactose. The virus specificity is directed to the sialyl residue and to the C'4 axial OH and the C'6 CH2OH of the galactose present in this structure.

Selective modification of Sendai virus hemagglutinin neuraminidase by pyridoxal 5′-phosphate: evidence for an allosteric modulation of neuraminidase activity

Incubation of Sendai virus with pyridoxal 5′-phosphate (PLP) causes inhibition of hemolytic activity, a slight reduction of hemagglutinating activity, and an increase in neuraminidase activity. The effects on hemagglutination and neuraminidase are prevented by the presence in the incubation mixture of sialyl lactose, a substrate of hemagglutinin-neuraminidase. Incubation with PLP of the water-soluble enzymatic domain of the neuraminidase has no effect on enzymatic activity, while the allosteric inhibition (Dallocchio et al. (1991) Biochem. Int. 25, 663–668) disappears. Both virus-bound and solubilized neuraminidase are selectively modified by PLP at the lysine-553. Our data suggest that PLP inactivates a previously undetected inhibitory site on the viral neuraminidase, and that a physiological effector is present on the viral envelope.

Sialidase from different sources compared for electrophoretically separating serum alkaline phosphatase fractions from liver and bone.

We compared sialidase (neuraminidase; EC 3.2.1.18) from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, seeking to improve the electrophoretic separation of the liver and bone isoenzymes of alkaline phosphatase (EC 3.1.3.1) on cellulose acetate membranes. Resolution is decisively determined by the type and activity of sialidase used in the preincubation of serum sample. Sialidase from Arthrobacter ureafaciens is not suited for this method. For optimal separation of the two isoenzymes we recommend the use of sialidase from Vibrio cholerae, determination of its activity with a standard procedure such as described here (mucin or sialyl lactose as substrates), and a final concentration of sialidase activity of 2.0 or 2.9 U/L (measured with mucin or sialyl lactose) in the incubation mixture.

Binding of Escherichia coli S fimbriae to human kidney epithelium

Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbriae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(alpha 2-3)lactose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as well as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomeruli and of the renal interstitium. No binding to connective tissue elements was observed. The results suggest that the biological function of S fimbriae is to mediate the adhesion of E. coli to human epithelial and vascular endothelial cells.

Relationship between urinary sialylated saccharides, serum amyloid A protein, and C-reactive protein in rheumatoid arthritis and systemic lupus erythematosus.

The urinary excretion of sialic-acid-containing oligosaccharides, total sialic acid, serum amyloid A protein (SAA), and C-reactive protein (CRP) has been studied in 48 patients with rheumatoid arthritis (RA) and in 17 patients with systemic lupus erythematosus (SLE). Linear regression analysis revealed a close positive correlation between serum SAA and CRP levels in both RA (r = 0.71, p less than 0.001) and SLE (r = 0.86, p less than 0.001). The urinary excretion of sialyl lactose showed a positive correlation with the serum levels of SAA and CRP in RA (r = 0.45 and r = 0.45, respectively, p less than 0.01) but not in SLE (r = 0.05 and r = 0.10 respectively). Changes in serum total sialic acid levels paralleled those in CRP and SAA in RA as well as in SLE. Patients with very active RA had higher urinary sialyl oligosaccharide excretion (p less than 0.001), higher CRP levels (p less than 0.01), and higher SAA levels ( p less than 0.05) than those with moderately active disease.

Nuclear magnetic resonance studies on wheat germ agglutinin-monomeric amino sugar interactions

The interaction of several N-acetyl- d-glucosamine analogs and of sialyl lactose with the lectin wheat germ agglutinin was studied by nuclear magnetic resonance. N- 2H 3-acetyl- d-gluocosamine was synthesized and found to displace the N-acetyl methyl signal toward its free chemical shift in N-acetylglucosamine and N-acetylneuraminic acid demonstrating common binding sites for the latter two compounds. The N-acetyl methyl signal of the α-methylglucoside of N-acetylglucosamine could be titrated but a 3-deoxy analog could not, the latter exhibiting very weak binding and demonstrating the importance of the 3-OH group in the binding process. Sialyl lactose (an N-acetylneuraminic acid analog) was rather tightly bound to the lectin. N-F 3-acetyl- d-glucosamine was synthesized and its binding to the lectin was studied at pH 4, 4.5, 5.1 by 19F NMR. The two anomers were found to bind with nearly equal K d ′s but exhibited a pH and anomer dependent Δ (total bound chemical shift). The -CF 3 analog was found to bind considerably stronger to the lectin than the -CH 3 compound. The clear resolution of the α and β anomers of this molecule make it a very useful probe of the lectin binding site.

Effects of denervation on the neuraminidase activity of slow and fast muscles of rabbits

The presence in skeletal muscles of rabbits of neuraminidase activity on sialyl lactose was demonstrated. By comparing the specific activity of this enzyme in different control muscles, it was shown that it was 2.3 times higher in the slow than in the fast muscles. Seven days after deneration, we observed a significant increase of activity in fast muscle and a significant decrease of activity in slow muscle. These results, together with previous data, are in favour of the hypothesis that turnover of muscle glycoconjugates is under a type-specific neural control.