Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells.

Sialic acids are the most abundant terminal carbohydrate moiety on cell surface glycoconjugates in eukaryotic cells and are of functional importance for many biological ligand-receptor interactions. It is a widely accepted view that sialic acids cannot be efficiently taken up from the extracellular space by eukaryotic cells. To test this assumption, we cultivated two recently identified human hematopoetic cell lines which are hyposialylated due to a deficiency in de novo sialic acid biosynthesis in the presence of N-acetylneuraminic acid (NeuAc), the most frequently found sialic acid. Surprisingly, NeuAc medium supplementation rapidly and potently compensated for the endogenous hyposialylation in a concentration-dependent manner, resulting in the presentation of cell surface sialoglycans involved in cell adhesion, virus infection and signal transduction. We provide several lines of experimental evidence that all suggest that NeuAc was neither extracellularly incorporated nor degraded to a less complex sugar before uptake. Importantly, NeuAc induced a marked increase in intracellular CMP-NeuAc levels in both human cell lines and in primary cells regardless of the prior sialylation status of the cells. Studies employing 9-[3H]NeuAc revealed an uptake consistent with the observed incorporation of unlabeled NeuAc. We propose the existence of an efficient uptake mechanism for NeuAc in eukaryotic cells.

Fibronectin is a binding partner for the myelin-associated glycoprotein (siglec-4a).

The myelin-associated glycoprotein (MAG) mediates cell-cell interactions between myelinating glial cells and neurons. Here we describe the extracellular matrix glycoprotein fibronectin as a binding partner of MAG. It has been identified by affinity precipitation with MAG-Fc from NG108-15 cells and by microsequencing of two peptides derived from a 210-kDa protein band. Western blot analysis showed that fibronectin is also present in MAG binding partners isolated from N(2)A (murine neuroblastoma) cells, rat brain and rat spinal cord. Different fibronectin isoforms have been isolated from brains of young and adult rats, indicating that the expression of MAG binding fibronectin changes during development.

Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.

The N-acetylneuraminate lyase (NAL) sub-family of (beta/alpha)(8) enzymes share a common catalytic step but catalyse reactions in different biological pathways. Known examples include NAL, dihydrodipicolinate synthetase (DHDPS), d-5-keto-4-deoxyglucarate dehydratase, 2-keto-3-deoxygluconate aldolase, trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase and trans-2'-carboxybenzalpyruvate hydratase-aldolase. Little is known about the way in which the three-dimensional structure of the respective active sites are modulated across the sub-family to achieve cognate substrate recognition. We present here the structure of Haemophilus influenzae NAL determined by X-ray crystallography to a maximum resolution of 1.60 A, in native form and in complex with three substrate analogues (sialic acid alditol, 4-deoxy-sialic acid and 4-oxo-sialic acid). These structures reveal for the first time the mode of binding of the complete substrate in the NAL active site. On the basis of the above structures, that of substrate-complexed DHDPS and sequence comparison across the sub-family we are able to propose a unified model for active site modulation. The model is one of economy, allowing wherever appropriate the retention or relocation of residues associated with binding common substrate substituent groups. Our structures also suggest a role for the strictly conserved tyrosine residue found in all active sites of the sub-family, namely that it mediates proton abstraction by the alpha-keto acid carboxylate in a substrate-assisted catalytic reaction pathway.

Specificity of the binding site of the sialic acid-binding lectin from ovine placenta, deduced from interactions with synthetic analogues.

The specificity of the sialic acid-binding lectin from ovine placenta was examined in detail by haemagglutination inhibition assays applying a panel of 32 synthetic sialic acid analogues. The carboxylic acid group is a prerequisite for the interaction with the lectin, the alpha-anomer of the methyl glycoside is only a little more effective as an inhibitor than the beta-anomer and the most potent inhibitor was 9-deoxy-10-carboxylic acid Neu5Ac, followed by 4-oxo-Neu5Ac. In contrast to the majority of known sialic acid-binding lectins, the N-acetyl group of Neu5Ac is not indispensable for binding, neither is the hydroxyl group at C-9 since substitutions at this carbon atom are well tolerated. Furthermore, all sulfur-containing substituents at C-9 enhanced the affinity of the lectin. This is the first sialic acid-binding lectin found to strongly bind thio derivatives.

The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3.

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.

Transfer of 131I and fluoresceinyl sialic acid derivatives into the oligosaccharide chains of IgG: a new method for site-specific labeling of antibodies.

Biochemical modifications of IgG can help to avoid damages caused by oxidation or reduction. Terminal groups of the saccharide structures, located in the Fc-portion of IgG molecules, were modified by enzymatic reactions. IgG was pretreated with sialidase, to cleave bound sialic acid, and with galactosyltransferase, to increase the number of acceptor sites for transfer reactions. Afterward, modified sialic acid derivatives were transferred enzymatically into the oligosaccharide chains of IgG. Labeling was possible with sialic acids modified in either position 5 or position 9. The usefulness of this method was demonstrated for radioactive and fluoresceinylated reagents, with yields up to 90% in 1 h. Immunological investigations have shown no influence on the immunoreactivity by the described modification of saccharide structures.

Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT.

In contrast to extracellular, long chain ceramides which comprise a structural component of the epidermal water barrier, intracellular ceramides originating from sphingomyelin hydrolysis have been shown to inhibit proliferation and to induce apoptosis in different cell populations. To further elucidate the possible role of intracellular ceramides in human epidermis, two new cell-permeable ceramide analogues, N-thioacetylsphingosine (C2-Cer=S) and 4-dodecanoylamino-decan-5-ol (FS-5), were synthesized and tested for their ability to suppress cell growth and to induce apoptosis in immortalized human keratinocytes. It was shown that the well-investigated ceramide analogue N-acetylsphingosine (C2-Cer=O), as well as the new compound C2-Cer=S inhibited proliferation of HaCaT cells with half-inhibitory concentrations (IC50) of 20 microg/ml and 10 microg/ml, respectively, whereas FS-5 has been potent with an IC50>40 microg/ml. Overall, all three ceramide analogues induced apoptosis in HaCaT cells as assessed by DNA-fragmentation using ELISA technique and in situ nick end labelling, thereby confirming the importance of ceramide signalling in keratinocytes.

Apoptosis and activation of the sphingomyelin-ceramide pathway induced by oxidized low density lipoproteins are not causally related in ECV-304 endothelial cells.

Oxidized low density lipoproteins (oxLDL) are thought to play a central role in the development of atherosclerosis. Toxic concentrations of mildly oxidized LDL elicit massive apoptosis of endothelial cells (Escargueil-Blanc, I., Meilhac, O., Pieraggi, M. T. , Arnal J. F., Salvayre, R., Nègre-Salvayre, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 331-339). Since the lipid mediator ceramide emerged as a potent inducer of apoptosis, we aimed at investigating the occurrence of ceramide formation and its potential role in oxLDL-induced apoptosis. In ECV-304 endothelial cells, toxic concentrations of oxLDL triggered an early activation of the sphingomyelin-ceramide pathway, as shown by both sphingomyelin hydrolysis and ceramide formation. N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and dichloroisocoumarin (DCIC), two serine-protease inhibitors (serpins), blocked the oxLDL-induced ceramide generation but, unexpectedly, did not inhibit the oxLDL-induced apoptosis. Conversely, treatment of endothelial cells by bacterial sphingomyelinase, under conditions effectively generating ceramide, did not induce apoptosis. In contrast, short-chain permeant C2- and C6-ceramides elicited apoptosis of ECV-304. However, the mechanisms of apoptosis triggered by C2-ceramide and by oxLDL were (at least in part) different, because C2-ceramide-induced apoptosis was calcium-independent, whereas oxLDL-induced apoptosis was calcium-dependent. In conclusion, it is suggested that oxLDL-induced apoptosis is calcium-dependent but independent of the activation of the sphingomyelin-ceramide pathway and that the toxic effect of short chain permeant ceramides is calcium-independent and does not mimic the effect of natural ceramides induced by oxLDL.

Functional groups of sialic acids involved in binding to siglecs (sialoadhesins) deduced from interactions with synthetic analogues.

The siglecs, formerly called sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free alpha-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength.

Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain.

The mammalian hepatic asialoglycoprotein receptor, a member of the C-type animal lectin family, displays preferential binding to N-acetylgalactosamine compared with galactose. The structural basis for selective binding to N-acetylgalactosamine has been investigated. Regions of the carbohydrate-recognition domain of the receptor believed to be important in preferential binding to N-acetylgalactosamine have been inserted into the homologous carbohydrate-recognition domain of a mannose-binding protein mutant that was previously altered to bind galactose. Introduction of a single histidine residue corresponding to residue 256 of the hepatic asialoglycoprotein receptor was found to cause a 14-fold increase in the relative affinity for N-acetylgalactosamine compared with galactose. The relative ability of various acyl derivatives of galactosamine to compete for binding to this modified carbohydrate-recognition domain suggest that it is a good model for the natural N-acetylgalactosamine binding site of the asialoglycoprotein receptor. Crystallographic analysis of this mutant carbohydrate-recognition domain in complex with N-acetylgalactosamine reveals a direct interaction between the inserted histidine residue and the methyl group of the N-acetyl substituent of the sugar. Evidence for the role of the side chain at position 208 of the receptor in positioning this key histidine residue was obtained from structural analysis and mutagenesis experiments. The corresponding serine residue in the modified carbohydrate-recognition domain of mannose-binding protein forms a hydrogen bond to the imidazole side chain. When this serine residue is changed to valine, loss in selectivity for N-acetylgalactosamine is observed. The structure of this mutant reveals that the beta-branched valine side chain interacts directly with the histidine side chain, resulting in an altered imidazole ring orientation.

Preparation and characterization of differently aggregated colorectal carcinoma cell subpopulations from surgical specimens.

The loss of intercellular adhesion within the primary tumor is one of the key events leading to metastasis. Although a number of adhesion molecules involved in intercellular adhesion have been described in experimental systems, the clinical relevance of many of these molecules still has to be determined. We tried to assess the contribution of membrane-bound carbohydrates and of E-Cadherin, CEA, and Sia-LeA for intercellular adhesion of cells isolated from colorectal carcinoma tissue directly obtained from the surgeon. A subpopulation of nonaggregating cells was prepared by means of slowly passing of freshly isolated cells through a series of sieves with decreasing mesh widths. Nonaggregating cells differed mainly in two aspects from aggregated cells: (i) determination of lectin binding and of specific sialytransferase activities revealed enhanced alpha2,6-sialylation of nonaggregating cells, and (ii) staining with specific antibodies documented a loss of E-Cadherin reactivity of such cells. An enhanced activity of beta-galactoside alpha2,6-sialytransferase (ST6Gal 1) was found in metastasizing colorectal carcinomas; however, its biological function has to be shown. Our results suggest that ST6Gal 1 is responsible for reduced homotypic aggregation of colorectal carcinoma cells and may thus facilitate the release of single cells from the primary tumor.

Partial characterization and enrichment of a membrane-bound sialidase specific for gangliosides from human brain tissue.

Gangliosides, constituents of surfaces of vertebrate cells, modulate important cellular functions. Ganglioside-specific sialidases that possibly control these processes have been observed in a number of tissues, but their characterization has proved difficult due to their low abundance and lability. Here we describe the partial isolation and characterization of a ganglioside sialidase from human brain grey matter. After membrane extraction with octylglucoside, the enzyme was purified about 1300-fold by ion-exchange, affinity and gel-permeation chromatographies. Although PAGE still showed several protein bands, specific photoaffinity labelling with iodinated 5-N-acetyl-9-(4-azidosalicoylamido)-2,9-dideoxy-2,3-didehydrone uraminic acid identified a single polypeptide of 60 kDa likely to contain the active site of the sialidase. In the presence of 0.4% octylglucoside, the purified sialidase desialylated gangliosides G(M3), G(D1a), G(D1b) and G(T1b), but was inactive towards G(M1), G(M2), colominic acid, sialyl-(alpha2-3)-lactose, 2-(4-methylumbelliferyl)-neuraminate, or the glycoprotein fetuin. The ganglioside sialidase activity was strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, heparin and heparan sulfate. Because of its substrate and inhibitor profiles, the purified enzyme resembles the activity characterized previously in the plasma membrane of human neuroblastoma cells, but is distinct from a lysosomal activity. The purified brain sialidase thus appears to function in the selective desialylation of gangliosides with terminal sialic acid residues.

Bcl-2 antagonizes apoptotic cell death induced by two new ceramide analogues.

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N-thioacetylsphingosine and FS-5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations > or = 14 microM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly-(ADP-ribose) polymerase (PARP). Additional experiments in bcl-2-transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl-2. Furthermore, neither N-thio-acetylsphingosine nor FS-5 induced PARP cleavage in bcl-2-transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.

The alpha-galactosyl specific lectin from Artocarpus integrifolia distinguishes between two lymphoma lines with different metastatic potential.

A lectin purified from the seeds of the Vietnamese Artocarpus integrifolia distinguishes between the mouse T-cell lymphoma cell lines Eb and ESb, with low and high metastatic potential, respectively. It agglutinates Eb cells as well as human erythrocytes, but not ESb cells or the human colon carcinomas cells HT29. The haemagglutinin is specific for alpha-galactosyl residues and has a molecular mass of 62 kDa.

Enhancement of Sia-lb1 antigenicity by sulphur-containing substituents at C-5 of free N-acetylneuraminic acid.

The Sia-lb1 epitope, recognized by anti-Sia-lb1 cold agglutinins, is unique since it is represented by the alpha-N-acetylneuraminic acid (alpha NeuNAc) monosaccharide. Chemical modifications of the chain at C-5 of alpha NeuNAc have shown that the natural 2-carbon and the artificial 3-carbon chains are optimal for anti-Sia-lb1 binding. Sia-lb1 antigenicity of alpha NeuNAc could be tenfold enhanced by replacement of the carbonyl oxygen by sulphur. The structural requirements of the Sia-lb1 epitope for optimal antibody binding were identified.

Transfer of an esterase-resistant receptor analog to the surface of influenza C virions results in reduced infectivity due to aggregate formation.

A synthetic sialic acid, N-acetyl-9-thioacetamidoneuraminic acid (9-ThioAcNeu5Ac), is recognized by influenza C virus as a receptor determinant but-in contrast to the natural receptor determinant, N-acetyl-9-O-acetylneuraminic acid-is resistant to inactivation by the viral acetylesterase. This sialic acid analog was used to analyze the importance of the receptor-destroying enzyme of influenza C virus in keeping the viral surface free of receptor determinants. Enzymatic transfer of 9-ThioAcNeu5Ac to the surface of influenza C virions resulted in the loss of the hemagglutinating activity. The ability to agglutinate erythrocytes was restored when the synthetic sialic acid was released from the viral surface by neuraminidase treatment. Infectivity of influenza C virus containing surface-bound 9-ThioAcNeu5Ac was reduced about 20-fold. Sedimentation analysis as well as electron microscopy indicated that virions resialylated with the esterase-resistant sialic acid analog formed virus aggregates. These results indicate that the receptor-destroying enzyme of influenza C virus is required to avoid the presence of receptor determinants on the virion surface and thus to prevent aggregate formation and a reduction of the infectious titer.

Substrate specificity of rainbow trout testis CMP-3-deoxy-D-glycero-D-galacto-nonulosonic acid (CMP-Kdn) synthetase: kinetic studies of the reaction of natural and synthetic analogues of nonulosonic acid catalyzed by CMP-Kdn synthetase.

In this report we present kinetic data of the activation reaction of several synthetic 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac) analogues catalyzed by the rainbow trout testis CMP-Kdn synthetase. This enzyme showed broad substrate specificity in terms of substitutions at C4 or C5 position of Kdn and Neu5Ac. In contrast, calf brain CMP-N-acylneuraminic acid synthetase had narrow substrate specificity, being active only on various N-acyl analogues of Neu5Ac and only slightly active on Kdn derivatives. Usefulness of the trout testis enzyme for synthesis of various CMP-sialate analogues, which could be donor substrates for sialyltransferases, was demonstrated.

Enzymatic transfer of sialic acids modified at C-5 employing four different sialyltransferases.

We present kinetic studies on the enzymatic transfer of several synthetic sialic acid analogues, modified at C-5, to distinct glycoprotein glycans by sialyltransferases differing in acceptor- and linkage-specificity. Biochemical properties of sialic acids were modified by introducing formyl-, trifluoroacetyl-, benzyloxy-carbonyl-, and aminoacetyl-groups to the amino group at C-5 of neuraminic acid. The latter substitution renders the corresponding alpha-glycoside resistant towards sialidases. The respective CMP-sialic acid analogues were prepared by CMP-sialic acid synthase with a yield of 13-55%. The kinetic parameters of several sialyltransferases for the 5-substituted CMP-glycosides differed significantly. Relative to parent CMP-NeuAc, reaction rates of human- and rat liver Gal beta 1, 4GlcNAc alpha 2,6-sialyltransferases ranged from 50 to 170%, of GalNAc alpha 2,6-sialyltransferases from 40-140%, and of Gal beta 1,3Gal-NAc alpha 2,3-sialyltransferase from 20-50%. Resialylation of asialo-alpha 1-acid glycoprotein by 5-N-formyl- and 5-N-aminoacetyl-neuraminic acid employing rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase proceeded to about 80% of galactose sites which is identical to the extent achieved with parent NeuAc. According to our data, neosialoglycoconjugates which carry sialic acids modified at the N-acetyl group can be prepared for structure-function analysis, as this position seems crucial for recognition of adhesion proteins and influenza viruses.

A synthetic sialic acid analog that is resistant to the receptor-destroying enzyme can be used by influenza C virus as a receptor determinant for infection of cells.

A synthetic sialic acid analog, N-acetyl-9-acetamido-9-deoxy-neuraminic acid, can be used by influenza C virus as a receptor determinant for attachment to cells. In contrast to the natural determinant, N-acetyl-9-O-acetylneuraminic acid, the synthetic sialic acid is resistant to the action of the receptor-destroying acetylesterase of this virus. The sialic acid analog was enzymatically transferred to the surface of Madin-Darby canine kidney cells that are resistant to infection because of a lack of receptors. Influenza C virus was able to infect the modified cells through requiring a 10-fold larger amount of the sialic acid analogue on the cell surface compared to the natural receptor determinant. The quantitative difference is accounted for mainly by a less efficient binding of influenza C virus to the analog. Thus, in our system, inactivation of the receptor by the viral esterase is not required for the initiation of an influenza C virus infection.

Suppression of influenza virus infection by an N-thioacetylneuraminic acid acrylamide copolymer resistant to neuraminidase.

We have previously shown that alpha-2-O-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2Me) has a higher affinity to bromelain-treated hemagglutinin (HA) of influenza A virus than sialic acid from natural sources (Machytka et al., 1993, FEBS Lett. 334, 117-120). We have now compared the inhibitory effects of alpha-Neu5thioAc2Me and other sialic acid analogs on receptor binding and plaque formation of intact influenza A viruses. When alpha-Neu5thioAc2Me was polymerized by conjugation to polyacrylamide, its affinity to HA increased 10(3)-fold. When analyzed by plaque reduction, the alpha-Neu5thioAc2 polymer was about 10 times more efficient as an inhibitor of virus replication than the alpha-Neu5Ac2 polymer, stressing the importance of sulfur at C5. The S-glycoside alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) had the same affinity to HA as alpha-Neu5thioAc2Me, but was resistant to neuraminidase. The alpha-Neu5thioAc2S polymer interfered with the replication of a wider spectrum of influenza A virus subtypes than the alpha-Neu5thioAc2 polymer. The results indicate that the alpha-Neu5thioAc2S polymer has the potential to be used as an inhibitor of influenza virus infection.