Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.     

Mobilization of neutrophil sialidase activity desialylates the pulmonary vascular endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium

The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dose-dependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor-activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.     

A direct evidence for the early membrane desialylation in cobalt-irradiated mouse lymphocytes

Using the sensitive periodate-borotritide radioassay to quantify the membrane sialic acid amount, we show that the $\text{in vitro}$ [⁶⁰Co]-irradiation of mouse splenocytes (10–50 Gy) significantly decrease their membrane sialic acid amount. The results show that the irradiation-induced desialylation is a very early phenomenon since the periodate oxidation is done immediately after the irradiation. A short incubation of cells at 25° C does not increase the extent of the desialylation. This membrane alteration might explain the rapid and drastic decrease in lymphocyte counts in mammals exposed $\text{in vivo}$ to irradiation.     

Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors

Despite numerous reports suggesting that beta(1) integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that beta(1) integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the beta(1) integrin subunit becomes hyposialylated, suggesting that the beta(1) integrin is a substrate for this enzyme. The expression of hyposialylated beta(1) integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified alpha(5)beta(1) integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated beta(1) integrins are more adhesive to fibronectin, although desialylation of alpha(5) subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the beta(1) integrin family of cell adhesion receptors.     

Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction

The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.     

Anticancer peptide NK-2 targets cell surface sulphated glycans rather than sialic acids

Abstract Some antimicrobial peptides have emerged as potential anticancer agents. In contrast to chemotherapeutics, they act primarily by physical disruption of the cancer cell membrane. Selective targeting of these cationic peptides still remains elusive. We focus on the interaction of α-helical peptides NK-2, cathelicidin LL32, and melittin with PC-3 prostate cancer cells, and we provide strong evidence that, amongst the anionic glycans covering the cell surface, sulphated carbohydrates rather than sialic acids are the preferred interaction sites of the peptides. To test the significance of cell surface carbohydrates, a glycan microarray screen with fluorescently labelled peptides has been performed. Amongst 465 mammalian glycan structures on the chip, more than 20 different sulphated glycans were detected as the preferred binding partners of the peptide NK-2. The amount of peptide bound to sialic acid containing oligosaccharides was close to background level. These findings were consistent with microcalorimetric experiments revealing high and low binding enthalpies of peptides to sulphated carbohydrates and to sialic acid, respectively. Enzymatic desialylation of PC-3 cells did not affect peptide-mediated changes in cell metabolism, cell membrane permeabilisation, killing rate, and kinetics. Finally, the cytotoxicity of all peptides could be drastically impaired through the competitive inhibition by chondroitin sulphate, but not by sialic acid and sialylated fetuin.     

Lectin-dependent neutrophil cytotoxicity: Enhanced susceptibility of desialylated red cells

Lectin-dependent neutrophil cytotoxicity against autologous human red cells was studied using an ¹¹¹In(indium)-release assay. Human red cells were not readily killed by neutrophils in the presence of phytohemagglutinin (PHA). However, removal of red cell membrane sialic acids (desialylation) markedly enhanced their susceptibility to PHA-dependent neutrophil cytotoxicity. This neutrophil cytotoxicity was dependent on the energy supplied by anaerobic glycolysis, but it was independent of erythrophagocytosis. Catalase, superoxide dismutase, KCN, and Na azide did not inhibit PHA-dependent neutrophil cytotoxicity. Neutrophils from a patient with chronic granulomatous disease, in the presence of PHA, also killed desialylated red cells normally. On the other hand, desialylation of neutrophils had no effect on the expression of their cytotoxic effect. The results suggest that desialylated red cells are much more susceptible to lectin-dependent neutrophil cytotoxicity than normal red cells, and that lectin-dependent neutrophil cytotoxicity is independent of reactive oxygen species.     

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.     

Selective ganglioside desialylation in the plasma membrane of human neuroblastoma cells*

Gangliosides of the plasma membrane are important modulatorsof cellular functions. Previous work from our laboratory hadsuggested that a plasma membrane sialidase was involved in growthcontrol and differentiation in cultured human neuroblastomacells (SK-N-MC), but its substrates had remained obscure. Wenow performed sialidase specificity studies in subcellular fractionsand found ganglioside GM3 desialylating activity in presenceof Triton X-100 to be associated with the plasma membrane, butabsent in lysosomes. This Triton-activated plasma membrane enzymedesialylated also gangliosides GDla, GD1b, and GT1b, therebyforming GM1; cleavage of GM1 and GM2, however, was not observed.Sialidase activity towards the glycoprotein fetuin with modifiedC-7 sialic acids and towards 4-methylumbelliferyl neuraminatewas solely found in lysosomal, but not in plasma membrane fractions. The role of the plasma membrane sialidase in ganglioside desialylationof living cells was examined by following the fate of [³H]galactose-labelledindividual gangliosides in pulse-chase experiments in absenceand presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminicacid. When the plasma membrane sialidase was inhibited, radioactivityof all gangliosides chased at the same rate. In the absenceof inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degradedat a considerably faster rate in confluent cultures, whereasthe GM1-pool seemed to be filled by the desialylation of highergangliosides. The results thus suggest that the plasma membranesialidase causes selective ganglioside desialylation, and thatsuch surface glycolipid modification triggers growth controland differentiation in human neuroblastoma cells. ganglioside neuroblastoma cells plasma membrane sialidase     

Cellular sialic acid level and phenotypic expression in B16 melanoma cells: comparison of spontaneous variations and bromodeoxyuridine- and theophylline-induced changes

The relation of the total cellular content of sialic acid to phenotypic expression of B16 mouse melanoma cells was examined by using phenotype-modifying reagents and more than 10 cloned cell lines with spontaneous phenotypic variations. The sialic acid content changed in a growth phase-dependent manner with a peak in the early log phase of growth. This peak completely disappeared when cells were treated with 5-bromodeoxyuridine (BrdU), suggesting its relation to quasi-normal phenotypes of the treated cells. BrdU treatment also reduced the cellular sialic acid content itself and resulted in the suppression of the activity of tyrosinase, the key enzyme for melanogenesis, and a considerable increase in cell-to-substratum adhesiveness. Treatment with theophylline, in contrast, markedly elevated the sialic acid content, which was accompanied by dramatic increments in tyrosinase activity and pigmentation as well as a slight increase in adhesiveness. The results show a correlation of sialic acid level with tyrosinase expression but not with cell adhesion. From comparison of spontaneous phenotypic variations, the correlation of sialic acid level with tyrosinase activity was confirmed, while there was only a slight correlation with adhesiveness. It is thus suggested that sialylation/desialylation, being reflected as variations in cellular sialic acid content, is implicated in melanoma cell differentiation in terms of tyrosinase expression.     

Level of sialic acid residues in platelet proteins in diabetes,aging, and Hodgkin's lymphoma: a potential role of free radicals in desialylation

The present study evaluated the levels of sialic acid and carbonylation in platelet proteins from diabetes, old age, and Hodgkin's lymphoma cases and explored the role of free radicals in desialylation. Sialic acid and carbonylation were estimated in platelet suspension obtained from 10 healthy adult control subjects, 20 elderly healthy persons, 20 cases with diabetes, and 10 with Hodgkin's lymphoma. The level of sialic acid residues in platelets was found to be significantly lower and the carbonylation of proteins was higher in old age, diabetes, and lymphoma cases in comparison to controls. The level of carbonylation correlated with desialylation in these cases. In vitro treatment of platelets with free radicals was found to cause desialylation and to increase the carbonyl content. Addition of 2-mercaptoethanol, an antioxidant, significantly attenuated the above effects. The study indicates the possible role of free radicals in desialylation of platelet proteins in the above clinical conditions.     

Recruitment of murine neutrophils in vivo through endogenous sialidase activity

Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface. This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis. We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo. A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activities in vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8. We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitment in vivo. The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes. Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response.     

Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

ABSTRACT: BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. RESULTS: We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, MAA and SNA, could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. CONCLUSIONS: In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.     

db—cAMP对转化细胞钙调素基因表达与细胞骨架的影响

We have demonstrated that the distribution of microtubules (MT), microfilaments (MF) and fibronectin (FN) were diminished, while the gene expression of the calmodulin and c-fos enhanced in the transformed C3 H10 T1/2 cells. After treatment with 1 mM db-cAMP for 1 hr. and 2 hrs., there was an early and rapidly reduced in gene expression of calmodulin and c-fos respectively. After db-cAMP treatment for 4-5 days, the number of Capping cells of ConA binding decreased significantly and the cell surface microvilli decreased also. The growth of treated cells was inhibited markedly. By using 4F1 cDNA probe, which is preferentially expressed in G1 phase, we have found that the db-cAMP treated cells were accumulated at G1 phase. Of particular interest is the fact that the distribution of microtubules, microfilaments and fibronectin were recovered after treatment with 1 mM db-cAMP for 6 days. It is suggested that the inhibition of proliferation, alteration of phenotype and recovery of cytoskeleton in transformed cells after treatment with db-cAMP are related to the inhibition of gene expression of calmodulin.     

Sialoforms of dipeptidylpeptidase IV from rat kidney and liver

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.     

α2,6 Sialylation associated with increased β1,6-branched N-oligosaccharides influences cellular adhesion and invasion

Expression of β1,6-branched N-linked oligosaccharides have a definite association with invasion and metastasis of cancer cells. However, the mechanism by which these oligosaccharides regulate these processes is not well understood. Invasive variants of B16 murine melanoma, B16F10 (parent) and B16BL6 (highly invasive variant) cell lines have been used for these studies. We demonstrate that substitution of α2,6-linked sialic acids on multiantennary structures formed as a result of β1,6-branching modulate cellular adhesion on both extracellular matrix (ECM) and basement membrane (BM) components. Removal of α2,6 sialic acids either by enzymatic desialylation or by stably down-regulating the ST6Gal-I (enzyme that catalyses the addition of α2,6-linked sialic acids on N-linked oligosaccharides) by lentiviral driven shRNA decreased the adhesion on both ECM and BM components and invasion through reconstituted BM matrigel.     

Phenotypic changes induced by expression of beta-galactoside alpha2,6 sialyltransferase I in the human colon cancer cell line SW948

Beta-galactoside alpha2,6 sialyltransferase (ST6Gal.I), the enzyme which adds sialic acid in alpha2,6-linkage on lactosaminic termini of glycoproteins, is frequently overexpressed in cancer, but its relationship with malignancy remains unclear. In this study, we have investigated the phenotypic changes induced by the expression of alpha2,6-sialylated lactosaminic chains in the human colon cancer cell line SW948 which was originally devoid of ST6Gal.I. Clones derived from transfection with the ST6Gal.I cDNA were compared with untransfected cells and mock transfectants. The ST6Gal.I-expressing clones show (1) increased adherence to fibronectin and collagen IV but not to hyaluronic acid. Treatment with Clostridium perfrigens neuraminidase reduces the binding to fibronectin and collagen IV of ST6Gal.I-expressing cells but not that of ST6Gal.I-negative cells; (2) accumulation and more focal distribution of beta1 integrins on the cell surface; (3) different distribution of actin fibers; (4) flatter morphology and reduced tendency to multilayer growth; (5) improved ability to heal a scratch wound; (6) reduced ability to grow at the subcutaneous site of injection in nude mice. Our data suggest that the presence of alpha2,6-linked sialic acid on membrane glycoconjugates increases the binding to extracellular matrix components, resulting in a membrane stabilization of beta1 integrins, further strengthening the binding. This mechanism can provide a basis for the flatter morphology and the reduced tendency to multilayer growth, resulting in a more ordered tissue organization. These data indicate that in the cell line SW948, the effect of ST6Gal.I expression is consistent with the attenuation of the neoplastic phenotype.     

Enhancement of the Influenza A Hemagglutinin (HA)-Mediated Cell-Cell Fusion and Virus Entry by the Viral Neuraminidase (NA)

Background

The major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity.

Methodology/Principal Findings

The role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogeneous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA.

Conclusion/Significance

The NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.     

Uptake of sialic acid by human erythrocyte. Characterization of a transport system

Upon incubation of human red blood cells (RBC) with [4-9-14C] N-acetylneuraminic acid, the cells incorporated this sugar, as demonstrated by the identification of labelled N-acetylmannosamine in the cytosol, as a result of the action of the sialic acid pyruvate-lyase we discovered previously (Biochimie 84 (2002) 655). The mechanism is saturable and indicates the presence of a limited number of transporter molecules in the RBC membrane. This transport process may have relevance to the desialylation of membrane glycoconjugates which occurs during ageing of erythrocytes.     

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3[emsp4 ]h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized 2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied 2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2[emsp4 ]h of etoposide treatment. Moreover, the decrease in 2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.