Removal Of Sialic Acid Research Articles

Differential Sialylation Modulates Voltage-gated Na+ Channel Gating throughout the Developing Myocardium

Voltage-gated sodium channel function from neonatal and adult rat cardiomyocytes was measured and compared. Channels from neonatal ventricles required an ∼10 mV greater depolarization for voltage-dependent gating events than did channels from neonatal atria and adult atria and ventricles. We questioned whether such gating shifts were due to developmental and/or chamber-dependent changes in channel-associated functional sialic acids. Thus, all gating characteristics for channels from neonatal atria and adult atria and ventricles shifted significantly to more depolarized potentials after removal of surface sialic acids. Desialylation of channels from neonatal ventricles did not affect channel gating. After removal of the complete surface N-glycosylation structures, gating of channels from neonatal atria and adult atria and ventricles shifted to depolarized potentials nearly identical to those measured for channels from neonatal ventricles. Gating of channels from neonatal ventricles were unaffected by such deglycosylation. Immunoblot gel shift analyses indicated that voltage-gated sodium channel α subunits from neonatal atria and adult atria and ventricles are more heavily sialylated than α subunits from neonatal ventricles. The data are consistent with approximately 15 more sialic acid residues attached to each α subunit from neonatal atria and adult atria and ventricles. The data indicate that differential sialylation of myocyte voltage-gated sodium channel α subunits is responsible for much of the developmental and chamber-specific remodeling of channel gating observed here. Further, cardiac excitability is likely impacted by these sialic acid–dependent gating effects, such as modulation of the rate of recovery from inactivation. A novel mechanism is described by which cardiac voltage-gated sodium channel gating and subsequently cardiac rhythms are modulated by changes in channel-associated sialic acids.

The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation

The trans-sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans-sialidase-mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans-sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans-sialidase-apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans-sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.

Lysosomal Sialidase (Neuraminidase-1) Is Targeted to the Cell Surface in a Multiprotein Complex That Facilitates Elastic Fiber Assembly

We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of beta-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surface-targeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.

107. α2, 3 and α2,6 N-Linked Sialic Acid Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Type 1 and 6

Recombinant adeno-associated viruses (AAV) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be associated with distribution of their receptors on target cells. Of the eleven well -characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and 4 & 5 respectively. In this report, we identify primary attachment molecules for the two closely related serotypes, AAV1 and AAV6. First we demonstrate use of similar receptor for AAV 1 & 6 through competition experiments after co-infection. Second, similar to removal of heparin sulfate for AAV2, enzymatic or genetic removal of sialic acid significantly reduced AAV1 and AAV6 binding and transduction. Third, contrasting AAV5, which uses only |[alpha]|2,3 sialic acid as the major attachment factor for transduction, lectin staining and lectin competition assay identified both |[alpha]|2,3-linked and |[alpha]|2,6-linked sialic acid for efficient AAV 1 & 6 transduction. Inhibitor of O-linked glycosylation did not block transduction, however, significantly reduction was observed on N-glycan deficient Lec1 cells, indicating that N-linked, but not O-linked sialic acid is required for AAV1 and AAV6 transduction. This was confirmed by resialylation experiment using sialic acid deficient Lec2 cells. Furthermore, mucin which binds to O-linked sialic acid and inhibits AAV4 transduction had no inhibition effect on AAV1 and AAV6 transduction. Finally, proteinase K treatment compared to glycolipid inhibitor treatment supports sialic acid modification of glycoproteins rather than glycolipids with respect to AAV1 and AAV6 transduction. Taken together, the data support a glycoprotein with N-linked |[alpha]|2,3 and /or |[alpha]| 2,6 sialic acid serving as a primary attachment molecule for AAV1 and AAV6 transduction.

Sialidase Activity of Influenza A Virus in an Endocytic Pathway Enhances Viral Replication

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-alpha-N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.

Terminal Sialic Acid Residues on Human Glycophorin A Are Recognized by Porcine Kupffer Cells

We have previously shown that recognition of human erythrocytes by porcine Kupffer cells is mediated by a carbohydrate-dependent mechanism. The present study explores the possible ligands existing on human glycophorin A and tests their ability to inhibit erythrocyte rosette formation. Human erythrocytes were tested for ABO and MN specificity and used as targets in a 51Chromium quantitative erythrocyte rosette assay. Monosaccharides present on human glycophorin A, neuraminyl lactoses, bovine and porcine submaxillary mucins (BSM and PSM), and hyaluronic acid as well as proteoglycan N-linked glycosidase F(PNGaseF)- and sialidase A-treated human erythrocyte glycoproteins (hEGP) and human erythrocytes were all tested for inhibitory potential in the rosetting assay. Porcine Kupffer-cell recognition of human erythrocytes was insensitive to differences in blood groups A, B, O, or MN. At 30 mM, the monosaccharide, N-acetylneuraminic acid, and the trisaccharide mixture, neuraminyl lactoses, disrupted human erythrocyte recognition by 25% and 30%, respectively. A dilution of BSM but not PSM inhibited the rosetting assay by 17% (.2 mg/mL), 33% (1 mg/mL), and 53% (2 mg/mL). The same dilution of hyaluronic acid had no effect on rosetting. Removal of N-linked oligosaccharides from hEGP with PNGaseF did not impair its ability to inhibit the rosetting assay. In contrast, removal of sialic acid completely abrogated its inhibitory ability. Treatment of whole human erythrocytes with sialidase A likewise prevented recognition by porcine Kupffer cells. Terminal sialic acid on human erythrocytes is a target recognized by porcine Kupffer cells, suggesting a role for a sialic-acid receptor in innate cellular recognition of xenogeneic epitopes. Inasmuch as this work reveals a carbohydrate-recognition mechanism for cellular rejection, we shed light on a potential new boundary that will need to be overcome within xenotransplantation.

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Glycosylation Pattern and Cell Attachment‐Inhibiting Property of Human Salivary Mucins

Whole human saliva (WHS) and its high molecular weight mucin constituent (Muc) inhibit fibroblast attachment and might influence periodontal and peri-implant wound healing. The aim of this work was to study the potential role of glycosylation of Muc in fibroblast attachment-inhibiting property and to examine in vitro the effect of WHS and Muc on epithelial cell attachment. Muc was isolated from WHS by CsCl density gradient ultracentrifugation; covalently immobilized on polystyrene; and subjected to enzymic digestion by N-glycanase, O-glycanase, and sialidase, or chemical desulfation and periodate treatment. Wells of tissue culture microtiter plates were incubated with WHS, Muc, or buffer as control; suspensions of normal human oral keratinocytes, spontaneously immortalized human keratinocytes, or human gingival fibroblasts were applied; and cell attachment determined using methylene blue assay. While enzymic cleavage of N-linked carbohydrates showed no effect, selective removal of O-linked residues and sialic acid as well as desulfation and periodate oxidation resulted in statistically significant reduction of cell attachment-inhibiting property of immobilized Muc. Significantly lower numbers of attached cells of each cell type were found in wells pretreated with WHS or Muc. Inhibition of cell attachment may be mediated by the carbohydrate residues suggesting specific interactions between the salivary constituent and the cell surface. Exposure of root and implant surfaces to saliva during early wound healing events might influence healing by inhibiting surface colonization by oral keratinocytes and fibroblasts, and enhancing wound repair in the form of long junctional epithelium rather than regeneration.

Influence of sialic acid removal on MUC1 antigenic reactivity in head and neck carcinoma.

To investigate the influence of sialic acid removal on MUC1 peptidic and carbohydrate epitope reactivity in head and neck squamous cell carcinoma (HNSCC), tumor samples belonging to 24 HNSCC patients were studied by standard immunohistochemistry (IHC) with and without desialylation with 0.1 U/ml neuraminidase. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and Western blotting (WB). Three monoclonal antibodies (MAbs) were used: C595 MAb directed to MUC1 protein core, an anti-Tn hapten MAb, and an anti-sTn hapten MAb; a comparative analysis between desialylated and sialylated samples was performed. By IHC without neuraminidase treatment, 19 of 24 samples reacted with anti-MUC1 peptidic epitope, while Tn hapten was not detected and sTn was found in 1 of 24 cases. Desialylation increased either the number of reacting cells or the intensity of the reaction with C595 and anti-Tn MAbs, and some negative samples became positive. On the other hand, sTn expression decreased with desialylation. By WB, several bands from >200 to 25 kDa were found; desialylation increased high-molecular-weight bands, diminishing the detection of low-molecular-weight ones. The use of desialylation is a suitable treatment that contributes to the exposure of MUC1-associated epitopes, which may be related to the spreading of HNSCC.

Babesia caballi and Babesia equi: Implications of host sialic acids in erythrocyte infection

The present study investigated the involvement of host sialic acids in the erythrocyte infection by two equine Babesia parasites, Babesia equi and Babesia caballi. We observed that the in vitro growth of both parasites is influenced by the removal of sialic acids from the surface of equine erythrocytes (RBC). When the parasites were cultured with neuraminidase (Nm, EC 3.2.1.18)-treated RBC, in which α2–3-linked sialic acid residues were removed from four membrane proteins of the RBC, B. caballi showed a significant inhibition of the erythrocyte invasion, while the intracellular development of B. equi seemed to be significantly affected. The possible involvement of host sialic acid in the erythrocyte invasion by B. caballi was also supported by a significant reduction in the parasite growth accompanied by an increased number of extracellular merozoites after the addition of exogenous 3′-sialyllactose (Neu5Acα(2–3)Galβ(1–4)Glc) into the culture. These results suggest that the α2–3-linked sialic acid residues on host RBC play important roles in the erythrocyte infections by B. caballi and B. equi.

High-Pressure-Induced Hemolysis in Papain-Digested Human Erythrocytes Is Suppressed by Cross-Linking of Band 3 via Anti-Band 3 Antibodies

Upon exposure of human erythrocytes to a high pressure of 200 mPa, both hemolysis and vesiculation occur. The hemolysis of erythrocytes at 200 mPa was enhanced by removal of sialic acids from the membrane surface with papain. However, such enhancement was suppressed by cross-linking of band 3 via an anti-band 3 antibody (AB3A), which recognizes the exofacial domain of band 3, or by clustering of band 3 via Zn2+. On the other hand, the size of high-pressure-induced vesicles increased from 423 to 525 nm in diameter upon exposure to papain of erythrocytes, but decreased to 444 nm with following treatment with AB3A. In these vesicles, the content of spectrin relative to band 3 was almost the same. Furthermore, the band 3-cytoskeleton interactions in erythrocyte membranes remained unaltered upon treatment with papain and AB3A. Flow cytometric analysis demonstrated that papain-pretreated erythrocytes mainly produce open ghosts at 200 mPa and that the production of such open ghosts is suppressed by AB3A. Thus, upon removal of negative charges from the membrane surface, open ghosts are readily produced due to the release of larger vesicles under pressure. Upon cross-linking of band 3 via AB3A, however, the release of smaller vesicles at 200 mPa is facilitated so that high-pressure-induced hemolysis is suppressed.

Sialic acid as a protective barrier against neointima development

Arterial sialic acid (SA) has been shown to attenuate the binding of fibrinogen and low-density lipoproteins (LDL) to the vessel wall, presumably protecting against atherosclerosis. This study was aimed to assess the effect of changes in SA content in intimal thickening, an early step in the development of atherosclerosis. New Zealand white rabbits were subjected to bilateral carotid periarterial collaring, followed by in situ-perfusion with neuroaminidase (random artery) and with vehicle (contralateral control artery). The efficiency of SA removal was evaluated in perfusates and arterial homogenates, and arterial tissue samples were obtained 7 and 14 days after the intervention to assess morphological changes. Neuraminidase significantly reduced SA by 16.7%. Arterial desialylation was associated with a significantly increased neointimal formation. Proliferation of smooth muscle cells (SMCs), assessed by incorporation of bromo-2′-deoxyuridine into replicating DNA was also significantly increased in desialylated arteries. In addition, immunohistochemical studies showed a slightly stronger oxidized-LDL (ox-LDL) immunostaining in neointima of desialylated arteries than in control vessels. A mild reduction of SA increases intimal thickening, at least partly due to an enhanced proliferation of SMCs, and may facilitate the accretion of atherogenic lipoproteins, providing evidence for the potential role of SA in the protection against neointimal development.

Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.

17 FLOW CYTOMETRY ANALYSIS OF THE TURKEY SPERM GLYCOCALYX: EFFECTS OF NEURAMINIDASE TREATMENT

The surface of all eucaryotic cells consists of a carbohydrate-rich zone known as the glycocalyx which mediates a variety of specific cell-recognition processes, including sperm-egg interactions. Several carbohydrate residues are known to be necessary for poultry sperm to traverse the female reproductive tract and for sperm-egg recognition. Alteration of the sperm glycocalyx during semen storage could adversely impact fertility. Accordingly, the aim of this work was to elucidate the sugar residues existing in the surface of turkey spermatozoa as a first step in understanding the deleterious effects of semen storage on sperm physiology and function. Nine turkey toms were randomly assigned to 1 of 3 groups (3 males/group) and semen was pooled for each group. Seminal plasma was removed and isolated sperm were resuspended in Tris-NaCl buffer at either (1) pH 7.4 for immediate staining with 1 of 16 FITC-conjugated lectins (100 �g/mL; 30 min; 25�C; 100 � 106 cells/mL), or (2) pH 6.0 for neuraminidase treatment (30 min; 37�C; 1 IU/109 cells), followed by washing (700 g, 5 min; 4 times) and lectin staining. The control consisted of sperm cells incubated only in Tris-NaCl buffer. Stained and control samples were washed twice (700 g, 5 min) and counterstained with 12 �M propidium iodide (PI); the green fluorescence intensity of PI-negative cells was assessed using a Coulter EPICS XL-MCL" Flow Cytometer (Beckman Coulter, Inc., Fullerton, CA, USA) and single-parameter histogram analysis (6 replicates). Median fluorescence intensity (MdFI) was used to assess differences in the presence of sugar residues among the control, non-neuraminidase-treated, and neuraminidase-treated samples (Kruskal-Wallis test; Tukey's HSD test in post-hoc comparisons). Lectin staining occurred in the PI-positive subpopulation, or membrane-disrupted cells, of all samples. By contrast, only intact cells were labeled by LPA, ConA, RCA-I, and s-WGA lectins in the non-neuraminidase-treated samples, indicating that only residues of sialic acid, �-mannose/�-glucose, �-galactose, and N-acetylglucosamine oligomers were on the surface. As expected, removal of sialic acid significantly (P &lt; 0.05) decreased the fluorescence intensity of LPA-labeled cells. This treatment significantly (P &lt; 0.05) increased the affinity of ConA, RCA-I, and s-WGA lectins, and exposed sugar residues previously undetected using ECA (N-acetyllactosamine), GNA (�-mannose), GS-I (�-galactose), GS-II (N-acetylglucosamine monomers), LOTUS (�-l-fucose), PNA (�-galactose), SBA (�-N-acetylgalactosamine), and WFA (�-N-acetylgalactosamine) lectins. Low levels of MdFI were observed for cells labeled by JAC (�-galactose), PSA (�-mannose/�-glucose), STA (N-acetylglucosamine polymers) and UEA-I (�-l-fucose) irrespective of neuraminidase treatment (P &gt; 0.05). These observations suggest that (1) the carbohydrate content of turkey sperm glycocalyx is extensively masked by sialic acid molecules, (2) it contains a diversity of sugar residues, and (3) discrimination between intact and damaged cells needs to be done in order to properly identify surface binding.

Decreased Dependence on Receptor Recognition for the Fusion Promotion Activity of L289A-Mutated Newcastle Disease Virus Fusion Protein Correlates with a Monoclonal Antibody-Detected Conformational Change

It has been shown that the L289A-mutated Newcastle disease virus (NDV) fusion (F) protein gains the ability to promote fusion of Cos-7 cells independent of the viral hemagglutinin-neuraminidase (HN) protein and exhibits a 50% enhancement in HN-dependent fusion over wild-type (wt) F protein. Here, we show that HN-independent fusion by L289A-F is not exhibited in BHK cells or in several other cell lines. However, similar to the results in Cos-7 cells, the mutated protein plus HN does promote 50 to 70% more fusion above wt levels in all of the cell lines tested. L289A-F protein exhibits the same specificity as the wt F protein for the homologous HN protein, as well as NDV-human parainfluenza virus 3 HN chimeras. The mutated F protein promotes fusion more effectively than the wt when it is coexpressed with either the chimeras or HN proteins deficient in receptor recognition activity. In addition, its fusogenic activity is significantly more resistant to removal of sialic acid on target cells. These findings are consistent with the demonstration that L289A-F interacts more efficiently with wt and mutated HN proteins than does wt F by a cell surface coimmunoprecipitation assay. Taken together, these findings indicate that L289A-F promotes fusion by a mechanism analogous to that of the wt protein with respect to the HN-F interaction but is less dependent on the attachment activity of HN. The phenotype of the mutated F protein correlates with a conformational change in the protein detectable by two different monoclonal antibodies. This conformational change may reflect a destabilization of F structure induced by the L289A substitution, which may in turn indicate a lower energy requirement for fusion activation.

Constitutive repressor activity of CD33 on human monocytes requires sialic acid recognition and phosphoinositide 3‐kinase‐mediated intracellular signaling

Human CD33 is a myeloid-restricted transmembrane protein of the sialic acid-binding Ig-like lectin (Siglec) family. While structural analysis predicts an inhibitory function, it remains unknown under which circumstances CD33 may operate as an inhibitory molecule. Here we show that treatment of human monocytes with anti-CD33 mAb induces the production of the proinflammatory cytokines IL-1 beta, TNF-alpha, and IL-8. However, decreased CD33 surface expression obtained by RNA interference using cognate small interfering RNA (siRNA) was specifically paralleled by spontaneous cytokine production. Similarly, sialic acid (CD33 ligand) removal from the monocyte surface by neuraminidase resulted in IL-1 beta up-regulation, while the addition of red blood cells or sialyllactosamine (but not lactosamine) reversed the effect of neuraminidase treatment, thus demonstrating the importance of ligand recognition by CD33 for repression of monocyte activation. Finally, inhibition of phosphoinositide 3-kinase (PI3K) dramatically enhanced the IL-1 beta response to anti-CD33 and neuraminidase, while inhibition of p38 mitogen-activated protein kinase (MAPK) abolished it. Simultaneous addition of both inhibitors resulted in low levels of IL-1 beta, suggesting that CD33 exerts an inhibitory role mediated by PI3K, while p38 MAPK signaling is required for IL-1 beta production. These data indicate that by controlling monocyte activation, CD33 is a key molecule in the inflammatory response, depending on the sialic acid microenvironment for its repressor activity.

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

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

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

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

Second sialic acid binding site in Newcastle disease virus hemagglutinin-neuraminidase: implications for fusion.

Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we present evidence for a second sialic acid binding site at the dimer interface of HN and present a model for its involvement in cell fusion. Three different crystal forms of NDV HN now reveal identical tetrameric arrangements of HN monomers, perhaps indicative of the tetramer association found on the viral surface.

Calcium binding to biliary mucins is dependent on sodium ion concentration: relevance to cystic fibrosis

Because hypersecretion of gallbladder (GB) mucus occurs in gallstone formation and because binding of Ca 2+ to biliary lipids only accounts for 50% of the total Ca 2+ in GB bile, we investigated the binding of Ca 2+ to human biliary mucin. Biliary mucin was purified from GB bile and binding to Ca 2+ studied. Scatchard plot analysis suggested two binding sites. Removal of sialic acid by neuraminidase resulted in 10% reduction of Ca 2+ binding, whereas, sulfatase treatment reduced Ca 2+ binding by 30%. Using a hypotonic NaCl solution, Ca 2+ binding to mucin increased curvilinearly with mucin concentration. However, binding decreased with increasing ionic strength of the NaCl solution. We conclude that binding of Ca 2+ to mucin is effected mainly through sulfate. Binding to Ca 2+ can be displaced by Na +. Ca 2+ binding to mucins is enhanced in the setting of low Na + concentrations. This phenomenon has pathophysiologic implications for the formation of thick mucus in cystic fibrosis epithelia.