Removal Of Surface Sialic Acid Research Articles

Synovial Fibroblast Sialylation Regulates Cell Migration and Activation of Inflammatory Pathways in Arthritogenesis.

Synovial fibroblasts have emerged as critical underlying factors to perpetuate chronic joint inflammation in Rheumatoid Arthritis. Like any other cell, synovial fibroblasts are covered with a complex layer of glycans that can change in response to extracellular signals, such as inflammation. We have previously shown that inflammatory synovial fibroblasts show decreased levels of sialic acid, but our understanding of sialic acid-dependent pathophysiological pathways in these stromal cells is still very limited. In this report, we used in vivo and in vitro studies with exogenous sialidases and RNA sequencing to investigate the responses of murine synovial fibroblasts upon desialylation. Our results show that hyposialylated fibroblasts present a dysregulated migratory ability and an activated phenotype characterized by the expression of inflammatory mediators, such as cytokines and chemokines, and anti-viral related mechanisms. Removal of surface sialic acid also affected the expression of sialyltransferases, revealing the existence of a positive feedback to sustain reduced sialylation. Moreover, we demonstrate that synovial fibroblasts subsets have distinct sialyltransferase expression profiles, both in healthy and arthritic mice. These findings underline the ability of sialic acid to modulate homeostatic and inflammatory responses in non-immune synovial fibroblasts, suggesting that sialylation plays a key role in perpetuating local inflammation in the arthritic joint.

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

Various PfRH5 polymorphisms can support Plasmodium falciparum invasion into the erythrocytes of owl monkeys and rats

Aotus nancymaae, the owl monkey, provides a useful laboratory model for research to develop drugs and vaccines against human falciparum malaria; however, many Plasmodium falciparum parasites are unable to invade A. nancymaae erythrocytes, rendering the parasites noninfective to the monkeys. In previous work, we identified a key polymorphism that determined the inheritance of erythrocyte invasion in a genetic cross of two P. falciparum clones that were virulent (GB4) or noninfective (7G8) to A. nancymaae. This polymorphism, an isoleucine-to-lysine polymorphism at position 204 (I204K) of the GB4 erythrocyte binding protein PfRH5, was nevertheless not found in several other P. falciparum lines that could also invade A. nancymaae erythrocytes. Alternative PfRH5 polymorphisms occur at different positions in these virulent parasites, and additional polymorphisms are found in P. falciparum parasites that cannot infect A. nancymaae. By allelic replacement methods, we have introduced the polymorphisms of these A. nancymaae-virulent or noninfective parasites at codons 204, 347, 358, 362, 410, and 429 of the endogenous PfRH5 gene in the noninfective 7G8 line. 7G8 transformants expressing the polymorphisms of the A. nancymaae-virulent parasites show neuraminidase-sensitive (sialic acid-dependent) invasion into the monkey erythrocytes, whereas 7G8 transformants expressing the PfRH5 alleles of noninfective parasites show little or no invasion of these erythrocytes. Parasites harboring PfRH5 polymorphisms 204K or 204R are also able to invade rat erythrocytes and are differentially sensitive to the removal of surface sialic acids by neuraminidase. These studies offer insights into the PfRH5 receptor-binding domain and interactions that support the invasion of various primate and rodent erythrocytes by P. falciparum.

Significance of glycosylation patterns of rat Zajdela ascitic hepatocellular carcinoma: Effect of enzymatic removal of surface sialic acid on humoral response

Rat Zajdela ascitic hepatocellular carcinoma (ZAH) is a malignant cell type with some properties in common with rat hepatocytes. The aim of this study was to determine the glycosylation patterns of surface glycoproteins of two cell lines C and D of ZAH by lectin binding for identification and characterization of tumor-specific markers. Plasma membrane proteins from these cells were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and probed using radio-iodinated lectins. We observed a decrease in the binding of concavalin A, and an increase in the binding of wheat germ agglutinin (WGA) to glycoproteins from the tumor cell lines as compared with those from normal liver cells. We showed that the increase in binding of WGA was mainly due to increased sialylation of the surface glycoproteins of the tumor cells. The major sialylated glycoproteins of the tumor cells contained O-linked carbohydrate chains. It was also shown that removal of surface sialic acid by neuraminidase significantly decreased the lethality of the tumor and led to increased survival of tumor-bearing animals. The decreased lethality of the tumor appears to be due to increased antigenicity of the desialylated tumor cells. Taken together, the presence of highly sialylated O-linked glycosylation of gp120 in ZAH tumor cells and its absence in normal liver cells is of significance with respect to the biological properties of this tumor.

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.

Expression of Thomsen-Friedenreich (TF) antigens on lymphocytes: II. Loss of cryptic TF antigens during mitogenic activation of human T and B lymphocytes

Human peripheral blood lymphocytes (HPBL) were examined for the presence of cryptic Thomsen-Friedenreich (TF) antigens as detected by PNA or an anti-TF mAb (49H8) after neuraminidase treatment of the cell surface. Neither PNA nor the mAb bound to the cells before treatment with neuraminidase. After removal of surface sialic acid, all lymphocytes were PNA-reactive, and 85% of HPBL reacted with the mAb 49H8. Seventy-seven percent of nylon wool (NW)-eluted T cells (96% Leu 1 +), 80% of enriched helper T cells (83% Leu 3a +), and 78% of suppressor/cytotoxic T cells (63% Leu 2a +) carried the cryptic TF determinant recognized by the mAb 49H8. Ninety-one percent of NW-adherent cells (68% Leu 10 +, 5% Leu 1 +) were also TF positive. In contrast to NW-eluted T cells which showed low to moderate mAb 49H8 binding, 48% of NW-adherent cells revealed strong binding of anti-TF mAb. With progressive activation of T cells by PHA, binding of mAb to the cryptic TF antigen completely disappeared on blast cells. The presence of TF antigens on small cells in the culture was only poorly affected. The same was observed for activation of B cells with PWM. On the other hand, binding sites for PNA did not change during blastogenesis. The disappearance of the particular, mAb 49H8-reactive TF antigen on T blast cells is not due to the loss of antigen in a distinct T cell subset, but occurs to an equal extent in the helper and suppressor/cytotoxic T cell subpopulations. Thus, the majority of peripheral T and B lymphocytes carries cryptic TF antigens. Activated T or B cell blasts, on the other hand, are deficient for the particular TF antigen detected by the mAb 49H8. We interpret these data as a modulation of certain TF antigens on effector cells in the course of lymphocyte activation.

The sensitivity of placental trophoblast cells to intraplacental and allogeneic cytotoxic lymphocytes.

The NK-susceptibility of trophoblast cells to allogeneic and autologous intraplacental natural killer (NK), antibody-dependent (K), and mitogen-induced cell-mediated cytotoxicity was studied, using untreated and neuraminidase-treated trophoblast cells from normal, full-term deliveries. The work was preceded by systematic studies of placental cell separation and labelling techniques, and the effects of these techniques on the NK target, K562. The results indicated that maternal NK cells are present among intraplacental lymphocytes, but that their activity is lower than that of peripheral blood lymphocytes and they are not stimulated by interferon to the same extent as peripheral blood lymphocytes (PBL). Trophoblast cells were rarely susceptible to allogeneic NK cells, with low cytotoxicity at high effector-target cell ratios in only two of five experiments. Interferon (IF)-boosted NK cells mediated some cytolysis of trophoblasts in three of four experiments, but high effector/target cell ratios were also required for the effect to be observed. The trophoblast cells could be lysed, however, by K cells and lectin-induced cytotoxicity. Removal of surface sialic acid by neuraminidase treatment of the trophoblast cells had little effect on the susceptibility of these cells to unstimulated NK cells (one of four experiments), but resulted in susceptibility to IF-boosted NK cells in four of four experiments. Normal trophoblast cells did not compete in IF-NK(K562) assays and neuraminidase-treated cells competed weakly in only one of three such experiments, indicating that the NK "target structure" is only weakly expressed on human trophoblast cells. Intraplacental lymphocytes lysed autologous trophoblast cells to a lower extent than allogeneic PBL.(ABSTRACT TRUNCATED AT 250 WORDS)