Cell Surface Sialoglycopeptide Metabolism and Surface Glycosyl Transferase Activity in the Ehrlich Ascites Cell

1975 ◽  
Vol 53 (8) ◽  
pp. 895-902 ◽  
Author(s):  
D. Irwin ◽  
T. P. Anastassiades
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Transferase Activity ◽  
Intact Cells ◽  
Neuraminidase Treatment ◽  
Glycosyl Transferase ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cell ◽  
Almost All

1. The in vivo incorporation of radioactivity from [3H]glucosamine into a trypsin labile, cell surface sialoglycopeptide fraction (SGP) of Ehrlich ascites cells was studied in the presence and absence of puromycin pretreatment. The results indicated a much more complete inhibition of incorporation into the surface SGP than in the average intracellular acid insoluble glycoproteins. No evidence of turnover of the carbohydrate portion of the surface SGP independent of protein synthesis could be obtained.2. However, when intact cells were incubated with labelled uridine 5′-diphosphate – N-acetyl glucosamine or cytidine 5′-monophosphate (CMP) – sialic acid there was some incorporation largely into acid insoluble material, suggesting the presence of glycosyl transferase activity at the surface. Further evidence for surface activity was obtained when neuraminidase pretreatment of intact cells stimulated incorporation of labelled CMP – sialic acid sixfold and almost all of the incorporated counts could be released by subsequent neuraminidase treatment. Furthermore, a much greater proportion of the incorporated counts could be released by papain than by trypsin treatment of the intact cells. These results suggest that the surface acceptor for exogenously added CMP – sialic acid is not identical to the endogenously synthesized trypsin labile surface SGP.

scholarly journals Sialic acid-Dependent Binding and Viral Entry of SARS-CoV-2

2021 ◽  
Author(s):  
Linh Nguyen ◽  
Kelli McCord ◽  
Duong Bui ◽  
Kim Bouwman ◽  
Elena Kitova ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Receptor Binding ◽  
Viral Entry ◽  
Receptor Binding Domain ◽  
Artificial Membrane ◽  
Neuraminidase Treatment ◽  
S Protein ◽  
Knock Out

Abstract Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral co-receptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Electrophoretic and Chemical Characterization of the Charged Groups at the Surface of Murine CL3 Ascites Leukaemia Cells

1969 ◽  
Vol 4 (2) ◽  
pp. 289-298
Author(s):  
P. D. WARD ◽  
E. J. AMBROSE
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Chemical Characterization ◽  
Surface Protein ◽  
Subsequent Treatment ◽  
Surface Proteins ◽  
Carboxyl Groups ◽  
Neuraminidase Treatment ◽  
Ionic Nature ◽  
Ph Range

The electrophoretic characteristics of the murine CL3 ascites tumour were investigated. Treatment of the cells with formaldehyde raised the electrophoretic mobility (E.P.M.) from - 1.06 to - 1.28 µ/sec/V/cm; subsequent treatment with diazomethane reduced their mobility to zero. The E.P.M. of the diazomethane-treated cells did not alter over the pH range 3.0-8.0. This proved that the only ionic groups at this cell surface were amino and carboxyl groups. The absence of phosphate groups, another possibility, was confirmed by the lack of calcium-ion binding from 10 mM Ca2+ solutions. Neuraminidase treatment reduced the E.P.M. from -1.06 to -0.55 µ/sec/V/cm and free sialic acid was identified in the enzyme supernatant. Subsequent treatment of the cells with formaldehyde raised the mobility to -1.22 µ/sec/V/cm indicating that the change in E.P.M. on neuraminidase treatment was not due solely to the removal of the carboxyl groups of sialic acid but also to a change in the ionic nature of the surface. This change is ascribed to a change in the conformation of the surface protein. The reason for this change and a suggestion for the possible role of sialic acid at the cell surface are mentioned. Treatment of the cells with trypsin did not affect the viable cells in any way, suggesting that the surface proteins lack the basic amino acids lysine and arginine. Pronase treatment served only to show that much of the sialic acid was bound to protein; the total amount was not determined.

scholarly journals Metastatic potential severely altered by changes in tumor cell adhesiveness and cell-surface sialylation.

1983 ◽  
Vol 157 (1) ◽  
pp. 371-376 ◽  
Author(s):  
M Fogel ◽  
P Altevogt ◽  
V Schirrmacher
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Tumor Cell ◽  
Metastatic Potential ◽  
Lectin Receptors ◽  
Lectin Receptor ◽  
Receptor Sites ◽  
Variant Line

A plastic adherent variant line (ESb-M) of a highly invasive and metastatic murine T cell lymphoma (ESb) was found to have lost its metastatic potential while still being tumorigenic in normal syngeneic hosts. The variant retained most of its ESb-derived antigenic and biochemical characteristics but differed at binding sites for certain lectins with specificity for terminal N-acetylgalactosamine residues. Whereas such sites were masked by sialic acid on metastatic ESb cells, they became unmasked on the adherent variant line. Metastatic revertants of ESb-M cells did not express the respective lectin receptor sites because these were again masked by sialic acid. It is suggested that the masking of specific lectin receptors sites on the tumor cell surface is of crucial importance for metastatis. If freely exposed, these sites may change adherence characteristics of the cells possibly not only in vitro (to plastic) but also in vivo.

scholarly journals Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

1996 ◽  
Vol 134 (4) ◽  
pp. 1089-1096 ◽  
Author(s):  
A R Günthert ◽  
J Sträter ◽  
U von Reyher ◽  
C Henne ◽  
S Joos ◽  
...  
Keyword(s):  
Cell Surface ◽  
Colon Carcinoma ◽  
Substantial Reduction ◽  
Carcinoma Cells ◽  
Surface Adhesion ◽  
Intact Cells ◽  
Surface Receptors ◽  
Colon Carcinoma Cells ◽  
Soluble Cd44

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

scholarly journals Characterization of a Glycosyl Transferase Inactivating Macrolides, Encoded by gimA from Streptomyces ambofaciens

1998 ◽  
Vol 42 (10) ◽  
pp. 2612-2619 ◽  
Author(s):  
Anne Gourmelen ◽  
Marie-Hélène Blondelet-Rouault ◽  
Jean-Luc Pernodet
Keyword(s):  
Gene Replacement ◽  
In Vivo Studies ◽  
Transferase Activity ◽  
Susceptible Host ◽  
Reading Frame ◽  
Glycosyl Transferase ◽  
Production Studies ◽  
Cell Extracts ◽  
Streptomyces Ambofaciens

ABSTRACT In Streptomyces ambofaciens, the producer of the macrolide antibiotic spiramycin, an open reading frame (ORF) was found downstream of srmA, a gene conferring resistance to spiramycin. The deduced product of this ORF had high degrees of similarity to Streptomyces lividans glycosyl transferase, which inactivates macrolides, and this ORF was called gimA. The cloned gimA gene was expressed in a susceptible host mutant of S. lividans devoid of any background macrolide-inactivating glycosyl transferase activity. In the presence of UDP-glucose, cell extracts from this strain could inactivate various macrolides by glycosylation. Spiramycin was not inactivated but forocidin, a spiramycin precursor, was modified. In vivo studies showed that gimA could confer low levels of resistance to some macrolides. The spectrum of this resistance differs from the one conferred by a rRNA monomethylase, such as SrmA. InS. ambofaciens, gimA was inactivated by gene replacement, without any deleterious effect on the survival of the strain, even under spiramycin-producing conditions. But the overexpression of gimA led to a marked decrease in spiramycin production. Studies with extracts from wild-type and gimA-null mutant strains revealed the existence of another macrolide-inactivating glycosyl transferase activity with a different substrate specificity. This activity might compensate for the effect of gimA inactivation.

scholarly journals Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein

2017 ◽  
Vol 114 (40) ◽  
pp. E8508-E8517 ◽  
Author(s):  
Wentao Li ◽  
Ruben J. G. Hulswit ◽  
Ivy Widjaja ◽  
V. Stalin Raj ◽  
Ryan McBride ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Middle East ◽  
Cell Surface ◽  
Human Erythrocytes ◽  
Middle East Respiratory Syndrome ◽  
Host Cells ◽  
Dromedary Camel ◽  
Differential Distribution ◽  
Neuraminidase Treatment ◽  
Intact Virus

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D. Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B. We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A. When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus–Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.

scholarly journals Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae.

1979 ◽  
Vol 82 (3) ◽  
pp. 708-714 ◽  
Author(s):  
P P De Bruyn ◽  
S Michelson
Keyword(s):  
Endothelial Cell ◽  
Sialic Acid ◽  
Cell Surface ◽  
Random Distribution ◽  
Colloidal Iron ◽  
Neuraminidase Treatment ◽  
Endothelial Cell Surface ◽  
Anionic Charge ◽  
Sinusoidal Endothelium ◽  
Ph Range

Diaphragmed fenestrae (DF) are sites of increased vascular permeability. The anionic charge distribution at the luminal aspect of the DF of the endothelium of the bone marrow vessels has been studied after aldehyde fixation by means of colloidal iron (CI), native ferritin (NF), and polycationic ferritin (PCF). At pH 1.8, these cationic agents are bound by the nonmodified luminal endothelial cell surface but not at the sites of the DF. PCF was used over a pH range of 1.8--7.2 (CI is unstable at higher pH levels, whereas NF which has a pI of 4.5 is anionic above this point). PCF shows increased binding at the DF from pH 3.5 upwards. PCF binding at pH 1.8 at the nonmodified luminal cell surface is significantly diminished by neuraminidase treatment which, however, does not perceptibly reduce PCF binding at the higher pH levels. It is concluded that there are exposed sialic acid groups at the lunimal cell surface which are absent or significantly fewer at the sites of the DF, whereas other anionic materials possibly with a pKa higher than that of sialic acid (pKa 2.6) are present both at the DF and at the nonmodified endothelial cell surface.

A thin hydrogel barrier linked onto cell surface sialic acids through covalent bonds induces cancer cell death in vivo

2020 ◽  
Vol 8 (2) ◽  
pp. 577-585 ◽  
Author(s):  
Kimika Ono ◽  
Yuka Sanada ◽  
Yuka Kimura ◽  
Seika Aoyama ◽  
Natsumi Ueda ◽  
...  
Keyword(s):  
Cell Death ◽  
Sialic Acid ◽  
Cell Surface ◽  
Cancer Cell ◽  
Sialic Acids ◽  
Cell Surfaces ◽  
Covalent Bonds ◽  
Novel Technique ◽  
Cancer Cell Death

A novel technique to form a thin hydrogel barrier on aberrantly expressed sialic acid residues on cancer cell surfaces was developed.

scholarly journals Reovirus Binding to Cell Surface Sialic Acid Potentiates Virus-Induced Apoptosis

2001 ◽  
Vol 75 (9) ◽  
pp. 4029-4039 ◽  
Author(s):  
Jodi L. Connolly ◽  
Erik S. Barton ◽  
Terence S. Dermody
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Cultured Cells ◽  
Cell Types ◽  
Biochemical Properties ◽  
Viral Protein Synthesis ◽  
Efficient Induction ◽  
Critical Requirement ◽  
Induced Apoptosis

ABSTRACT Reovirus induces apoptosis in cultured cells and in vivo. Genetic studies indicate that the efficiency with which reovirus strains induce apoptosis is determined by the viral S1 gene, which encodes attachment protein ς1. However, the biochemical properties of ς1 that influence apoptosis induction are unknown. To determine whether the capacity of ς1 to bind cell surface sialic acid determines the magnitude of the apoptotic response, we used isogenic reovirus mutants that differ in the capacity to engage sialic acid. We found that T3SA+, a virus capable of binding sialic acid, induces high levels of apoptosis in both HeLa cells and L cells. In contrast, non-sialic-acid-binding strain T3SA− induces little or no apoptosis in these cell types. Differences in the capacity of T3SA− and T3SA+ to induce apoptosis are not due to differences in viral protein synthesis or production of viral progeny. Removal of cell surface sialic acid with neuraminidase abolishes the capacity of T3SA+ to induce apoptosis. Similarly, incubation of T3SA+ with sialyllactose, a trisaccharide comprised of lactose and sialic acid, blocks apoptosis. These findings demonstrate that reovirus binding to cell surface sialic acid is a critical requirement for the efficient induction of apoptosis and suggest that virus receptor utilization plays an important role in regulating cell death.

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