scholarly journals EVIDENCE FOR CELL-SURFACE GLYCOSYLTRANSFERASES

1971 ◽  
Vol 51 (2) ◽  
pp. 536-547 ◽  
Author(s):  
Stephen Roth ◽  
Edward J. McGuire ◽  
Saul Roseman
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Extracellular Fluid ◽  
Neural Retina ◽  
Uridine Diphosphate ◽  
Intact Cells ◽  
Cellular Recognition ◽  
Uridine Diphosphate Galactose ◽  
Lines Of Evidence

Intact chicken embryo neural retina cells have been shown to catalyze the transfer of galactose-14C from uridine diphosphate galactose (UDP-galactose) to endogenous acceptors of high molecular weight as well as to exogenous acceptors. Four lines of evidence indicate that the galactosyltransferases catalyzing these reactions are at least partly located on the outside surface of the plasma membrane: (a) there is no evidence for appreciable uptake of sugar-nucleotides by vertebrate cells nor did unlabeled galactose, galactose 1-phosphate, or UDP-glucose interfere with the radioactivity incorporated during the reaction; (b) the cells remained essentially intact during the course of the reaction; (c) there was insufficient galactosyltransferase activity in the cell supernatants to account for the incorporation of galactose-14C into cell pellets; and (d) the intact cells could transfer galactose to acceptors of 106 daltons, and the product of this reaction was in the extracellular fluid. Appropriate galactosyl acceptors interfered with the adhesive specificity of neural retina cells; other compounds, which were not acceptors, had no effect. These results suggested that the transferase-acceptor complex may play a role in cellular recognition.

scholarly journals Quantification of ganglioside GM1 synthetase activity on intact chick neural retinal cells.

1982 ◽  
Vol 93 (1) ◽  
pp. 76-81 ◽  
Author(s):  
M Pierce
Keyword(s):  
Cell Surface ◽  
Neural Retina ◽  
Synthetase Activity ◽  
Chick Embryos ◽  
Uridine Diphosphate ◽  
Ganglioside Gm1 ◽  
Intact Cells ◽  
Retinal Cells ◽  
Dorsoventral Axis ◽  
Specific Activities

Neural retinal cells from 9-d-old chick embryos were assayed for uridine diphosphate (UDP)-galactose:ganglioside GM2 galactosyltransferase, or GM1 synthetase, activity using the oligosaccharide fragment of GM2, oligo-GM2, oligo-GM2, as the exogenous acceptor. The results demonstrated that this enzyme activity was present on the external surfaces of intact cells. Little difference between the specific activities of cell surface GM1 synthetase could be detected when cells derived from dorsal and ventral segments of the neural retina were compared. These results suggested that this cell-surface enzyme was not present in a concentration gradient along the dorsoventral axis of the neural retina.

Polylactosaminoglycan modification of a small integral membrane glycoprotein, influenza B virus NB

1988 ◽  
Vol 8 (3) ◽  
pp. 1186-1196
Author(s):  
M A Williams ◽  
R A Lamb
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Gel Filtration ◽  
Cell Types ◽  
Influenza B Virus ◽  
Influenza B ◽  
Membrane Glycoprotein ◽  
Intact Cells ◽  
B Virus ◽  
Carbohydrate Chains

The structure of the carbohydrate components of NB, the small integral membrane glycoprotein of influenza B virus, was investigated. The carbohydrate chains of NB are processed from the high-mannose form (NB18) to a heterogeneous form of much higher molecular weight, designated NBp. Selection of this carbohydrate-containing form of NB with Datura stramonium lectin, its susceptibility to digestion by endo-beta-galactosidase, and determination of the size of NBp glycopeptides by gel filtration chromatography suggested that the increase in molecular weight is due to processing to polylactosaminoglycan. Investigation of the polypeptides produced by influenza B/Lee/40 virus infection of several cell types and another strain of influenza B virus suggested that the signal for modification to polylactosaminoglycan is contained in NB. Expression of mutants of NB lacking either one or both of the normal N-terminal sites of asparagine-linked glycosylation indicated that both carbohydrate chains are modified to contain polylactosaminoglycan. NBp and a small amount of unprocessed NB18 are expressed at the infected-cell surface, as determined by digestion of the surfaces of intact cells with various endoglycosidases. Unglycosylated NB, expressed either in influenza B virus-infected cells treated with tunicamycin or in cells expressing the NB mutant lacking both N-linked glycosylation sites, was expressed at the cell surface, indicating that NB does not require carbohydrate addition for transport.

Assembly of Active High Molecular Weight CD39 Complex: Role of Proteolysis and Endocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2110-2110
Author(s):  
Kim Olson ◽  
Aaron j Marcus
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Enzymatic Activity ◽  
Sucrose Gradient ◽  
Early Endosome ◽  
Full Length ◽  
Membrane Cholesterol ◽  
293 Cells ◽  
Proportional Decrease

Abstract Abstract 2110 HUVECs, neutrophils, monocyte/macrophages and T cell subsets all express ectonucleoside triphosphate diphosphohydrolase 1 (CD39) on the cell surface. In addition, they all express both P2X and P2Y receptors and dose dependently respond to ATP. ATP enhances superoxide production in activated neutrophils and supports chemotaxis of macrophages responding to a chemoattractant. ATP also induces apoptosis of anti-inflammatory T regulatory cells and supports the differentiation of pro-inflammatory Th17 cells. Lastly, ADP drives thrombus formation by activation of platelet P2Y12 receptors. An increase in the activity of expressed CD39 would result in increased metabolism of these pro-thrombotic and pro-inflammatory nucleotides. We examined the reported relationship between CD39 cleavage and cell surface enzymatic activity. We cloned N-terminal and C-terminal V5 and VP16 tagged CD39 into eukaryotic expression vectors as well as lentiviral genomic vectors for analysis of CD39 expression in 293 cells and HUVECs. Western blots of membrane fractions prepared from HUVECs and transiently and stably transfected 293 cells identified the previously described CD39 cleavage fragments. Streptavidin precipitation of biotinylated membrane proteins demonstrated that both fragments are present on the cell surface of both transduced HUVECs and transfected 293 cells. We next separated membranes on a discontinuous sucrose gradient to yield ER, Golgi/plasma membrane, early endosome and late endosome enriched membrane fractions. Surprisingly, full length CD39 and the N- and C-terminal fragments appeared in both the Golgi/plasma membrane and early endosome fractions. In addition, the early endosome CD39 exhibited enzyme activity equal to that of the Golgi/plasma membrane CD39. We then prepared membranes from cells treated with either chloroquine or bafilomycin, reagents known to interfere with endosomal acidification and/or maturation. In each case, we observed a decrease in the fractional cleavage of full length CD39 and a proportional decrease in associated enzymatic activity. When sonicated membranes were resolved on a continuous sucrose gradient, the N- and C-terminal fragments and a fraction of full length CD39 as well as maximal enzymatic activity were found in the low density, “raft” fractions. These results suggest that the formation of an enzymatically active CD39 complex requires N- and C-terminal CD39 fragments as well as membrane cholesterol. When 293 cells that stably expressed CD39 were transiently transfected with dominant negative dynamin 2, we observed a decrease in fractional cleavage as well as a proportional decrease in enzymatic activity. This result suggested that the cleavage event occurs following endocytosis of plasma membrane expressed CD39. Finally, we treated both stably transfected 293 cells and HUVECs with the cell permeable cysteine protease inhibitor zLLY.fmk. Prepared membranes analyzed by Western blot showed a decrease in fractional cleavage of full length CD39. Apyrase assays showed a corresponding decrease in ATPase and ADPase activity. We then examined the cholesterol dependence of CD39 activity by depleting membrane cholesterol with MβCD. As expected, ATPase activity decreased in a dose dependent manner. The predominant “active” species appeared as a 1.4 megadalton complex on a 3–12% BN gel of Digitonin solubilized membranes prepared from cultured cells treated with the cleavable cross-linker DTSSP. As a result of membrane cholesterol depletion, there was a proportional decrease in the amount of full length CD39 and N- and C-terminal fragments present in the DTSSP cross-linked HMW complex. Interestingly, there was a marked increase in the abundance of lower molecular weight complexes in cholesterol depleted cells. In conclusion, we provide evidence that CD39 enzymatic activity resides in a megadalton complex formed by protein-protein interactions between full length CD39 and C- and N-terminal fragments generated by cleavage of the full length molecule. Assembly of the oligomeric protein complex requires membrane cholesterol and likely occurs on intracellular membranes. Moreover, approximately 50% of the active enzyme complex remains sequestered on intracellular membranes. These results suggest that up-regulation of CD39 metabolism of pro-thrombotic and pro-inflammatory nucleotides involves pathways independent of gene transcription. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes.

1993 ◽  
Vol 177 (5) ◽  
pp. 1287-1298 ◽  
Author(s):  
U Frevert ◽  
P Sinnis ◽  
C Cerami ◽  
W Shreffler ◽  
B Takacs ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Target Cell ◽  
Surface Membrane ◽  
Heparan Sulfate Proteoglycans ◽  
Circumsporozoite Protein ◽  
Cell Specificity ◽  
Region Ii

During feeding by infected mosquitoes, malaria sporozoites are injected into the host's bloodstream and enter hepatocytes within minutes. The remarkable target cell specificity of this parasite may be explained by the presence of receptors for the region II-plus of the circumsporozoite protein (CS) on the basolateral domain of the plasma membrane of hepatocytes. We have now identified these receptors as heparan sulfate proteoglycans (HSPG). The binding of CS to the receptors is abolished by heparitinase treatment, indicating that the recognition of region II-plus is via the glycosaminoglycan chains. We have purified and partially characterized the CS-binding HSPGs from HepG2 cells. They have a molecular weight of 400,000-700,000, are tightly associated with the plasma membrane, and are released from the cell surface by very mild trypsinization, a property which the CS receptors share with the syndecan family of proteoglycans.

scholarly journals Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium

1989 ◽  
Vol 259 (3) ◽  
pp. 645-650 ◽  
Author(s):  
C W Taylor ◽  
M J Berridge ◽  
A M Cooke ◽  
B V L Potter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Calcium ◽  
Racemic Mixture ◽  
Synthetic Analogue ◽  
Intact Cells ◽  
Surface Receptors ◽  
Prolonged Stimulation ◽  
Crude Preparation ◽  
Intracellular Messenger

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.

scholarly journals Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules

2018 ◽  
Vol 115 (40) ◽  
pp. E9353-E9361 ◽  
Author(s):  
F. Tudor Ilca ◽  
Andreas Neerincx ◽  
Mark R. Wills ◽  
Maike de la Roche ◽  
Louise H. Boyle
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Receptor ◽  
Target Cells ◽  
Intact Cells ◽  
Mhc I ◽  
Immunogenic Peptides ◽  
Presentation Pathway ◽  
Peptide Exchange

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future.

scholarly journals Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

1991 ◽  
Vol 112 (1) ◽  
pp. 39-54 ◽  
Author(s):  
S G Miller ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Secretory Granules ◽  
Intact Cells ◽  
Transport Reaction ◽  
Permeabilized Cells ◽  
Transmembrane Glycoprotein ◽  
Transport Vesicles ◽  
Constitutive Secretion

Regulated exocytosis in many permeabilized cells can be triggered by calcium and nonhydrolyzable GTP analogues. Here we examine the role of these effectors in exocytosis of constitutive vesicles using a system that reconstitutes transport between the trans-Golgi region and the plasma membrane. Transport is assayed by two independent methods: the movement of a transmembrane glycoprotein (vesicular stomatitis virus glycoprotein [VSV G protein]) to the cell surface; and the release of a soluble marker, sulfated glycosaminoglycan (GAG) chains, that have been synthesized and radiolabeled in the trans-Golgi. The plasma membrane of CHO cells was selectively perforated with the bacterial cytolysin streptolysin-O. These perforated cells allow exchange of ions and cytosolic proteins but retain intracellular organelles and transport vesicles. Incubation of the semi-intact cells with ATP and a cytosolic fraction results in transport of VSV G protein and GAG chains to the cell surface. The transport reaction is temperature dependent, requires hydrolyzable ATP, and is inhibited by N-ethylmaleimide. Nonhydrolyzable GTP analogs such as GTP gamma S, which stimulate the fusion of regulated secretory granules, completely abolish constitutive secretion. The rate and extent of constitutive transport between the trans-Golgi and the plasma membrane is independent of free Ca2+ concentrations. This is in marked contrast to fusion of regulated secretory granules with the plasma membrane, and transport between the ER and the cis-Golgi (Beckers, C. J. M., and W. E. Balch. 1989. J. Cell Biol. 108:1245-1256; Baker, D., L. Wuestehube, R. Schekman, and D. Botstein. 1990. Proc. Natl. Acad. Sci. USA. 87:355-359).

scholarly journals Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase

1988 ◽  
Vol 254 (3) ◽  
pp. 765-771 ◽  
Author(s):  
D Allan ◽  
P Quinn
Keyword(s):  
Staphylococcus Aureus ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Fluid Phase ◽  
Intact Cells ◽  
Prolonged Incubation ◽  
Bhk Cells ◽  
Streptolysin O ◽  
Incubation Experiments ◽  
Original Cell

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.

scholarly journals N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins

2014 ◽  
Vol 306 (10) ◽  
pp. C943-C960 ◽  
Author(s):  
Jing Li ◽  
Fan Xia ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Complex Form ◽  
Cell Surface Expression ◽  
Intact Cells ◽  
Hek 293 ◽  
Permeabilized Cells ◽  
Glycosylation Sites ◽  
High Mannose

The human solute carrier ( SLC26) family of anion transporters consists of 10 members ( SLCA1–11, SLCA10 being a pseudogene) that encode membrane proteins containing ∼12 transmembrane (TM) segments with putative N-glycosylation sites (-NXS/T-) in extracellular loops and a COOH-terminal cytosolic STAS domain. All 10 members of the human SLC26 family, FLAG-tagged at the NH2 terminus, were transiently expressed in HEK-293 cells. While most proteins were observed to contain both high-mannose and complex oligosaccharides, SLC26A2 was mainly in the complex form, SLC26A4 in the high-mannose form, and SLC26A8 was not N-glycosylated. Mutation of the putative N-glycosylation sites showed that most members contain multiple N-glycosylation sites in the second extracytosolic (EC) loop, except SLC26A11, which was N-glycosylated in EC loop 4. Immunofluorescence staining of permeabilized cells localized the proteins to the plasma membrane and the endoplasmic reticulum, with SLC26A2 highly localized to the plasma membrane. N-glycosylation was not a necessary requirement for cell surface expression as the localization of nonglycosylated proteins was similar to their wild-type counterparts, although a lower level of cell-surface biotinylation was observed. No immunostaining of intact cells was observed for any SLC26 members, demonstrating that the NH2-terminal FLAG tag was located in the cytosol. Topological models of the SLC26 proteins that contain an even number of transmembrane segments with both the NH2 and COOH termini located in the cytosol and utilized N-glycosylation sites defining the positions of two EC loops are presented.

scholarly journals Polylactosaminoglycan modification of a small integral membrane glycoprotein, influenza B virus NB.

1988 ◽  
Vol 8 (3) ◽  
pp. 1186-1196 ◽  
Author(s):  
M A Williams ◽  
R A Lamb
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Gel Filtration ◽  
Cell Types ◽  
Influenza B Virus ◽  
Influenza B ◽  
Membrane Glycoprotein ◽  
Intact Cells ◽  
B Virus ◽  
Carbohydrate Chains

The structure of the carbohydrate components of NB, the small integral membrane glycoprotein of influenza B virus, was investigated. The carbohydrate chains of NB are processed from the high-mannose form (NB18) to a heterogeneous form of much higher molecular weight, designated NBp. Selection of this carbohydrate-containing form of NB with Datura stramonium lectin, its susceptibility to digestion by endo-beta-galactosidase, and determination of the size of NBp glycopeptides by gel filtration chromatography suggested that the increase in molecular weight is due to processing to polylactosaminoglycan. Investigation of the polypeptides produced by influenza B/Lee/40 virus infection of several cell types and another strain of influenza B virus suggested that the signal for modification to polylactosaminoglycan is contained in NB. Expression of mutants of NB lacking either one or both of the normal N-terminal sites of asparagine-linked glycosylation indicated that both carbohydrate chains are modified to contain polylactosaminoglycan. NBp and a small amount of unprocessed NB18 are expressed at the infected-cell surface, as determined by digestion of the surfaces of intact cells with various endoglycosidases. Unglycosylated NB, expressed either in influenza B virus-infected cells treated with tunicamycin or in cells expressing the NB mutant lacking both N-linked glycosylation sites, was expressed at the cell surface, indicating that NB does not require carbohydrate addition for transport.

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