scholarly journals The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum.

1994 ◽  
Vol 127 (2) ◽  
pp. 333-341 ◽  
Author(s):  
J Vidugiriene ◽  
A K Menon
Keyword(s):  
Cell Surface ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Proteinase K ◽  
Con A ◽  
Membrane Bilayer ◽  
Gpi Anchor ◽  
Assembly Pathway ◽  
Cytoplasmic Face ◽  
External Face

Glycosylphosphatidylinositol (GPI) membrane protein anchors are synthesized from sugar nucleotides and phospholipids in the ER and transferred to newly synthesized proteins destined for the cell surface. The topology of GPI synthesis in the ER was investigated using sealed trypanosome microsomes and the membrane-impermeant probes phosphatidylinositol-specific phospholipase C, Con A, and proteinase K. All the GPI biosynthetic intermediates examined were found to be located on the external face of the microsomal vesicles suggesting that the principal steps of GPI assembly occur in the cytoplasmic leaflet of the ER. Protease protection experiments showed that newly GPI-modified trypanosome variant surface glycoprotein was primarily oriented towards the ER lumen, consistent with eventual expression at the cell surface. The unusual topographical arrangement of the GPI assembly pathway suggests that a biosynthetic intermediate, possibly the phosphoethanolamine-containing anchor precursor, must be translocated across the ER membrane bilayer in the process of constructing a GPI anchor.

scholarly journals Identification of the major lectin-binding surface proteins of human neutrophils and alveolar macrophages

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1624-1632
Author(s):  
NP Christiansen ◽  
KM Skubitz
Keyword(s):  
Cell Surface ◽  
Alveolar Macrophages ◽  
Cell Function ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Lectin Affinity Chromatography ◽  
Con A ◽  
Major Surface

Concanavalin A (Con A) and wheat germ agglutinin (WGA) are frequently used as stimuli of neutrophils and macrophages. While the effects of these lectins on cell function are presumably mediated by interaction with cell-surface molecules, the target structures on the cell surface involved are not well defined. We have used the techniques of lactoperoxidase catalyzed cell-surface iodination, lectin affinity chromatography, monoclonal antibody immunoprecipitation, and NaDodSO4- polyacrylamide gel electrophoresis to study the surface proteins of human neutrophils and alveolar macrophages that react with six lectins including Con A and WGA. We found that several major surface-labeled proteins of neutrophils bound Con A. Four of these proteins were identified by immunoprecipitation as members of the LFA-1/HMac- 1/gp150,95 adhesion glycoprotein family. Con A also bound CR1 and a 135- kd surface-labeled protein recognized by CD15 monoclonal antibodies. WGA also bound many of these proteins, but had a much lower avidity for CR1. All three of the major surface-labeled proteins of human alveolar macrophages bound to Con A, including the 183-kd mannose receptor and the 30-kd smoking-associated protein. WGA also bound the 183-kd macrophage protein, but not the 30-kd protein. These results should aid the understanding of studies using these lectins as stimuli.

scholarly journals Migrating endothelial cells are distinctly hyperglycosylated and express specific migration-associated cell surface glycoproteins.

1992 ◽  
Vol 119 (2) ◽  
pp. 483-491 ◽  
Author(s):  
H G Augustin-Voss ◽  
B U Pauli
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Surface Glycoprotein ◽  
Endothelial Cell Migration ◽  
Con A ◽  
Neuraminidase Treatment ◽  
Cell Surface Glycoprotein ◽  
Cell Surface Glycoproteins ◽  
Surface Glycoproteins

Migration of endothelial cells is one of the first cellular responses in the cascade of events that leads to re-endothelialization of an injured vessel and neovascularization of growing tissues and tumors. To examine the hypothesis that endothelial cells express a specific migration-associated phenotype, we analyzed the cell surface glycoprotein expression of migrating bovine aortic endothelial cell (BAECs). Light microscopic analysis revealed an upregulation of binding sites for the lectins Concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin after neuraminidase treatment (N-PNA) on migrating endothelial cells relative to contact-inhibited cells. These findings were confirmed and quantitated with an enzyme-linked lectin assay (ELLA) of circularly scraped BAEC monolayers. The expression of migration-associated cell surface glycoproteins was also analyzed by SDS-PAGE. The overall expression of cell surface glycoproteins was upregulated on migrating BAECs. Migrating BAECs expressed Con A- and WGA-binding glycoproteins with apparent molecular masses of 25 and 48 kD that were not expressed by contact-inhibited BAEC monolayers and, accordingly, disappeared as circularly scraped monolayers reached confluence. Subconfluent BAEC monolayers expressed the same cell surface glycoconjugate pattern as migrating endothelial cells. FACS analysis of circularly scraped BAEC monolayers showed that the phenotypic changes of cell surface glycoprotein expression after release from growth arrest occurred before the recruitment of the cells into the cell cycle (3 vs. 12 h). Suramin, which inhibits endothelial cell migration, abrogated the expression of the migration-associated phenotype and induced the expression of a prominent 28-kD Con A- and WGA-binding cell surface glycoprotein. These results indicate that endothelial cells express a specific migration-associated phenotype, which is characterized by the upregulation of distinct cellular glycoconjugates and the expression of specific migration-associated cell surface glycoproteins.

scholarly journals Host Complement Regulatory Protein CD59 Is Transported to the Chlamydial Inclusion by a Golgi Apparatus-Independent Pathway

2009 ◽  
Vol 77 (4) ◽  
pp. 1285-1292 ◽  
Author(s):  
Ayako Hasegawa ◽  
L. Farah Sogo ◽  
Ming Tan ◽  
Christine Sütterlin
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Regulatory Protein ◽  
Cytoplasmic Inclusion ◽  
Surface Proteins ◽  
Host Protein ◽  
Membrane Microdomains ◽  
Gpi Anchor ◽  
Inclusion Membrane ◽  
Chlamydial Inclusion

ABSTRACT Chlamydia is an obligate intracellular bacterium that grows and replicates inside a cytoplasmic inclusion. We report that a host protein, CD59, which regulates complement function at the surfaces of uninfected cells, can be detected at the membrane of the chlamydial inclusion. This localization to the inclusion membrane was specific for CD59 and not a general feature of other glycosylphosphatidylinositol (GPI)-anchored proteins or representative cell surface proteins. Using differential permeabilization studies, we showed that CD59 is localized to the luminal but not the cytoplasmic face of the inclusion membrane, consistent with membrane association via its GPI anchor. Furthermore, CD59 was present at the inclusion even when we prevented it from associating with membrane microdomains via the GPI anchor or when we inhibited general protein transport to the cell surface, indicating that a conventional Golgi apparatus-dependent trafficking mechanism was not involved. Based on these findings, we propose that selected host proteins are trafficked to the inclusion by a Golgi apparatus-independent pathway during a Chlamydia infection.

scholarly journals Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes

2011 ◽  
Vol 10 (7) ◽  
pp. 916-931 ◽  
Author(s):  
Ka Fai Leung ◽  
Fay S. Riley ◽  
Mark Carrington ◽  
Mark C. Field
Keyword(s):  
Cell Surface ◽  
Protein Turnover ◽  
Full Length ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
General Mechanism ◽  
Membrane Domain ◽  
Reporter Protein ◽  
Invariant Surface ◽  
Content Type

ABSTRACTThe cell surface ofTrypanosoma bruceiis dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, buttrans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic andtrans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulatingtrans-membrane domain surface proteins in trypanosomes.

scholarly journals Identification of the major lectin-binding surface proteins of human neutrophils and alveolar macrophages

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1624-1632 ◽  
Author(s):  
NP Christiansen ◽  
KM Skubitz
Keyword(s):  
Cell Surface ◽  
Alveolar Macrophages ◽  
Cell Function ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Lectin Affinity Chromatography ◽  
Con A ◽  
Major Surface

Abstract Concanavalin A (Con A) and wheat germ agglutinin (WGA) are frequently used as stimuli of neutrophils and macrophages. While the effects of these lectins on cell function are presumably mediated by interaction with cell-surface molecules, the target structures on the cell surface involved are not well defined. We have used the techniques of lactoperoxidase catalyzed cell-surface iodination, lectin affinity chromatography, monoclonal antibody immunoprecipitation, and NaDodSO4- polyacrylamide gel electrophoresis to study the surface proteins of human neutrophils and alveolar macrophages that react with six lectins including Con A and WGA. We found that several major surface-labeled proteins of neutrophils bound Con A. Four of these proteins were identified by immunoprecipitation as members of the LFA-1/HMac- 1/gp150,95 adhesion glycoprotein family. Con A also bound CR1 and a 135- kd surface-labeled protein recognized by CD15 monoclonal antibodies. WGA also bound many of these proteins, but had a much lower avidity for CR1. All three of the major surface-labeled proteins of human alveolar macrophages bound to Con A, including the 183-kd mannose receptor and the 30-kd smoking-associated protein. WGA also bound the 183-kd macrophage protein, but not the 30-kd protein. These results should aid the understanding of studies using these lectins as stimuli.

scholarly journals Lymphocyte mechanical response triggered by cross-linking surface receptors.

1985 ◽  
Vol 100 (3) ◽  
pp. 860-872 ◽  
Author(s):  
C Pasternak ◽  
E L Elson
Keyword(s):  
Cell Surface ◽  
Mechanical Response ◽  
Calcium Ionophore ◽  
Lateral Diffusion ◽  
Surface Proteins ◽  
Cell Stiffness ◽  
Cross Linking ◽  
Con A ◽  
Cell Surface Proteins ◽  
Hypertonic Medium

Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (sIg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of sIg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when sIg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was approximately 0.15 mdyn/micron; for cells treated with antibodies against sIg or with Con A the stiffness increased to approximately 0.6 or 0.4 mdyn/micron, respectively. The stiffness decreased after completion of the capping of sIg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against sIg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface.

Bloodstream form trypanosome plasma membrane proteins: antigenic variation and invariant antigens

Parasitology ◽  
2010 ◽  
Vol 137 (14) ◽  
pp. 2029-2039 ◽  
Author(s):  
ANGELA SCHWEDE ◽  
MARK CARRINGTON
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Adaptive Immune System ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Invariant Surface ◽  
Adaptive Immune ◽  
External Face

SUMMARYTrypanosoma bruceiis exposed to the adaptive immune system and complement in the blood of its mammalian hosts. The aim of this review is to analyse the role and regulation of the proteins present on the external face of the plasma membrane in the long-term persistence of an infection and transmission. In particular, the following are addressed: (1) antigenic variation of the variant surface glycoprotein (VSG), (2) the formation of an effective VSG barrier shielding invariant surface proteins, and (3) the rapid uptake of VSG antibody complexes combined with degradation of the immunoglobulin and recycling of the VSG.

scholarly journals Factors affecting the ability of glycosylphosphatidylinositol-specific phospholipase D to degrade the membrane anchors of cell surface proteins

1991 ◽  
Vol 279 (2) ◽  
pp. 483-493 ◽  
Author(s):  
M G Low ◽  
K S Huang
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Phospholipase D ◽  
Bovine Serum ◽  
Cell Membranes ◽  
Surface Proteins ◽  
Phospholipid Bilayer ◽  
Placental Alkaline Phosphatase ◽  
Gpi Anchor ◽  
Cell Surface Proteins

Mammalian serum and plasma contain high levels of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). Previous studies with crude serum or partially purified GPI-PLD have shown that this enzyme is capable of degrading the GPI anchor of several purified detergent-solubilized cell surface proteins yet is unable to act on GPI-anchored proteins located in intact cells. Treatment of intact ROS17/2.8, WISH or HeLa cells (or membrane fractions prepared from them) with GPI-PLD purified from bovine serum by immunoaffinity chromatography gave no detectable release of alkaline phosphatase into the medium. However, when membranes were treated with GPI-PLD in the presence of 0.1% Nonidet P-40 substantial GPI anchor degradation (as measured by Triton X-114 phase separation) was observed. The mechanism of this stimulatory effect of detergent was further investigated using [3H]myristate-labelled variant surface glycoprotein and human placental alkaline phosphatase reconstituted into phospholipid vesicles. As with the cell membranes the reconstituted substrates exhibited marked resistance to the action of purified GPI-PLD which could be overcome by the inclusion of Nonidet P-40. Similar results were obtained when crude bovine serum was used as the source of GPI-PLD. These data indicate that the resistance of cell membranes to the action of GPI-PLD is not entirely due to the action of serum or membrane-associated inhibitory factors. A more likely explanation is that, in common with many other eukaryotic phospholipases, the action of GPI-PLD is restricted by the physical state of the phospholipid bilayer in which the substrates are embedded. These data may account for the ability of endothelial and blood cells to retain GPI-anchored proteins on their surfaces in spite of the high levels of GPI-PLD present in plasma.

A novel method for measuring protein expression at the cell surface

1993 ◽  
Vol 106 (4) ◽  
pp. 1201-1209
Author(s):  
W. Stoorvogel ◽  
V. Oorschot ◽  
B. Neve
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Surface Proteins ◽  
Proteinase K ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Quantitative Measurements ◽  
Dab Cytochemistry

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3′-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of laminin and beta-1 integrin in embryonic, neonatal, and adult rat heart

1991 ◽  
Vol 49 ◽  
pp. 182-183
Author(s):  
R.L. Price ◽  
T.K. Borg ◽  
L. Terracio ◽  
M. Nakagawa
Keyword(s):  
Temporal Expression ◽  
Qualitative And Quantitative ◽  
Adult Rat ◽  
Cytoplasmic Face ◽  
Extracellular Matrix Components ◽  
Basement Membrane Component ◽  
Beta 1 Integrin ◽  
Quantitative Changes ◽  
Cytoskeletal Elements ◽  
External Face

Little is known about the temporal expression of extracellular matrix components (ECM) and its receptors during development of the heart. Recent reports have shown that ECM components undergo both qualitative and quantitative changes during development, and it is believed that ECM components are important in the regulation of cell migration and cell:cell and cell:ECM recognition and adhesion.Integrins are transmembrane glycoproteins which bind several ECM components on their external face and cytoskeletal elements on the cytoplasmic face. Laminin is a basement membrane component which has been recognized as an important site for cell adhesion. Both the integrins and laminin are expressed early in development and continue to be expressed in the adult heart. With their documented roles in cell recognition, and cell:cell and cell:ECM migration and adhesion these proteins appear to be important components in development of the heart, and their temporal expression may play a pivotal role in morphogenesis and myofibrillogenesis of the heart.

Sign in / Sign up

Export Citation Format

Share Document