Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor

2001 ◽  
Vol 354 (2) ◽  
pp. 331-336
Author(s):  
Sandrine SIFFROI-FERNANDEZ ◽  
Sabine COSTAGLIOLA ◽  
Sophie PAUMEL ◽  
Annie GIRAUD ◽  
J. Paul BANGA ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Molecular Mass ◽  
Cell Line ◽  
Ovary Cell ◽  
Thyrotropin Receptor ◽  
Complex Type ◽  
C Peptide ◽  
A Subunit

To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.

An analysis of Con A-mediated agglutination in a Chinese hamster ovary subclone which responds morphologically to growth in dibutyryl cyclic AMP. III. The role of microvilli in the agglutination process

1978 ◽  
Vol 34 (1) ◽  
pp. 103-115
Author(s):  
K.D. Noonan ◽  
T. Ukena
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Dibutyryl Cyclic Amp ◽  
Con A ◽  
A Cell ◽  
Ovary Cell Line

We have used the H-7w subclone of a Chinese hamster ovary cell line (K1) to investigate the role of cell surface architecture (specifically microvilli, blebs, and sheets) in determining the relative agglutinability of a cell line with Con A. Our evidence clearly demonstrates that no specific, immediately recognizable surface architecture is associated with the agglutinable or non-agglutinable phenotype. Our data suggest that the expression of microvilli on the cell surface is neither necessary to nor sufficient for the phenotype described by enhanced agglutinability with Con A. Furthermore our work demonstrates that cells covered with blebs are as agglutinable as cells covered with microvilli thereby suggesting that the intertwining of microvilli may not be an essential facet of the agglutination phenomenon.

A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components

1993 ◽  
Vol 104 (2) ◽  
pp. 391-398
Author(s):  
A. Koutoulis ◽  
M. Ludwig ◽  
R. Wetherbee
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Endomembrane System ◽  
Thin Sections ◽  
Specific Location ◽  
Whole Cells

Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.

Immune-Mediated Hemolytic Anemia

Hematology ◽  
2004 ◽  
Vol 2004 (1) ◽  
pp. 48-62 ◽  
Author(s):  
Wendell F. Rosse ◽  
Peter Hillmen ◽  
Alan D. Schreiber
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Hemolytic Anemia ◽  
Complement Activation ◽  
Autoimmune Hemolytic Anemia ◽  
Clinical Manifestations ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Remarkable Effect

Abstract Hemolytic anemia due to immune function is one of the major causes of acquired hemolytic anemia. In recent years, as more is known about the immune system, these entities have become better understood and their treatment improved. In this section, we will discuss three areas in which this progress has been apparent. In Section I, Dr. Peter Hillmen outlines the recent findings in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), relating the biochemical defect (the lack of glycosylphosphatidylinositol [GPI]-linked proteins on the cell surface) to the clinical manifestations, particularly hemolysis (and its effects) and thrombosis. He discusses the pathogenesis of the disorder in the face of marrow dysfunction insofar as it is known. His major emphasis is on innovative therapies that are designed to decrease the effectiveness of complement activation, since the lack of cellular modulation of this system is the primary cause of the pathology of the disease. He recounts his considerable experience with a humanized monoclonal antibody against C5, which has a remarkable effect in controlling the manifestations of the disease. Other means of controlling the action of complement include replacing the missing modulatory proteins on the cell surface; these studies are not as developed as the former agent. In Section II, Dr. Alan Schreiber describes the biochemistry, genetics, and function of the Fcγ receptors and their role in the pathobiology of autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura due to IgG antibodies. He outlines the complex varieties of these molecules, showing how they vary in genetic origin and in function. These variations can be related to three-dimensional topography, which is known in some detail. Liganding IgG results in the transduction of a signal through the tyrosine-based activation motif and Syk signaling. The role of these receptors in the pathogenesis of hematological diseases due to IgG antibodies is outlined and the potential of therapy of these diseases by regulation of these receptors is discussed. In Section III, Dr. Wendell Rosse discusses the forms of autoimmune hemolytic anemia characterized by antibodies that react preferentially in the cold–cold agglutinin disease and paroxysmal cold hemoglobinuria (PCH). The former is due to IgM antibodies with a common but particular structure that reacts primarily with carbohydrate or carbohydrate-containing antigens, an interaction that is diminished at body temperature. PCH is a less common but probably underdiagnosed illness due to an IgG antibody reacting with a carbohydrate antigen; improved techniques for the diagnosis of PCH are described. Therapy for the two disorders differs somewhat because of the differences in isotype of the antibody. Since the hemolysis in both is primarily due to complement activation, the potential role of its control, as by the monoclonal antibody described by Dr. Hillmen, is discussed.

scholarly journals A New Marker on Chicken Hematopoietic Cells is Defined by a Monoclonal Antibody Raised Against a VßChain of the Human TCR

1992 ◽  
Vol 2 (4) ◽  
pp. 273-284
Author(s):  
Anne-Sophie Lacoste-Eleaume ◽  
Christian Bleux ◽  
Catherine Corbel ◽  
Dominique Carriere ◽  
Philippe Poncelet ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
T Cell ◽  
Cell Surface ◽  
Cell Line ◽  
T Cell Receptor ◽  
Hematopoietic Cells ◽  
Apparent Molecular Weight ◽  
Cell Receptor ◽  
Cell Lysates

In this paper, we show that a mouse monoclonal antibody, 111-427, specific for the Vß5.3 chain of the human T-cell receptor (TCR) for antigen, also reacts with chicken hematopoietic cells. Our data indicate that the majority of 111-427 positive cells among peripheral blood leucocytes (PBL) are thrombocytes. This antibody also recognizes two in vitro cell lines, III-C5, an IL-2-dependent T-cell-line and HD11, a macrophage cell line. In addition, erythrocytes and a minor subpopulation of thymus and spleen cells are also stained by the monoclonal antibody (mAb). No specific immunoprecipitation could be detected from125I radiolabeled cell lysates. By Western blotting techniques, the 111- 427 mAb identifies a single band of apparent molecular weight 91 kD, unaffected by reduction, from III-C5 and HD11 cell lysates. This band is absent in negative cell control lysates. On thrombocytes, the apparent molecular weight of the band is shifted to 87 kD. These results indicate that the mAb does not recognize the chicken T-cell receptor for antigen, but a cell surface marker shared primarily between thrombocytes and erythrocytes. This new chicken cell marker is compared to other cell surface markers in avian or mammalian species that present some analogies in their tissue distribution.

Biosynthesis of N-Acetylneuraminic Acid in Cells Lacking UDP-N-Acetylglucosamine 2-Epimerase/ N-Acetylmannosamine Kinase

2001 ◽  
Vol 382 (2) ◽  
pp. 291-297 ◽  
Author(s):  
Stephan Hinderlich ◽  
Markus Berger ◽  
Oliver T. Keppler ◽  
Michael Pawlita ◽  
Werner Reutter
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Kinase Activity ◽  
Gel Filtration ◽  
Acetylneuraminic Acid ◽  
Biological Recognition ◽  
Functionally Impaired ◽  
Cell Surface Glycoconjugates

Abstract The first two steps in mammalian biosynthesis of Nacetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase. A subclone of the human B lymphoma cell line BJAB K20, lacking UDPNacetylglucosamine 2- epimerase/Nacetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains Nacetylmannosamine kinase activity. A gel filtration analysis of BJAB K88 control cells, which express UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase, revealed two Nacetylmannosamine kinase activity peaks, one coeluting with UDPNacetylglucosamine 2-epimerase activity and one coeluting with Nacetylglucosamine kinase. For this enzyme previous studies already showed ManNAc kinase activity in vitro. In contrast, the hyposialylated BJAB K20 subclone displayed only the Nacetylmannosamine kinase peak, comigrating with Nacetylglucosamine kinase. The CMPNacetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supple menting the medium with Nacetylmannosamine. This Nacetylmannosamineinduced increase was drastically reduced by cosupplementation with Nacetylglucosamine only in K20 cells. We therefore propose the phosphorylation of Nacetylmannosamine as a hitherto unrecognized role of Nacetylglucosamine kinase in living cells.

scholarly journals A cell-based assay for the detection of neutralizing antibodies against alemtuzumab

BioTechniques ◽  
2020 ◽  
Vol 68 (4) ◽  
pp. 185-190 ◽  
Author(s):  
Liaqat Ali ◽  
Gauri Saxena ◽  
Meleri Jones ◽  
Georgia R Leisegang ◽  
Luke Gammon ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Monoclonal Antibody ◽  
Cell Surface ◽  
Cell Line ◽  
Neutralizing Antibodies ◽  
Proof Of Concept ◽  
Alexa Fluor ◽  
Concept Study ◽  
A Cell ◽  
Relapsing Multiple Sclerosis

Aim: The humanized anti-CD52 monoclonal antibody alemtuzumab depletes lymphocytes and is currently used to treat relapsing multiple sclerosis. During treatment, anti-alemtuzumab antibodies may develop and reduce effective lymphocyte depletion in future treatment cycles. Results: Alemtuzumab–Alexa Fluor 488 conjugate binding to the CHO-CD52 cell surface was inhibited by anti-alemtuzumab antibodies. Conclusion: In this proof-of-concept study, a CHO-CD52 cell line has been developed and used to detect the presence of anti-alemtuzumab neutralizing antibodies. This platform provides the basis of an assay for routine screening of serum for neutralizing antibodies from patients treated with alemtuzumab.

scholarly journals The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule.

1982 ◽  
Vol 93 (2) ◽  
pp. 269-277 ◽  
Author(s):  
D A Herzlinger ◽  
T G Easton ◽  
G K Ojakian
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Cell Line ◽  
Surface Antigen ◽  
Mdck Cells ◽  
Distal Tubule ◽  
Cell Surface Antigen ◽  
Thick Ascending Limb ◽  
A Cell

Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues.

scholarly journals Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

2008 ◽  
Vol 154 (3) ◽  
pp. 305-315 ◽  
Author(s):  
Y. Mizutori ◽  
Y. Nagayama ◽  
D. Flower ◽  
A. Misharin ◽  
H. A. Aliesky ◽  
...  
Keyword(s):  
T Cell ◽  
Regulatory T Cell ◽  
Cell Depletion ◽  
Thyrotropin Receptor ◽  
T Cell Depletion ◽  
A Subunit
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