Asparagine-linked sugar chains of plasma membrane glycoproteins from healthy and common variable immunodeficiency B lymphoblastoid cell lines

Biotherapy ◽  
1992 ◽  
Vol 4 (4) ◽  
pp. 285-288
Author(s):  
Naomi Kondo ◽  
Fumiaki Motoyoshi ◽  
Tadao Orii
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Common Variable Immunodeficiency ◽  
Lymphoblastoid Cell Lines ◽  
Membrane Glycoproteins ◽  
Sugar Chains ◽  
Common Variable

scholarly journals B lymphoblastoid cell lines with normal and defective O-glycosylation established from an individual with blood group Tn

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 973-979 ◽  
Author(s):  
CG Gahmberg ◽  
L Peltokorpi ◽  
LC Andersson
Keyword(s):  
Blood Group ◽  
Cell Lines ◽  
Helix Pomatia ◽  
Lymphoblastoid Cell Lines ◽  
Membrane Glycoproteins ◽  
Leukocyte Antigen ◽  
Galactosyl Transferase ◽  
The Common ◽  
Common Leukocyte Antigen

Abstract Individuals with the Tn blood group contain terminal serine/threonine- linked N-acetylgalactosamine residues in their blood cells. This is due to lack of UDP-D-galactose: D-N-acetyl galactosamine beta-D-galactosyl transferase from part of their red cells and probably from their leukocytes. We have established B lymphoblastoid cell lines from such an individual by in vitro infection of his lymphocytes with Epstein- Barr virus. The original line contained a mixture of cells reactive and nonreactive with Helix pomatia lectin (Hp). These cells were subcloned after staining with fluorescent Hp by a fluorescence-activated cell sorter (FACS) into homogeneous, phenotypically stable lines of Hp- positive (Hp+) and Hp-negative (Hp-) cells. The molecular differences between the membrane glycoproteins were characterized by carbohydrate- specific surface labeling techniques, Hp affinity chromatography, polyacrylamide slab gel electrophoresis and glycopeptide/oligosaccharide analysis. The major O-glycosidic membrane glycoprotein (GP105) was retained on Hp-Sepharose columns only from Hp+ cells, whereas the common leukocyte antigen (GP160–200) was partially retained on Hp columns from both lines. These proteins were isolated by immune precipitation with monoclonal antibodies and characterized. The results show that the GP105 glycoprotein from Hp+ cells contains terminal N-acetylgalactosamine residues but also more complex oligosaccharides. The common leukocyte antigen showed different electrophoretic mobilities in Hp+ and Hp- cells. UDP-galactose D-N- acetyl galactosamine beta-galactosyl transferase was almost absent in the Hp+ cells. These cell lines are useful for studies on the functional role and regulation of the biosynthesis of O-glycosidic carbohydrates.

scholarly journals B lymphoblastoid cell lines with normal and defective O-glycosylation established from an individual with blood group Tn

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 973-979
Author(s):  
CG Gahmberg ◽  
L Peltokorpi ◽  
LC Andersson
Keyword(s):  
Blood Group ◽  
Cell Lines ◽  
Helix Pomatia ◽  
Lymphoblastoid Cell Lines ◽  
Membrane Glycoproteins ◽  
Leukocyte Antigen ◽  
Galactosyl Transferase ◽  
The Common ◽  
Common Leukocyte Antigen

Individuals with the Tn blood group contain terminal serine/threonine- linked N-acetylgalactosamine residues in their blood cells. This is due to lack of UDP-D-galactose: D-N-acetyl galactosamine beta-D-galactosyl transferase from part of their red cells and probably from their leukocytes. We have established B lymphoblastoid cell lines from such an individual by in vitro infection of his lymphocytes with Epstein- Barr virus. The original line contained a mixture of cells reactive and nonreactive with Helix pomatia lectin (Hp). These cells were subcloned after staining with fluorescent Hp by a fluorescence-activated cell sorter (FACS) into homogeneous, phenotypically stable lines of Hp- positive (Hp+) and Hp-negative (Hp-) cells. The molecular differences between the membrane glycoproteins were characterized by carbohydrate- specific surface labeling techniques, Hp affinity chromatography, polyacrylamide slab gel electrophoresis and glycopeptide/oligosaccharide analysis. The major O-glycosidic membrane glycoprotein (GP105) was retained on Hp-Sepharose columns only from Hp+ cells, whereas the common leukocyte antigen (GP160–200) was partially retained on Hp columns from both lines. These proteins were isolated by immune precipitation with monoclonal antibodies and characterized. The results show that the GP105 glycoprotein from Hp+ cells contains terminal N-acetylgalactosamine residues but also more complex oligosaccharides. The common leukocyte antigen showed different electrophoretic mobilities in Hp+ and Hp- cells. UDP-galactose D-N- acetyl galactosamine beta-galactosyl transferase was almost absent in the Hp+ cells. These cell lines are useful for studies on the functional role and regulation of the biosynthesis of O-glycosidic carbohydrates.

scholarly journals Identification of a Subset of Common Variable Immunodeficiency Patients with Impaired B-Cell Protein Tyrosine Phosphorylation

1999 ◽  
Vol 6 (6) ◽  
pp. 856-860 ◽  
Author(s):  
Rivka Schwartz ◽  
Yael Ben-Anat Porat ◽  
Zeev Handzel ◽  
Zeev Sthoeger ◽  
Ben-Zion Garty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Common Variable Immunodeficiency ◽  
Cell Protein ◽  
Normal Donor ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Common Variable

ABSTRACT The mechanisms responsible for common variable immunodeficiency syndrome (CVID) are as yet unknown. In the present study, we show that the B-cell dysfunction in a subset of CVID patients is caused by defective protein tyrosine phosphorylation (PTP). We demonstrated that the PTP level and immunoglobulin (Ig) secretion malfunctions can be successfully repaired when normal plasma membrane components are implanted into these patients’ B cells. Stimulation of CVID patients’ peripheral blood mononucleated cells with anti-Ig antibody revealed that 7 of 11 patients had lower PTP levels than those found in the normal donor cells. Plasma membrane implantation to the cells of these patients resulted in elevated PTP levels which reached normal levels upon stimulation with anti-human Ig antibody. The results revealed two distinct groups of CVID patients. The first group included patients whose B cells expressed low PTP levels after Ig stimulation. In these patients the plasma membrane implantation restored the normal PTP level as well as the ability to secrete IgM and/or IgG after B-cell stimulation. In the second group, patients whose B cells expressed a normal PTP level after Ig stimulation, with no restoration of their ability to secrete Ig upon plasma membrane implantation and lipopolysaccharide stimulation. We conclude that the first group has an early signal transduction defect located in the B-cell plasma membrane, while in the second group the defect is located elsewhere.

The Asparagine-Linked Sugar Chains of Plasma Membrane Glycoproteins of K-562 Human Leukaemic Cells: A Comparative Study with Human Erythrocytes1

1982 ◽  
Vol 91 (1) ◽  
pp. 233-246 ◽  
Author(s):  
Hideo YOSHIMA ◽  
Nobuyuki SHIRAISHI ◽  
Akira MATSUMOTO ◽  
Sakan MAEDA ◽  
Taketoshi SUGIYAMA ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Comparative Study ◽  
Membrane Glycoproteins ◽  
Leukaemic Cells ◽  
Sugar Chains

scholarly journals Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins.

1989 ◽  
Vol 108 (2) ◽  
pp. 339-353 ◽  
Author(s):  
J Hearing ◽  
E Hunter ◽  
L Rodgers ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Membrane Glycoproteins

A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.

PRODUCTION OF HUMAN MONOCLONAL ANTIBODIESSPECIFIC FOR PLATELET MEMBRANE GLYCOPROTEIN IIIa

1987 ◽  
Author(s):  
Kenichi Furihata ◽  
Diane J Nugent ◽  
Amy L Bissonette ◽  
Elizabeth Vokac ◽  
Thomas J Kunicki
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Peripheral Blood Lymphocytes ◽  
Platelet Membrane ◽  
Lymphoblastoid Cell Lines ◽  
Membrane Glycoproteins ◽  
Human Monoclonal Antibodies ◽  
Microtiter Plates ◽  
Culture Supernatants ◽  
Limiting Dilution

Human monoclonal antibodies specific for platelet membrane glycoproteins (GPs) arepotentially important reagentsfor studies of the immunogenicity of membrane glycoproteins. A human monoclonalautoantibody, 5E5, reactive with plateletGPIIIa has been developed (Nugent, et al.,Blood, 1987, in press). In this report, we describe the production of additional human monoclonal antibodies specific for GPIIIa. Peripheral blood lymphocytes fromone patient with post-transfusion purpur(PTP) and one woman who had delivered an infant with neonatal alloimmune thrombocytopenic purpura (NATP) were used as a source of antigen-specific lymphocytes. A B-lympho-cyte-enriched population was transformed with Epstein Barr virus, strain B95-8, and cultured in microtiter plates. After two weeks, culture supernatants were screened by an antigen-capture ELISA wherein murine monoclonal antibody specificfor the GPIIb-IIIa complex was used to holdcorresponding antigen from a lysate ofnormal platelets. B-lymphoblastoid cell lines producing IgG and/or IgM antibodies were expanded and either cloned by limiting dilution technqiue or hybridized with a HAT-sensitive, ouabain-resistant heterohybrid fusion line, F6, using polyethylene glycol. Hybridomas were selected in medium containing HAT andouabain. After twoweeks, hybridomas producing anti-GPIIb and/or anti-GPIIIa antibody were cloned by limiting dilution. Culture supernatants from cloned B-lymphoblastoid cell lines and cloned hybridomas were rescreened by ELISA wherein affinity-purified GPIIIa or other platelet GP weredirectly conjugatedto microtiter plates. One IgM antibody produced by acloned B-lymphoblastoid cellline (CH16) andtwo IgG antibodies produced bycloned hybridomas(Del5.19 and Del5.23) were shownto react with GPIIIa but not other GP. Further characterization of these human monoclonal antibodies, produced continuouslyin culture now for four months, is in progress.

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