B lymphoblastoid cell lines with normal and defective O-glycosylation established from an individual with blood group Tn.
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.
D001402:B-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D001789:Blood Group Antigens; D001881:Borates; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005692:Galactose Oxidase; D005700:Galactosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D037102:Lectins; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000302:isolation & purification; D017350:N-Acetylgalactosaminyltransferases; D009844:Oligosaccharides / Q000097:blood*; D010504:Periodic Acid; D010641:Phenotype; D011233:Precipitin Tests
D001789:Blood Group Antigens; D001881:Borates; D006023:Glycoproteins; C038095:Helix lectin; D037102:Lectins; D008565:Membrane Proteins; D009844:Oligosaccharides; D010504:Periodic Acid; C010634:sodium borate; C009288:metaperiodate; D005692:Galactose Oxidase; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase