Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones.
To aid in studies of human glycoinositol-phospholipid (GPI) anchor pathway biochemistry in normal and affected paroxysmal nocturnal hemoglobinuria cells, GPI anchor-defective human K562 cell lines were derived by negative fluorescent sorting of anti-decay-accelerating factor (DAF) monoclonal antibody-stained cells either following or in the absence of ethylmethylsulfonate pretreatment. The resulting cloned cells showed deficiencies of both DAF and GPI-anchored CD59, some (designated group A) exhibiting total absence and some (designated group B) exhibiting approximately 10% levels of surface expression of the two proteins. In heterologous cell fusions, group A clones complemented defective Thy-1 expression by class A, B, C, E, and I Thy-1-negative lymphoma lines, but not H or D lines, the latter of which is defective in the Thy-1 structural gene. In contrast, group B clones complemented all previously described GPI anchor pathway-defective lymphoma classes. Immunoradiomatic assays of cells and supernatants and 35S biosynthetic labeling showed that group A cells degraded DAF protein while group B cells secreted it but failed to attach a GPI anchor structure. [3H]Man labeling of intact cells and UDP-[3H]GlcNAc and GDP-[3H]Man labeling of broken cell preparations demonstrated that group A cells failed to synthesize GlcNAc- and GlcN-PI (GPI-A and -B) as well as more polar mannolipids, whereas group B cells showed accumulation of GlcNAc-PI with approximately 10-fold diminished levels of GlcN-PI and more polar mannolipids. The failed assembly of GlcNAc-PI in group A cells and the reduced conversion of this intermediate to GlcN-PI in group B cells indicates that the former harbors a defect in UDP-GlcNAc transferase or in assembly of its PI acceptor, while the latter harbors a defect in GlcN-PI deacetylase activity.
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
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Hirose
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Mohney
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Mutka
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Ravi
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Singleton
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Perry
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Tartakoff
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Medof
D000911:Antibodies, Monoclonal; D018958:CD55 Antigens; D002459:Cell Fusion; D002460:Cell Line; D002999:Clone Cells; D005434:Flow Cytometry; D005816:Genetic Complementation Test; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008223:Lymphoma; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology; D016296:Mutagenesis; D010716:Phosphatidylinositols / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018958:CD55 Antigens; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D013462:Sulfur Radioisotopes; D014316:Tritium; D006155:Guanosine Diphosphate Mannose; D014537:Uridine Diphosphate N-Acetylglucosamine; D008358:Mannose