Quinine-dependent antibodies to neutrophils react with a 60-Kd glycoprotein on which neutrophil-specific antigen NB1 is located and an 85-Kd glycosyl-phosphatidylinositol-linked N-glycosylated plasma membrane glycoprotein.
We have previously described a 24-year-old woman with quinine-dependent antibodies that reacted with neutrophils, red blood cells (RBCs), platelets, and T lymphocytes. The drug-dependent neutrophil antibody was found to react with 85- and 60-Kd neutrophil membrane molecules. In these studies, we further characterized these molecules and found that both were glycosyl-phosphatidylinositol (GPI)-linked and contained sialic acid residues and N-linked carbohydrate side chains, but neither contained O-linked carbohydrates. The protein backbone of the 60-Kd molecule was 45 Kd, and the 85 Kd glycoprotein (GP) was made up of 33- and 31-Kd proteins. While some GPI-anchored neutrophil GPs are released by stimulated neutrophils, neither the 85- nor the 60-Kd GP was released by neutrophil stimulated with C5a, f-met-leu-phe (FMLP), or phorbol myristate acetate (PMA). Neutrophil-specific antigen NB1 is located on a 58- to 64-Kd GP. To determine if the quinine-dependent antibody and anti-NB1 recognize the same GP, immunoprecipitation studies were performed with the quinine-dependent antibody using neutrophils with varying NB1 phenotypes. The 60-Kd GP was detected on NB1-positive neutrophils from 11 of 12 donors tested, but not on NB1-negative neutrophils from two donors tested. After solubilized 125I-labeled neutrophils were absorbed with anti-NB1, the quinine-dependent antibody immunoprecipitated the 85-Kd GP, but not the 60-Kd GP. These results indicate that anti-NB1 and the quinine-dependent antibody identified the same GP. The 85-Kd GP was detected on neutrophils from all 14 donors tested. The electrophoretic mobility of the 85-Kd GP was similar to the electrophoretic mobility of the major 125I-labeled neutrophil protein.
University of Minnesota, Department of Laboratory Medicine and Pathology, Minneapolis.
D F
DF
Stroncek
R A
RA
Shankar
G P
GP
Herr
D000328:Adult; D000906:Antibodies / Q000097:blood* / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D002462:Cell Membrane / Q000378:metabolism; D015936:Complement C5a / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D017261:Glycosylphosphatidylinositols / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D010641:Phenotype; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D011803:Quinine; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase