Biochemical characterization of an Fc gamma receptor purified from human neutrophils.
The Fc receptor identified by mAb 3G8 (Fc gamma RIII) was isolated by mAb affinity chromatography from 0.5 to 2 x 10(10) neutrophils yielding 33 to 149 micrograms of protein. Iodination of the purified protein identified a polypeptide of broad electrophoretic mobility from Mr 47 to 70 kDa and occasionally a fainter polypeptide at 100 to 130 kDa, which may be dimerized receptor. Two-dimensional isoelectric focusing gel electrophoresis illustrated multiple diffuse polypeptides ranging from a pI of less than 4.7 to 6.5. Treatment of the purified receptor with neuraminidase shifted the mobility of these polypeptides to a more basic pI, ranging from 6 to 8, illustrating the presence of sialic acid residues on Fc gamma RIII. The glycoprotein nature of Fc gamma RIII was characterized by several criteria. The receptor bound to Con A-Sepharose. Treatment of Fc gamma RIII with endoglycosidase H or F, which cleave high mannose and biantennary complex N-linked oligosaccharides, respectively, failed to alter the electrophoretic mobility of the Fc gamma R. Peptide N:glycosidase F, which cleaves all classes of N-linked oligosaccharides, reduced the Mr of Fc gamma RIII by 60% to reveal two poorly resolved polypeptides centered at Mr 25 kDa and ranging from Mr 16 to 28 kDa. Chemical deglycosylation with trifluoromethanesulfonic acid, which cleaves O- and N-linked oligosaccharides except for the asparagine-linked N-acetylglucosamine, reduced the Mr of Fc gamma RIII to 21 to 36 kDa. These results demonstrate that Fc gamma RIII is an acidic complex sialoglycoprotein and suggest that there may be 8 to 15 N-linked oligosaccharide chains on Fc gamma RIII.
Fleit
H B
HB
Department of Pathology, State University of New York, Stony Brook 11794.
Kuhnle
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D008562:Membrane Glycoproteins / Q000302:isolation & purification; D008970:Molecular Weight; D009504:Neutrophils / Q000032:analysis / Q000276:immunology*; D011961:Receptors, Fc / Q000302:isolation & purification*; D017452:Receptors, IgG; D014357:Trypsin
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D011961:Receptors, Fc; D017452:Receptors, IgG; D002918:Chymotrypsin; D014357:Trypsin