Isolation and characterization of biologically active Fc receptors of human B lymphocytes

An experimental approach consisting of five steps was designed and tested to isolate Fc receptors (FcR) from human B cell lysates in a molecularly distinct form. The five steps were: (1) radioiodination of cell surface proteins by lactoperoxidase-catalyzed method; (2) lysis of radiolabeled cells by detergent treatment; (3) removal of intrinsic IgG by affinity chromatography on Protein A-Sepharose CL-4B gel; (4) extraction of FcR by affinity chromatography on heat-aggregated IgG and Fc fragments coupled to Sepharose 4B; and (5) gel filtration and isoelectric focusing. Our results with FcR + and FcR − cells indicated that affinity chromatography on heat-aggregated IgG and Fc fragments-Sepharose 4B (Step 4) extracted not only FcR proteins, but also small amounts of various other membrane components. However, they could be effectively separated by the combination of gel filtration and isoelectric focusing. By applying this procedure in large scale, FcR proteins were isolated from CLL cell lysates originated from three different patients. The isolated FcR molecules were found to be biologically active, as they produced inhibition of rosette formation between Rh-D antibody coated human O + red blood cells and human peripheral blood mononuclear cells. Such inhibition of EA rosette formation by isolated FcR proteins was also observed when EA preparation consisted of rabbit antibody and sheep erythrocytes confirming their lack of species specificity. Furthermore, F(ab') 2 fragments of rabbit antibody elicited against an isolated FcR preparation efficiently inhibited EA rosette formation. By affinity chromatography, it was shown that isolated FcR proteins bind with the Fc portion of IgG, but not to Fab or F(ab') 2 fragments. FcR proteins isolated from three different patient cell lysates were excluded from Sephadex G-100 and were focused in the natural pH gradient formed in the presence of 6 M urea at pH near 6.8-6.5; suggesting their molecular uniformity.