Analysis of gp 140, a C3b-binding membrane component present on Raji cells: a comparison with factor H

In a previous report (M. Barel et al. FEBS Lett., 1981. 136: 111) using radiolabeling methods, we characterized from the membrane of the human B lymphoblastoid cell line Raji, a 140 000-Mr glycoprotein (gp140) carrying a C3b-binding activity with 125I-labeled C3b or Sepharose-bound C3b. The facts of absence on Raji cells of CR1, the C3b receptor purified from human erythrocytes, the observations made by others that H-like activity (the 150 000 Mr C3b binding serum protein) was present in Raji cells and the same molecular weight range of H and gp140, led us to investigate the relationship between both antigens. A rabbit antibody anti-5.4 was prepared against gp140, highly purified from Raji cells. However, anti-H specificities were detected in crude anti-5.4 IgG, while anti-serum H IgG did not react with gp140 antigen. The crude anti-5.4 IgG fraction, anti-gp 140 IgG or F(ab')2 and anti-H specificities present in anti-5.4 IgG, separated by absorption on Sepharose-bound H, and anti-serum H IgG were tested on Raji cells by immunofluorescence techniques, by measuring the inhibition of specific cytotoxic assays and the inhibition of specific binding of soluble or particle-bound C3b to the cell surface and on solubilized antigens by immunoblotting techniques. All the data obtained support that: (a) anti-H specificities are not shared by antibodies bearing anti-gp 140 specificities and their presence in crude anti-5.4 IgG is more likely due to a contamination by H antigen of gp 140 antigen used in the immunization process, and (b) gp 140 antigen is highly expressed on Raji cell surface, whereas H antigen can not be detected under the same conditions. Molecular analysis of gp140 and H antigens confirmed differences between both antigens in molecular weight, trypsin sensitivity and charge properties. All the results presented herein support the notion that gp140 is not identical with the H molecule and that C3b binding to gp140 is not mediated by H. The relationship between gp140 and C3 receptors described by others is discussed.