Analysis of gp140, a C3b‐binding membrane component present on Raji cells: a comparison with factor H

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Abstract
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 140000‐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 150000 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‐gp140 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‐gp140 specificities and their presence in crude anti‐5.4 IgG is more likely due to a contamination by H antigen of gp140 antigen used in the immunization process, and (b) gp140 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.
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