Identification of a spontaneously shed fragment of B cell complement receptor type two (CR2) containing the C3d-binding site.
CR2 is a 140-kilodalton glycoprotein expressed on B lymphocytes which binds to both C3d and Epstein-Barr virus (EBV). The present study identified a 72-kilodalton C3d-binding protein (gp72) in the spent culture media of Raji B lymphoblastoid cells as a spontaneously shed fragment of the 140-kilodalton CR2 molecule. Both polyclonal and monoclonal antibodies (AB) were used in several assay systems to detect antigenic determinants shared between the gp72 fragment and CR2. Rabbit antibodies to either intact CR2 or gp72 blocked C3d receptor activity, and this inhibitory activity was removed by absorption of anti-CR2 with purified gp72.OKB7, a monoclonal anti-CR2 AB that blocks both C3d and EBV binding to CR2, reacted specifically with both CR2 and gp72, whereas both anti-B2 and HB-5 monoclonal anti-CR2 AB, that block neither of these receptor sites, were unreactive with gp72. The data suggested that the gp72 fragment was not present as such in intact cells, but rather was a product of cells generated by proteolysis of CR2. Thus, intrinsically labeled gp72 was isolated from Raji cell media by affinity chromatography on OKB7-Sepharose, but only intact CR2 was isolated from the Raji cell fraction solubilized in the presence of protease inhibitors. Several lines of evidence suggested that gp72 was not a second type of C3d receptor that was distinct from CR2. First, Raji cells expressed nearly identical amounts of OKB7 and HB-5 epitopes when analyzed by flow cytometry or radioimmune assay, excluding the possibility that B cells expressed OKB7 antigens in both CR2 and a distinct HB-5-negative C3d receptor. Second, all Raji cell surface C3d receptor activity was associated with HB-5-reactive CR2 molecules. We conclude that gp72 represents a spontaneously shed proteolytic fragment of CR2 that contains the C3d-binding site and the closely associated OKB7 epitope.
D001402:B-Lymphocytes / Q000276:immunology*; D001665:Binding Sites; D002460:Cell Line; D003175:Complement C2 / Q000378:metabolism*; D003176:Complement C3 / Q000378:metabolism*; D015933:Complement C3d; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D011951:Receptors, Complement / Q000302:isolation & purification*; D012995:Solubility
D003175:Complement C2; D003176:Complement C3; D006023:Glycoproteins; D011951:Receptors, Complement; D015933:Complement C3d