Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.
Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.
D001798:Blood Proteins / Q000494:pharmacology; D018958:CD55 Antigens; D003185:Complement C8 / Q000276:immunology; D003186:Complement C9 / Q000276:immunology; D003169:Complement Inactivator Proteins / Q000302:isolation & purification*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins; D004910:Erythrocyte Membrane / Q000032:analysis*; D006461:Hemolysis; D006801:Humans; D008081:Liposomes; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*; D009504:Neutrophils / Q000032:analysis
D001798:Blood Proteins; D018958:CD55 Antigens; D003185:Complement C8; D003186:Complement C9; D003169:Complement Inactivator Proteins; D015938:Complement Membrane Attack Complex; D008081:Liposomes; D008565:Membrane Proteins; D003165:Complement System Proteins