title: Molecular basis of complement resistance of human melanoma cells expressing the C3-cleaving membrane protease p65.
abstract: The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.
authors.affiliation: Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007.
authors.forename: M W
authors.initials: MW
authors.lastname: Ollert
authors.forename: J V
authors.initials: JV
authors.lastname: Kadlec
authors.forename: E C
authors.initials: EC
authors.lastname: Petrella
authors.forename: R
authors.initials: R
authors.lastname: Bredehorst
authors.forename: C W
authors.initials: CW
authors.lastname: Vogel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003167:Complement Activation / Q000502:physiology*; D015922:Complement C1q / Q000378:metabolism; D003176:Complement C3 / Q000378:metabolism / Q000502:physiology; D003179:Complement C3b / Q000378:metabolism* / Q000502:physiology; D003180:Complement C3b Inactivator Proteins / Q000494:pharmacology; D003181:Complement C4 / Q000378:metabolism / Q000502:physiology; D003182:Complement C5 / Q000378:metabolism / Q000502:physiology; D003186:Complement C9 / Q000378:metabolism / Q000502:physiology; D015938:Complement Membrane Attack Complex / Q000502:physiology; D003165:Complement System Proteins / Q000276:immunology*; D010450:Endopeptidases / Q000502:physiology*; D006801:Humans; D008545:Melanoma / Q000201:enzymology* / Q000378:metabolism / Q000503:physiopathology; D051379:Mice; D008807:Mice, Inbred BALB C; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D003176:Complement C3; D003180:Complement C3b Inactivator Proteins; D003181:Complement C4; D003182:Complement C5; D003186:Complement C9; D015938:Complement Membrane Attack Complex; D015922:Complement C1q; D003179:Complement C3b; D003165:Complement System Proteins; D010450:Endopeptidases