Activation of the fifth and sixth components of the human complement system: C6-dependent cleavage of C5 in acid and the formation of a bimolecular lytic complex, C5b,6a.

Acidification of C5 and C6 or serum to pH 6.4 at 0 degrees C, followed by neutralization, generates a factor-designated C(56)a that causes lysis of nonsensitized erythrocytes in the presence of C7, C8, and C9. C(56)a is functionally similar to alternative pathway-generated C5b,6 in respect to the formation of C5b,6,7 sites on cells, the potentiation of lytic activity by membrane-bound C3b or the membrane-active agent A2C, and the required species compatibilities between target membranes and terminal components for optimal activity. The formation of C(56)a complex from purified components C5 and C6 proceeds independently of the classical or alternative pathway C5 convertases and requires the simultaneous H+ ion treatment of the components. The generation of C(56)a from C5 and C6 and the physicochemical properties of the complex were studied in detail and compared with those of C5b,6. Acid generation of C(56)a is dose-dependent on C5 and C6 and its efficiency is similar to that of the conventional convertase in the production of lytic activity. Sucrose gradient ultracentrifugation of C(56)a containing activated 125I-C5 demonstrated a shift in sedimentation from that of native C5 to 11S, which is consistent with C5,6 complex formation. C(56)a sedimentation was identical to C5b,6, and both migrated coincident with lytic complex activity. These complexes, however, are not identical because unlike C5b,6, C(56)a is unstable at 37 degrees C, demonstrating a nonlinear decay curve. In the presence of C7, both complexes exhibit similar first order decay with a T1/2 of 3 min at 37 degrees C. SDS-PAGE autoradiographic analysis of the C5-subunit structure of 125I-C5 in C(56)a and the Zx-activated C5b,6 complex prepared from purified components showed similar alpha-chain cleavage to several fragments of 109,000, 100,000, and 58,000 daltons. Conversion to lower m.w. peptides by acid treatment was more extensive. Comparison of the 125I-C5 polypeptide chains in the membrane attack complex extracted from guinea pig erythrocyte membranes, prepared by acid activation or classical pathway lysis with whole serum, demonstrated similar C5 alpha-chain cleavage to a predominant subunit of 102,000 daltons. Acid activation also produced a 109,000 dalton C5 alpha'-fragment barely detectable with classical pathway activation. Low pH treatment of C5 alone did not inactivate C5 function, form a lytic complex on the subsequent addition of C6, or cleave the C5 alpha-chain. Thus, it is postulated that local high H+ ion concentration during simultaneous acidification of C5 and C6 allows complex formation with the concomitant C6-dependent cleavage of the C5 alpha-chain and the generation of lytic capacity.