Evidence for a two-domain structure of the terminal membrane C5b-9 complex of human complement.

Abstract

Lipid vesicles carrying the purified membrane C5b-9 complex [C5b-9(m)] of complement were analyzed immunochemically and in the electron microscope after treatment with a combination of trypsin and a-chymotrypsin. Under reducing conditions, the externally oriented annulus was removed. The remaining part of the C5b-9(m), representing approximately half of the total mass of the macromolecular complex, was visualized in the electron microscope as a hollow cylindrical structure with walls of 1-nm thickness. This structure remained tenaciously attached to the lipid bilayer, projecting 8-9 nm from the external membrane surface into the aqueous environment. Cleavage of C5b-9(m) by proteolysis and reduction resulted in a sharp reduction of its antigenic determinants. One hydrophilic protease-resistant C5 derivative was released from the membrane and recovered in the fluid phase. The membrane-bound residue almost totally lacked antigens precipitable with antisera to CS, C6, C9, and CSb-9(m). The membrane attack unit of complement (C) is a macromolecular C5b-9 protein complex (1, 2) that becomes assembled on and in a target membrane during the course of complement activation (3,4). The protein complex [termed C5b-9(m)] possesses the structure of an apparently hollow cylinder (5); one terminus is rimmed by an annulus, identical to the classical ultrastructural complement lesion (5-7). The other terminus possesses apolar lipid-binding surfaces through which intrusion into the membrane lipid bilayer is effected (8, 9). Current evidence indicates that a transmembrane channel walled by the inserted hollow C5b-9(m) is thus created (3, 4, 9, 10), and this has been proposed to constitute the basic molecular mechanism of cytolysis by complement (9,11,12).Earlier studies of Hammer et al. (13,14) indicated the existence of protease-resistant polypeptide chains derived from C5, C8, and C9 in guinea pig C5b-9(m), and the possibility has been raised that these polypeptide chains are buried within the lipid bilayer of the target membrane. In another attempt to characterize the membrane-bound portion of C5b-9(m) we have now employed liposomal membranes reconstituted with purified human C5b-9(m) as substrates for proteolytic dissection. We present immunochemical and ultrastructural evidence that combined treatment of membrane-bound C5b-9(m) with proteases and a reducing agent induces the removal of the externally oriented annulus, leaving a hollow cylindrical residue of low immunological reactivity firmly attached to the lipid bilayer.MATERIALS AND METHODS Unless otherwise stated, sources of reagents and antibodies were as previously given (5,8,9,15,16). Preparation of Lipid Vesicles Containing C5b-9(m). Preparation of native C5b-9(m) followed an improved procedure that will be detailed elsewhere. Native or proteolyzed C5b-9(m) was incorporated into artificial lipid vesicles basically as described (9), with the following modifications. Egg lecithin (Sigma, type V-E) was used instead of the mixture of sheep erythrocyte membrane li...

Molecular weight of the membrane C5b-9 complex of human complement: characterization of the terminal complex as a C5b-9 monomer.

“…A molecular weight of approximately 1,000,000 would be in good agreement with the ultrastructural data on the complex (7,9,35) and dictates that the macromolecule is truly hollow, because the molecular weight of a compact protein globule of the same dimensions (molecular radius, 11 nm) would be several times higher. The value of 1,000,000 is still higher than the sum of the individual molecular weights of C5-C9 components in a monomer complex.…”

Polymerization of the ninth component of complement (C9): formation of poly(C9) with a tubular ultrastructure resembling the membrane attack complex of complement.

“…2) and cytolysis (2,4,5), on the similar ultrastructure of poly(C9) (Fig. 1) and the MAC (13,14,16,17,19,20,31), and on the requirement for C9 binding in formation of ultrastructural complement lesions representing the membrane-bound MAC (13,14). The present study suggests that poly(C9) represents the structure previously ascribed to the C5b-9 complex (13,14,19,20,31).…”

“…1) and the MAC (13,14,16,17,19,20,31), and on the requirement for C9 binding in formation of ultrastructural complement lesions representing the membrane-bound MAC (13,14). The present study suggests that poly(C9) represents the structure previously ascribed to the C5b-9 complex (13,14,19,20,31). The structure ofC5b-8 (14) in the membrane attack complex (16) apparently is bound adjacent to the poly(C9) tubule without contributing to the ultrastructural appearance of the membrane lesion.…”

“…Subsequent studies (unpublished) indicated a tendency ofthe MAC to form MAC oligomers (18). In contrast, other.groups suggested a monomeric C5b-9 composition of the MAC (19,20).…”

Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water.

“…It is directly responsible for complement-dependent membrane damage and cytolysis. The manner in which the MAC exerts its cytolytic and membranolytic functions has been the object of many recent studies which included the determination of the apparent pore size of the MAC-induced membrane lesion (1)(2)(3)(4)(5)(6)(7)(8), the effect of the MAC on lipid bilayer structure (9)(10)(11)(12), and the structural analysis of the MAC itself (13)(14)(15)(16)(17)(18)(19). The aim ofthe present study was to identify those subunits of the MAC and its intermediate complexes that are in direct physical contact with the hydrocarbon core of the target membrane and thus are probably responsible for the expression of membranolytic activity.…”