(C5b-8)iC9i C9


3 nm

Figure 1 Diagrammatic illustration of the complement membrane attack pathway and electron microscopy of MAC protein complexes. Reaction sequence from activation of C5 through assembly of the complete MAC is shown. Interaction of complexes with a cell membrane is illustrated, with C8 depicted in black. Stippled areas indicate membrane pores of progressively larger size that develop in parallel with progression of MAC assembly. Arrows indicate loss of permeability barrier. Also shown are electron micrographs of membranes bearing C5b-7 (panels a and b), C5b-8 (panel c) and the fully assembled MAC (panel d, lateral view, and panel e, top view). (Electron micrographs are reproduced from RR Dourmashkin (1978), The structural events associated with the attachment of complement components to cell membranes in reactive lysis, Immunology 38: 205-212, with permission from the publisher.)

transient site is facilitated by the weak association of C5 and C6 in solution. The resultant C5b6 is stable and has a binding site for C7. C5b6 can remain loosely associated with C3b, which assists in directing C5b-7 to the adjacent membrane, or detach to the fluid phase. Once C7 incorporates into the complex a metastable binding site with a high affinity for phospholipids is produced. This site is very short-lived, less than 10 ms, and allows the C5b-7 complex, after detachment from C3b, to anchor in the external surface of the lipid bilayer, primarily through C7. The electron microscopic appearance of membrane-bound C5b-7 is that of a foliaceous particle (Figure 1). Assembly of the complex can also continue in the fluid phase; however, competition for the lipophilic binding site precludes attachment to a cell membrane. Vitronectin, clusterin, lipoproteins and C8 can all bind to fluid phase C5b-7 and prevent further association with membrane phospholipids.

Membrane-associated C5b-7 binds one molecule of C8, forming the C5b-8 complex, which then may bind multiple molecules of C9. The C5b-7 complex in the membrane bilayer binds a C8 molecule by interaction of the C8 (3 chain with C5b; possibly a second binding site on C7 promotes a conformational rearrangement in C8 resulting in the C8 a chain penetrating the lipid bilayer. Bound C5b-8 appears as a particle with a variable number of arms (Figure 1), with C5b and C8(3 most distal from the membrane. The resultant C5b-8 complex has a greater affinity for phospholipids than C5b-7, penetrates more deeply into the membrane and disrupts membrane integrity to produce small transient ion-pcrmeable channels. C9 binds to the C8 a chain in the C5b-8 complex and undergoes a conformational change that allows C9 to insert through the bilayer and expose additional binding sites for C9 polymerization. The C5b-9 complex has a greater affinity for phospholipids than C5b-8 and the membrane channel becomes more stable. The channels are formed by the disruption of the bilayer structure as membrane phospholipids attach to the complex. Additional molecules of C9 incorporate into the complex in a circular configuration and the size of the channel continues to increase (Figure 1). Incorporation of 12-18 molecules of C9 results in formation of the closed ring, tubular polyC9 structure, which may function as a protein-walled channel. The ultrastructure of this complex on top view corresponds to a ring with an internal diameter of 10 nm and an external diameter of 21 nm. On a lateral view there is a pedicle attached to polyC9 that represents the majority of the C5b-8 complex and extends 16 nm beyond the ring which has a height of 15 nm. Penetration of a membrane by the C5b-9 complex results in loss of membrane phospholipids and a reduction in membrane fluidity. Phospholipids and cholesterol in red cell membranes that have been exposed to the MAC are resistant to dissociation by caotropic agents.

MAC assembly is controlled by several regulatory mechanisms that protect tissues from damage by physiologic activation of autologous complement. Inhibition of early events of complement activation reduces formation of C5 convertases and thus prevents generation of C5b and MAC. Even after C5b has been produced there are mechanisms that control

MAC assembly. Intrinsic control is provided by the lability of the exposed binding sites in C5b and C5b-7. There are also membrane-bound and plasma inhibitors that prevent further assembly of complement proteins into the complex. The membrane-associated inhibitors are homologous restriction factor (HRF) and CD59 (also called protectin, MAC inhibitory factor or HRF20) and they are more effective to protect a cell from lysis by complement of the same species than by complement from other species (homologous restriction). CD59 and HRF inhibit formation of the MAC through interaction with membrane-bound C5b-8 to impair the binding of C9 and with C5b-9 to block additional binding and polymerization of C9. These inhibitors are expressed on most cells that are in contact with blood or other biological fluids containing complement. These proteins as well as the inhibitor of the C3 and C5 convertases decay-accelerating factor (DAF) are anchored to the membrane through a glycosyl phos-phatidylinositol moiety, although in certain cells they may also exist as transmembrane proteins. There are also plasma proteins that control MAC formation. Vitronectin (S protein) and clusterin (SP-40,40) are multifunctional proteins that interact with late-act-ing complement components as complexes assemble in plasma, and inhibit binding of the protein complex to cell membranes. In addition, apolipoproteins A-I and A-II may inhibit MAC formation by interfering with polymerization of C9.

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