Cell-killing mechanisms are essential components of host defense against infectious agents, parasites, and malignant cells. We distinguish humoral and cellular killing mechanisms. Complement utilizes specific proteins to assemble its killer molecule. Macrophages generate active oxygen radicals that injure target membranes. Lymphocytes, like complement, use protein molecules as tools in the cellular cytotoxicity reaction. As will be suggested at the conclusion of this chapter, considerable similarity between the structure of the cytolytic apparatus of complement and that of lymphocytes has recently come to light. An in-depth knowledge of the structure and function of the killer molecule of complement may therefore facilitate elucidation of the cell-killing mechanism of lymphocytes. Complement encompasses 20 proteins, not including the various cell­ surface complement receptors and regulatory proteins. Only 5 of these 20 proteins participate directly in cell killing-C5, C6, C7, C8, and C9-and none has enzymatic activity, proteolytic or lipolytic. Upon activation of C5, the 5 proteins interact in a sequential manner and fuse into a macromo­ lecular organization, called the membrarie attack complex or MAC. Fusion brings forth hydrophobic sites through which the complex inserts itself into the hydrocarbon core of lipid membranes. There it forms transmembrane channels, the largest of which constitutes tubular poly C9. Activation of C5 is accomplished by a highly specific serine protease, C5 convertase, itself an assembly of three protein molecules. Thus, formation of the MAC is initiated enzymatically, but this enzyme does not participate in actual membrane attack, which is entirely a physicochemical process.

The membrane attack complex of complement.

Damage to mammalian cells by proteins that form transmembrane pores

Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations.

Membrane damage by complement

Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.

Immune-Mediated Pore-Forming Pathways Induce Cellular Hypercitrullination and Generate Citrullinated Autoantigens in Rheumatoid Arthritis

The molecular arrangement of the membrane attack mechanism of complement was explored. The molar ratios of the components within the C5-9 assembly on the target cell surface were determined using human complement proteins in highly purified and radiolabeled form. With the aid of monospecific complement antisera it was possible to probe the spatial relationships between the components of the assembly. C5 and C6, in the presence of C7, were bound to EAC1-3 in equimolar quantities irrespective of the amounts and the relative proportions of C5, C6, and C7 offered. The amount of C8 bound to EAC1-7 increased with input and at saturation of all C8 binding sites the molar ratio of bound C8/bound C5 approached 1.0. Uptake of C9 by EAC1-8 increased with input and at saturation of all C9 binding sites the molar ratio of bound C9/bound C8 became 6.0. However, calculations suggest that the binding of three C9 molecules to one C8 molecule is sufficient to achieve a full hemolytic effect. Evidence was obtained indicating that binding and hemolytic function of C9 depends upon cooperative interaction of multiple C9 molecules. Binding of C8 to EAC1-7 and the generation of hemolytic C8 sites were inhibited by antibody to either C5, C6, or C7. Uptake of C9 by EAC1-8 and the generation of hemolytic C9 sites were strongly inhibited by anti-C8 and to a lesser degree by anti-C5. Binding of C9 (but not hemolysis) was also reduced by antibody to C6 or C7. The data are consistent with the concept that the fully assembled membrane attack mechanism of complement consists of a decamolecular complex: a trimolecular arrangement composed of C5, C6, and C7 forms the binding site for one C8 molecule which in turn furnishes binding sites for six C9 molecules, saturation of three sites apparently being sufficient for expression of full cytolytic activity of the complex. This work made it possible to design a simple molecular model.

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Molecular analysis of the membrane attack mechanism of complement

Molecular analysis of the m embrane attack mechanism of complement

Reversible interactions in free solution were demonstrated to occur (a) between C5 and C8, (b) between C5, 6, 7 and C8, and (c) between C8 and C9. No interaction was observed between C8 and C6 or C7 and between C9 and C5, 6, 7. Interactions between C8 and C9 were enhanced at lowered ionic strength (0.05) and a molar excess of C8 over C9. Complex formation was independent of pH over the range of 6.5–8.5. Under optimal conditions the C8, 9 complex had a sedimentation coefficient of 10.2–10.6S, while native C8 and C9 sedimented at 8.5 and 4.8S, respectively. Specificity and reversibility of these interactions were established. In spite of the limited number of interactions observed, all five of the native proteins of the membrane attack mechanism interacted to form an association product that sedimented at 10.8–11.2S. Demonstration of this product in free solution supports the concept that C5-9 on acquisition of cytolytic activity assemble into a stable multimolecular complex.

https://rupress.org/jem/article-pdf/138/2/428/1085529/428.pdf

James A. Haxby

Papers

Molecular analysis of the membrane attack mechanism of complement

Molecular analysis of the m embrane attack mechanism of complement

The membrane attack mechanism of complement reversible interactions among the five native components in free solution

REVERSIBLE ~NTERACTIONS AMONG TlYE FIVE NATIVE COMPONENTS IN FREE SOLUTION*