The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies.

Infectious diseases associated with complement deficiencies.

Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity.

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.

Publisher Summary This chapter focuses on the activation and regulation of the first complement component. . The chapter outlines the history of the classical pathway of the complement system. Clq and C1 are bound and activated by immune complexes or aggregates containing IgG or IgM but not by those containing IgA, IgD, or IgE . Among IgG subclasses, IgG3 is most reactive followed by IgG1, and IgG2; intact IgG4 is minimally reactive, although its Fc region binds C1. C1-In does not efficiently regulate C1 activation induced by immune complexes. It, thus, appears that immune complex dependent activation is not under positive host regulation. This is apparently largely true. However, C1 activation by small immune complexes or complexes formed with nonavid antibody or with ratios of antigen to antibody far from equivalence, all of which are poor C1 activators, may well be regulated by C1-In.

The classical complement pathway: activation and regulation of the first complement component.

Publisher Summary This chapter discusses the use of retroviral vectors for gene transfer and expression Retroviruses have evolved a highly efficient gene transfer capability that provides the basis for one of the most effective gene transfer systems available to date The retroviral vector system has proved useful for the transfer of genes into many cell types, such as hematopoietic cells and other primary cells that are difficult to transduce by using other methods Most attempts to make virus from a particular vector have been straightforward, resulting in high-titer virus carrying the unrearranged vector In some cases, it has proved difficult to generate high-titer virus from vectors carrying specific genes or cDNAs However, the presence of the intron in reverse orientation resulted in aberrant vector transcription and low vector titers Some reports suggest that the titer of retroviral vectors can be dramatically increased by the cocultivation of a vector-producing cell line with a packaging cell line having a different host range However, no more than a 2- to 10-fold increase has been found in titer by using this method, with the disadvantages of increased probability of helper virus generation and vector rearrangement

Use of retroviral vectors for gene transfer and expression.

Moloney leukemia virus activated both the classical and alternative pathways of human complement. About 500,000 virions were required to detect activation of the classical pathway whereas 5,000 times as many virions were necessary to initiate the alternative pathway, indicating that in this system only the former is of biological significance. Disruption of the virus with Triton X-100 destroyed its ability to initiate the alternative pathway without affecting its ability to activate the classical pathway. After ultracentrifugation of disrupted virus the active component could be recovered in the supernate and was isolated by isoelectric focusing in granulated gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic and analysis and cyanogen bromide digestion studies revealed that the activity resided in a methionine-containing protein having a pI of 7.5 and a molecular weight of approximately equal to 15,000 daltons. The purified protein interacts strongly with Clq and efficiently activates Cl. RNase and lipolytic enzymes had no effect on the isolated protein but incubation with trypsin resulted in loss of activity. Enzymatic digestion studies of surface-labeled virus indicate that the active protein is a viral membrane protein. On the basis of these results it is concluded that the complement receptor of Moloney leukemia virus is the surface protein p15E.

/pdf/lysis-of-oncornaviruses-by-human-serum-isolation-of-the-fqgqg8zfna.pdf

Lysis of oncornaviruses by human serum. Isolation of the viral complement (C1) receptor and identification as p15E.

The interaction between the membrane attack complex (MAC) of complement and flat lipid bilayers was investigated. Using spin-labeled derivatives of phospholipids and cholesterol and electron paramagnetic resonance spectroscopy, we measured the penetration of the MAC into bilayers and its influence on the order of bilayers. The MAC precursor components C5b--6, C7, C8, and C9 did not exert any measurable influence on lipid membranes. Functional C5b--7 was shown to interact strongly with the bilayer surface without deep penetration into the bilayer. Formation of C5b--8 and especially C5b--9 caused a marked change in the anisotropy of spectra from probes located within the hydrocarbon phase. The spectral changes are not caused by changes in probe rotation and, in the case of the cholesterol probes, are not due to direct probe--protein interactions. For these reasons we interpret the spectral changes to be the result of reorientation of ordered bilayer lipids effected by strong binding of phospholipids to MAC proteins.

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

This study was conducted to gain insight into the process of assembly of the membrane attack complex (MAC) of complement through structural analysis. Four intermediate complexes and the MAC were examined by electron microscopy and by sucrose density-gradient ultracentrifugation. The C5b-6 complex has a sedimentation rate of 11S, an elongated, slightly curved shape and dimensions of 160 x 60 x 60 A. At protein concentrattions greater than 1 mg/ml, and physiologic ionic strength and pH, the complex forms paracrystals that have the appearance of parallel strands. Equimolar quantities of C5b-6 and C7 mixed in the absence of lipids or detergents give rise to C5b-7 protein micelles which are soluble in aqueous media and have a sedimentation rate of 36S, suggesting a tetrameric composition. Ultrastructurally, C5b-7 protein micelles consist of four half-rings, each measuring 200 x 50 A, which are connected to one another by short stalks extending from the convex side of the half-rings. C5b-7 bound to dioleoyl lecithin (DOL) vesicles has a similar ultrastructural appearance. After extraction with deoxycholate (DOC), C5b-7 has a sedimentation velocity of 36S which further suggests the occurrence of C5b-7 in the form of tetrameric protein micelles. Attachment of C8 to vesicle-bound C5b-7 results in dissociation of the protein micelles. An individual C5b-8 complex appears as a half-ring attached to the DOL-vesicle via a 100-A-long and 30-A-wide stalk. After extraction from the DOL-vesicles with DOC, C5b-8 has a sedimentation velocity of approximately 18S. Binding of C9 to DOL-vesicle bound C5b-8 induces the formation of the typical ultrastructural complement lesions. C5b-9 extracted from the vesicles with DOC has a sedimentation rate of 33S, which is characteristic of the C5b-9 dimer. It is concluded that dimerization is a function of C9. C5b-9 monomers are visualized when a single C5b-9 complex or an odd number of complexes were bound per DOL-vesicle. The C5b-9 monomer has an ultrastructural appearance that is theoretically expected of a half-dimer: a 200- x 50-A half-ring which is attached to the DOL-vesicle by a 100- x 80-A appendage. Extracted with DOC, the C5b-9 monomer has a sedimentation rate of 23S. At a higher multiplicity of MAC per DOL-vesicle, large structural defects in the lipid bilayer are seen which are attributed to direct physical destruction of membranes by the known lipid-binding capacity of the MAC. It is proposed that protein micelle formation at the C5b-7 stage of MAC assembly and dissociation of these micelles upon binding of C8 are events that facilitate dimerization of C5b-9 and thus MAC formation.

https://rupress.org/jem/article-pdf/151/2/301/1090658/301.pdf

Membrane attack complex of complement: a structural analysis of its assembly.

A new method for the isolation of C6 and C7 by affinity chromatography of human serum with anti-C6 and anti-C7 coupled to Sepharose is described. C6 and C7 prepared by this method are hemolytically fully active, homogeneous proteins obtained in 25% yield. A comparison of the properties of isolated C6 and C7 gave the following results: The amino acid composition of the two proteins is very similar. The m.w. calculated from the amino acid content is 124,800 for C6 and 120,800 for C7. Both components are single chain glycoproteins migrating upon electrophoresis at pH 8.6 as beta 2-globulins, Both proteins are polymorphic as detected by isoelectrofocusing in polyacrylamide gels and range in their isoelectric points from pH 6.15 to 6.7. The UV spectra reveal only minor differences; the extinction coefficients are: EC6 = 1.71 cm2 X mg-1 and EC7 = 1.92 cm2 X mg-1. CD-spectra show 8% alpha-helix and 10% beta-structure for C6 and 10% alpha-helix and 14% beta-structure for C7. The structural similarities of C6 and C7 suggest their evolution from a common ancestral gene.

Structural similarities between C6 and C7 of human complement.

During the routine examination of a healthy 31-yr-old woman, we found an incomplete deficiency of the 9th component of complement (C9). By hemolytic assay her serum C9 activity was 10 to 15% of normal. Limited family studies suggested that she inherited the deficiency as an autosomal codominant trait. She had no history of unusual or severe infections. When tested for bactericidal activity against serum-sensitive Neisseria gonorrhoeae and N. meningitidis, her serum reacted comparably to normal serum. Normal serum depleted immunochemically of C9 and sera from congenitally C9-deficient patients were also bactericidal against serum-sensitive Neisseria but required 120 min to kill the same numbers of gonococci that intact serum killed within 30 min. In the electron microscope, N. gonorrhoeae incubated with C9-depleted serum were fragmented but lacked the typical C lesions. Therefore, serum lacking C9 can kill serum-sensitive Neisseria, unlike sera deficient in the other terminal C components.

A F Esser

Papers

Lysis of oncornaviruses by human serum. Isolation of the viral complement (C1) receptor and identification as p15E.

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

Membrane attack complex of complement: a structural analysis of its assembly.

Structural similarities between C6 and C7 of human complement.

The role of C9 in complement-mediated killing of Neisseria.