Protein ultrastructure and the nanoscience of complement activation.

The complement system constitutes an important barrier to infection of the human body. Over more than four decades structural properties of the proteins of the complement system have been investigated with X-ray crystallography, electron microscopy, small-angle scattering, and atomic force microscopy. Here, we review the accumulated evidence that the nm-scaled dimensions and conformational changes of these proteins support functions of the complement system with regard to tissue distribution, molecular crowding effects, avidity binding, and conformational regulation of complement activation. In the targeting of complement activation to the surfaces of nanoparticulate material, such as engineered nanoparticles or fragments of the microbial cell wall, these processes play intimately together. This way the complement system is an excellent example where nanoscience may serve to unravel the molecular biology of the immune response.

Constrained solution scattering modelling of human antibodies and complement proteins reveals novel biological insights.

X-ray and neutron-scattering techniques characterize proteins in solution and complement high-resolution structural studies. They are useful when either a large protein cannot be crystallized, in which case scattering yields a solution structure, or a crystal structure has been determined and requires validation in solution. These solution structures are determined by the application of constrained modelling methods based on known subunit structures. First, an appropriate starting model is generated. Next, its conformation is randomized to generate thousands of models for trial-and-error fits. Comparison with the experimental data identifies a small family of best-fit models. Finally, their significance for biological function is assessed. We illustrate this in application to structure determinations for secretory immunoglobulin A, the most prevalent antibody in the human body and a first line of defence in mucosal immunity. We also discuss the applications to the large multi-domain proteins of the complement system, most notably its major regulator factor H, which is important in age-related macular degeneration and renal diseases. We discuss the importance of complementary data from analytical ultracentrifugation, and structural studies of protein-protein complexes. We conclude that constrained scattering modelling makes useful contributions to our understanding of antibody and complement structure and function.

Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions.

The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid was purified from saline extracts of aortic atherosclerotic lesions by sucrose density gradient centrifugation followed by molecular sieve chromatography on Sepharose 2B. It contained little protein other than albumin, was 100-500 nm in size, exhibited an unesterified to total cholesterol ratio of 0.58 and an unesterified cholesterol to phospholipid ratio of 1.2. The lipid, termed lesion lipid complement (LCA), activated the alternative pathway of complement in a dose-dependent manner. Lesion-extracted low density lipoprotein (LDL) obtained during the purification procedure failed to activate complement. Specific generation of C3a desArg and C5b-9 by LCA indicated C3/C5 convertase formation with activation proceeding to completion. Biochemical and electron microscopic evaluations revealed that much of the C5b-9 present in atherosclerotic lesions is membraneous, rather than fluid phase SC5b-9. The observations reported herein establish a link between lipid insudation and inflammation in atherosclerotic lesions via the mechanism of complement activation.

Comparison of channels formed by poly C9, C5b-8 and the membrane attack complex of complement.

The channels formed by poly C9, C5b-8 and C5b-9 were examined using the liposome swelling assay. By plotting the relative rate of swelling of C5b-8-containing liposomes vs the molecular weight of the sugar solute and by applying the Renkin equation, the size of the C5b-8 channel was estimated to be 1.5 mm radius. As increasing amounts of C9 were added during the formation of C5b-9, in C8:C9 ratios of 1:1, 1:2, 1:6 and 1:12, the size of the function channel increased. Poly C9 had a pore that was somewhat larger than C5b-9 at a C8:C9 ratio of 1:12. Using molecular sieving experiments with four different iodinated protein size markers, the channel diameter of poly C9 was estimated at between 90 and 100 A. Monoclonal antibodies to different complement proteins were added to the liposomes to see which might inhibit the channels. C5b-8 containing liposomes could be inhibited by antibodies to C8. Liposomes containing C5b-9 could be inhibited slightly by antibodies to C9 and most strongly by antibodies to the neoantigen of poly C9.

SC5b-7, SC5b-8 and SC5b-9 complexes of complement: ultrastructure and localization of the S-protein (vitronectin) within the macromolecules.

Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all antisera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)n and (C5b-8)n, negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 less than 27 S) appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.

Complement attack of altered outer membrane areas synthesized after inhibition of the 3-deoxy-D-manno-octulosonate pathway leads to cell death.

Salmonella typhimurium containing specific genes coding for either temperature-sensitive (TS) 3-deoxy-D-manno-octulosonate (KDO) 8-phosphate synthetase or TS cytidine monophosphate-KDO synthetase grow normally when incubated at 30 degrees C and are resistant to C-mediated killing. However, bacteria become avirulent and sensitive to C-mediated killing upon thermal inhibition of TS KDO-8-phosphate synthetase (incubation at 38 degrees C) or TS cytidine monophosphate-KDO synthetase (incubation at 42 degrees C). Such thermal inhibition concurrently causes synthesis of an altered outer membrane which we now show is the site that renders cells susceptible to C-mediated killing. After incubation of cells in serum, the altered outer membrane area contains C9 in a trypsin-resistant state and membrane attack complex (MAC) lesions observable by electron microscopy. Trypsin-resistant C9 and MAC lesions were also observed in the inner membrane fraction from such serum-treated cells. In contrast, little C9 and few MAC lesions were associated with unaltered outer membrane areas present on these same serum treated cells. Control cells, grown at 30 degrees C and treated with serum (1) bound one-fifth as much C9 as was bound to cells incubated at 42 degrees C, (2) contained only a rare MAC lesion in the outer membrane, and (3) no observable MAC lesions in the inner membrane. We conclude that the altered outer membrane area is the site that renders cells susceptible to insertion of the MAC into both the outer and inner membrane resulting in cell death.

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)