Fluorometric detection of low temperature thermal transitions in the C1Q component of human complement.
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Complement Activating Enzymes↗
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Complement activation and C1q binding activity in haemodialysis.
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Antigen-Antibody Complex↗
Effects of soluble aggregates of IgG on the binding, uptake and degradation of the C1q subcomponent of complement by adherent guinea pig peritoneal macrophages.
Earlier studies have indicated that C1q, the first subcomponent of complement component C1, is bound to lymphocytes via specific C1q receptor sites. We have recently shown that adherent guinea pig peritoneal exudate macrophages express specific receptors for C1q (Veerhuis, R. et al., Immunology 1985. 54: 801). The present studies were performed to determine whether binding of 125I-labeled human C1q (125I-C1qhu) to adherent guinea pig peritoneal exudate macrophages would also result in ingestion and subsequent degradation of 125I-C1qhu. The binding of 125I-C1qhu to adherent peritoneal macrophages at 4 degrees C is inhibited fully not only by C1qhu and guinea pig C1q (C1qgp) but also by pepsin fragments of C1qhu. The amount of trichloroacetic acid nonprecipitable radioactivity that appeared in the supernatant was used as a measure for the degradation of 125I-C1qhu. 125I-C1qhu is degraded initially into fragments of 25 kDa, after which it is degraded further into small molecular weight peptides. Ingestion of 125I-C1q by the macrophages occurs before the 125I-C1q is degraded. In the presence of limited amounts of soluble aggregates of guinea pig IgG2 (AIgG), a known activator of C1, part of the C1q is bound to the AIgG and all of the AIgG in turn is bound to the cellular Fc receptors leading to an enhanced binding of 125I-C1q to the cells, a binding that was maximal at near equimolar concentrations of 125I-C1qhu and 131I-AIgG. In the presence of a 30-fold excess of AIgG, however, only a small percentage of the AIgG binds to cellular Fc receptors and the interaction of C1q with its receptor is decreased due to competitive inhibition. The results presented in this report thus suggest that free C1q may be eliminated by specific interaction with C1q receptors present on circulating and tissue phagocytoses and, in addition, that in the presence of immune complexes modulation of elimination of C1q may be encountered.
Animals↗
Formation of complement subcomponent C1q-immunoglobulin G complex. Thermodynamic and chemical-modification studies.
The interaction between the complement subcomponent C1q and immunoglobulin G was investigated under a variety of experimental conditions. Formation of the subcomponent C1q--immunoglobulin G complex was shown to be an equilibrium process. Thermodynamic studies of the effect of varying the ionic strength indicate that over the salt range 0.15--0.225 M-NaCl the binding of subcomponent C1q to immunoglobulin aggregates releases 9--12 salt ions (Na+ and/or Cl-), illustrating the importance of ionic interactions for the formation of the complex. The effects of small peptide and organic ion inhibitors support this conclusion. Chemical modifications of carboxylate residues on immunoglobulin G by glycine ethyl ester/water-soluble carbodi-imide (up to 12 residues modified per whole molecule of immunoglobulin G) and of lysine residues by acetic anhydride (3 residues per whole molecule of immunoglobulin G) or methyl acetimidate (19 residues per whole molecule of immunoglobulin G) lowered the binding affinity of immunoglobulin for subcomponent C1q. Modification of arginine residues by cyclohexane-1,2-dione-1,2 (14 residues per whole molecule of immunoglobulin G) and of tryptophan by hydroxynitrobenzyl bromide (2 residues per whole molecule of immunoglobulin G), however, had little or no effect. The results are consistent with the proposal that the subcomponent-C1q-binding site on immunoglobulin G is to be found on the last two beta-strands of the Cv2 domain [Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek (1980) Nature (London) 288, 338--344].
Amino Acids↗
Purification and characterization of subcomponent C1q of the first component of bovine complement.
Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.
Amino Acids↗
Enhanced binding and degradation of the C1q subcomponent of complement by thioglycollate-stimulated guinea pig peritoneal macrophages.
Expression of C1q receptors on the plasma membrane of thioglycollate-stimulated guinea pig peritoneal exudate macrophages increased 1.54 times as compared to unstimulated controls. A Scatchard plot of the binding of 125I-C1q to the cells revealed that the binding is a result of an increase in the number of receptors and not to an increased affinity of the receptors. Thioglycollate-activated macrophages were found to be 1.6 times more active than nonactivated macrophages in the binding of 125I-C1q at 4 degrees C. The enhanced binding of 125I-C1q by activated peritoneal macrophages was reflected in an increase in the amount of 125I-C1q degraded by these cells as compared to resident peritoneal macrophages. This suggests that stimulation of phagocytic cells leads to an increase in the expression of C1q receptors and to a concomitant increase in the uptake and degradation of C1q.
Animals↗
Molecular basis of complement activation in ischemic myocardium: identification of specific molecules of mitochondrial origin that bind human C1q and fix complement.
Mitochondria may be a source of molecules that activate complement during ischemic injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. To identify specific molecules that activate the classical complement pathway, detergent lysates of canine cardiac mitochondria were fractionated by polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose paper (NCP). The NCP replicas of the gels were incubated with isolated C1q and fresh sera as a source of complement, washed briefly, and overlaid with sensitized sheep erythrocytes (RBC) in agarose. A cluster of four to six molecules between 45 and 53 kDa as well as four others, 34, 30, 26, and 23 kDa, consumed complement thereby preventing complement-mediated lysis of sensitized sheep RBC in the agarose overlay. Additional molecules reactive with C1 were identified by their ability to bind isolated human C1q and to serve as assembly sites for later acting complement components. Sites of localization of complement were demonstrated by incubating NCP replicas of fractionated mitochondria with antisera specific for C1q, C3, C5, and C9, followed by peroxidase-conjugated anti-immunoglobulin and substrate. A total of 12 C1q binding molecules ranging in size from 67 kDa to 23 kDa, which can fix later acting complement components, were identified. At least two of these reacted with antisera prepared against canine cardiac lymph collected in the first 3-4 hours after a 45-minute coronary artery occlusion. These studies present direct evidence that specific molecules, released from subcellular fractions of myocardial cells rich in mitochondria, can activate the complement cascade.
Animals↗
The asparagine-linked sugar chains of subcomponent C1q of the first component of human complement.
Human C1q, a subcomponent of the first component of complement, contains six asparagine-linked sugar chains in 1 molecule. The sugar chains are exclusively located in the COOH-terminal globular region which is composed of 330 amino acid residues. The sugar chains were liberated from the polypeptide portion by hydrazinolysis, and their structures were studied by the combination of sequential exoglycosidase digestion and methylation analysis. Based on the results, the structures NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 6(+/- NeuAcalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4(+/-Fucalpha1 leads to 6)GlcNAc were confirmed.
Asparagine↗
Chromatographic and electrophoretic studies of immune complexes in non-A, non-B hepatitis.
Immune complexes isolated from two patients with chronic non-A, non-B hepatitis, one patient with acute non-A, non-B hepatitis and one patient with juvenile rheumatoid arthritis were examined by means of a combined chromatographic and electrophoretic method. Both analyses showed the presence of complexes consisting of IgG, IgM, complement c1q factor and albumin; no antigen constituents were detected. The IgG-to-IgM ratio varied from 1:1 to 4:1, suggesting that one could be dealing with complexes of both IgG-IgM and IgG-IgG types. Moreover, the detectable presence of c1q factor might indicate that such complexes were capable of activating complement.
Antigen-Antibody Complex↗
Analysis of cell populations in crescentic glomerulonephritis.
The cell types present in the crescents were studied in 5 human patients with crescentic glomerulonephritis: two cases of systemic lupus erythematosus, one case of hemolytic uremic syndrome and two cases of rapidly progressive glomerulonephritis. Frozen sections of renal biopsies were studied by immunofluorescence, using murine monoclonal antibodies (orthoclones) against specific antigens on the membrane of human peripheral blood cells, and by histochemical methods. Monocytes (OKM1+, OKIa+ cells) but no lymphocytes (OKT+ cells), were detected in the crescentic glomeruli. Subsets of T lymphocytes (inducer-helper and cytotoxic-suppressor) were detected in the interstitium. Non-specific esterase-positive cells were observed in the glomeruli and in small numbers in the crescents. Fibrinogen deposits were present in the crescents of four of the five cases studied. No immunoglobulins (IgG, IgM, IgA) or complement (C1q, C3) deposits were detected in the crescents. Fibrinogen, immunoglobulins and complement were present in the glomerular tufts.
Antibodies, Monoclonal↗
[Effect of the C1q subcomponent of complement on thrombocyte adhesion and spreading].
In vitro experiments have shown that C1q at a concentration of 8-250 mkg/ml produced a 1.5-2-fold increase in platelet adhesion to glass. Low doses (4-60 mkg/ml) enhanced platelet splitting 2-3-fold. C1q did not cause platelet aggregation or change ADP-, adrenalin- and thrombin-induced aggregation. C1q participation in the induction of immune response is suggested.
Blood Platelets↗
Failure to detect deposition of complement and immunoglobulin in allergen-induced late-phase skin reaction in atopic subjects.
There still remains some controversy regarding the possible role of immune complexes in the pathogenesis of the late-phase skin reaction (LPSR). To assess this, skin biopsies were obtained from LPSR induced in atopic human subjects 6, 24 and 48 h after allergen challenge. Cryostat sections were stained by direct immunofluorescence for the presence of fibrinogen, immunoglobulin classes IgM and IgG and for the complement components C1q and C3c. Complement components were observed in only two of the 29 biopsies studied. In both instances, only C3c was detected. One of these subjects also had unequivocal IgG staining at 6 h. IgM staining was detected in two out of 10 subjects at 6 h but no significant deposition of immunoglobulins could be found at 24 or 48 h. Fibrinogen deposition was observed in about half of the biopsies at each time-point. This study suggests that substantial complement and immunoglobulin deposition are not overt features of the allergen-induced LPSR, although the presence of small amounts of immune complexes, below the sensitivity of the method employed cannot be excluded. Fibrin deposition occurs in the LPSR but does not appear to be a prerequisite for LPSR development.
Allergens↗
C1q binding and complement activation by capsular and cell wall components of S. pneumoniae type XIX.
Cell wall components (purified cell walls, teichoic acid and residual cell walls) from S. pneumoniae type XIX showed antibody independent C1q binding capacity, as assessed by C1q deviation test, with teichoic acid being the most efficient. Specific capsular substance did not bind C1q. All substances tested produced C1 activation in normal human serum, but not in hypo-gamma-globulinemic serum. Thus, teichoic acid showed high C1q binding capacity but did not activate C1 in the absence of antibodies. Teichoic acid was an effective activator of alternative pathway. Specific capsular substance did not activate the alternative pathway in C1q deficient serum or in Mg2+ -EGTA chelated normal serum.
Cell Wall↗
Segmental flexibility of the C1q subcomponent of human complement and its possible role in the immune response.
Fluorescence polarization techniques were used to study the rotational dynamics of the C1q subcomponent of human complement. C1q was covalently labeled with dansyl (DNS) chloride. Digestion of either C1q-DNS4.0 or C1q-DNS1.8 conjugates with pepsin showed that about 75% of the DNS probes were attached to the C1q globular heads and that the remainder were on the collagen-like stalk (peptic fragment). C1q-DNS conjugates readily agglutinated IgG-coated latex beads and combined with C1r2C1s2 to form hemolytically active 16 S C1-DNS. Both C1q-DNS and C1-DNS samples displayed steady-state rotational correlation time and fluorescence lifetime transitions near 48 degrees C. Hydrodynamic studies showed that C1q formed soluble aggregates near the transition temperature. In contrast, stalk samples with a DNS probe apparently attached to the large central fibril showed no thermal transitions or aggregation even when heated above 50 degrees C. Nanosecond fluorescence depolarization measurements detected restricted flexible motions of the C1q heads with an associated rotational correlation time, phi s, of about 25 ns. The C1q anisotropy decay was dominated, however, by a long component, phi L, of perhaps 1000 ns. Except for probe wiggle, the stalk-DNS anisotropy profile was essentially flat. The rapid rotations associated with phi s could represent restricted twisting motions of the arm-head segments or wobbling motions of the heads themselves. Such motions may facilitate binding of the C1q heads to immune complexes. Straightforward diffusion calculations indicated that phi L could represent either global tumbling of the entire C1q molecule or wagging motions of the individual arm-head segments, as suggested by electron micrographs. Upon binding of the C1q heads to an activator, some of the C1q segments may be held in a slightly more open or more closed conformation, which in turn may trigger activation of the C1 proenzymes. In conclusion, we suggest a plausible triggering mechanism for C1 activation that is compatible with the flexible properties of its subcomponents.
Complement Activating Enzymes↗
Identification of a collagen-like antigen different from C1Q component of complement in serum.
The problem of the proportions of hydroxyproline proteins in serum linked to collagen and to the C1Q component of complement was examined. The C1Q component from human and bovine serum, and acid soluble calf skin collagen were purified. Antibodies to soluble collagen were prepared. With these antibodies we found by immunoelectrophoresis that in serum two distinct antigens exist: one reacts with antiserum to C1Q and the other reacts with antiserum to collagen. The collagen-like antigen may be precipitated by sodium chloride 4.3 mol/1.
Animals↗
Rapid three-step purification procedure for isolation of the C1q component of human complement and its use in a solid-phase C1q binding assay.
A new procedure for the isolation of the C1q subcomponent of complement from human sera has been devised. The 3-step protocol employs DEAE Sephadex A-50, hydroxyapatite and Sephacryl S-200 chromatographies and can be performed within 9 h. It yields immunoglobulin-free homogeneous C1q protein with about 80% recovery. The isolated C1q protein is biologically active and may be used for the detection of circulating immune complexes in sera by the solid-phase C1q binding assay.
Antigen-Antibody Complex↗
Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.
The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.
Animals↗
Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3.
The present study was undertaken to examine and compare the direct effect of two Pseudomonas enzymes, elastase and alkaline protease, on the serum hemolytic complement as a whole, and on the two recognition molecules of complement, C1q and C3 in particular. The results of our study show that incubation of serum with 0-50 micrograms/ml elastase or protease (60 min, 37 degrees C) resulted in a dose-dependent depletion of hemolytic complement with the protease being 3-4 times more efficient than elastase. Incubation of highly purified C3 (20 hr, 37 degrees C) with protease (2% w/w) resulted in the conversion of the 190-kDa molecule to a 120-kDa fragment. When analyzed by SDS-PAGE under reducing conditions, the 120-kDa piece yielded three distinct bands: an intact 75-kDa beta-chain and two alpha-chain pieces of approximately 41- and 26-kDa. NH2-terminal end sequence analysis localized the 26-kDa fragment within the cysteine-rich 41-kDa, COOH-terminal piece. This in turn suggests that the 70-kDa fragment which is not accounted for on SDS-PAGE is derived from the NH2-terminal end of the alpha-chain molecule which is completely degraded into small fragments. While the degradation pattern obtained with elastase is similar to that of protease, the latter enzyme was found to be more efficient. Exposure of C1q (0-5 hr, 37 degrees C) to protease or elastase on the other hand appears to reveal preferential sensitivity of the 28-kDa A-chain and 24-kDa C-chain, of the C1q molecule, with the protease being more potent than the elastase. Since both C1q and physiologic fragments of C3 (C3b, iC3b, and C3dg) are important opsonins of varying efficiencies, degradation of these molecules by Pseudomonas enzymes may, in part, facilitate the survival and proliferation of the organism in plasma. Furthermore, degradation of the key recognition molecules of complement, C1q and C3, would enhance the virulence of this organism by aborting complement-mediated bacterial killing. In addition the results imply that during Pseudomonas bacteremia, PaAP may be a much more destructive enzyme than PaE with regards to C3 and C1q but combined, the synergistic effect may overwhelm not only the proteins of the complement system, but other proteins of the humoral immune defense system as well.
Amino Acid Sequence↗