Activation of the lectin complement pathway by H-ficolin (Hakata antigen).

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.

Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome.

Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE.

Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis.

C1q, a subunit of the first component (C1) of the classical complement pathway, and the pulmonary surfactant protein SP-A are structurally homologous molecules, each having an extended collagen-like domain contiguous with a non-collagenous domain. It is the collagen-like region of C1q that binds to mononuclear phagocytes and mediates the enhancement of phagocytosis of opsonized particles by these cells. Because SP-A enhances the endocytosis of phospholipids by alveolar type II cells and alveolar macrophages, we examined whether these two molecules were functionally interchangeable. The phagocytosis of sheep erythrocytes opsonized with IgG or with IgM and complement was enhanced by the adherence of monocytes or macrophages, respectively, to SP-A. The enhanced response was dependent on the concentration of SP-A used for coating the surfaces, similar to that seen when monocytes were adhered to C1q-coated surfaces. Both the percentage of cells ingesting the opsonized targets and the number of targets ingested per cell increased with increasing concentrations of SP-A. No such enhancement was seen with cells adhered to albumin, iron-saturated transferrin, or uncoated surfaces. However, SP-A did not substitute for C1q in the formation of hemolytically active C1. C1q did not stimulate lipid uptake by alveolar type II cells or alveolar macrophages and had only a slight inhibitory effect on the binding of SP-A to alveolar type II cells. Thus, these results suggested that a function which requires interactions of both the collagenous and the non-collagenous regions (i.e. initiation of the classic complement cascade) could not be mimicked by a protein sharing structural macromolecular similarity but lacking sequence homology in the non-collagen-like region. However, SP-A could substitute for C1q in stimulating a function previously shown to be mediated by the collagen-like domains of the C1q molecule.

[A method of isolating C1q from human serum and its use in the solid-phase immunoenzyme determination of immune complexes]. / Metod vydeleniia C1q iz syvorotki cheloveka i ispol'zovaniia ego v tverdofaznom immunofermentnom opredelenii immunnykh kompleksov.

C1q was isolated from human serum by dialysis in 0.24 M EDTA, followed by affinity chromatography on immobilized IgG and removal of IgG traces in a column with anti-IgG antibodies. Microplates were coated with C1q in PBS at 10-20 mg/l, nonspecific binding sites were saturated with human serum albumin. The sera were diluted 16-fold in 0.05 M PBS, 0.01 M EDTA, 0.05% Tween. After incubation with diluted samples the plates were treated with horseradish peroxidase--anti-human IgG conjugates. Enzymic activity was measured by adding p-phenylenediamine (0.2 g/l) in acetate buffer, pH 5.9, containing 0.05% H2O2. The sensitivity of the assay ranged between 2.5 and 300 mg/l.

Further evidence for dilution-dependent dissociation of C1q in human serum.

Dissociation of the C1q subcomponent in native C1 upon dilution was reexamined by using ultracentrifugation in a sucrose gradient and high performance liquid chromatography system with a size exclusion column for separating the dissociated C1q fractions. The antigenic content of C1q in each fraction was detected by ELISA and Western blotting; binding to erythrocyte antibody was also determined. The results confirmed a previous claim that C1q in native C1 dissociated as a function of dilution: up to 14.5% of C1q antigen was in the low molecular weight form (approximate S value: 4-5). Commercial preparations of purified C1q also contained C1q antigen in the low molecular weight form.

C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme.

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.

A mechanism for the spontaneous activation of the first component of complement, C1, and its regulation by C1-inhibitor.

We have developed a method to initiate spontaneous activation of the first component of complement in serum, by the removal of C1-inhibitor through complexation with added C1s. Preliminary experiments to test this method using C1 reconstituted from its purified subcomponents led to an unexpected result: pre-incubation of the reassembled subcomponents with C1-inhibitor, followed by its removal with C1s, altered the subsequent pattern of spontaneous activation. Thus, pre-incubation with C1-inhibitor at 37 degrees C for 1 h resulted in sigmoidal activation of C1 with a prolonged lag phase. In contrast, pre-incubation with C1-inhibitor on ice for the same time resulted in subsequent rapid, pseudo first order activation of C1 with a half-life of about 5 min. We have examined the activation kinetics under a variety of conditions, and our data are consistent with a model proposed by Lepow and coworkers in 1965, involving both spontaneous activation and C1 catalyzed activation: (1) C1----k1 C1 (2) C1----k2C1 C1 According to this model, the role of C1-inhibitor is to eliminate the second step by rapidly forming a tight complex with C1 which becomes irreversible at 37 degrees C. When C1s was added to normal human serum, activation at 37 degrees C was also sigmoidal, similar to that of reconstituted C1.

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K).

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.

Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain.

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.

C1q binding by a high affinity anti-fluorescein murine monoclonal IgM antibody and monomeric subunits.

Comparative interactions of purified rabbit C1q with 18-2-3, a high affinity (2-3 X 10(10) M-1) anti-fluorescein (anti-F1) murine monoclonal IgM antibody (pentamer) and constitutive monomeric subunits (IgMs) were studied. Using a solid phase radioimmunoassay (SPRIA), based on immobilized polyvalent antigen, it was shown that the mechanism of C1q binding to IgM was characteristically multiphasic while IgMs yielded monophasic binding curves. The latter compared qualitatively and quantitatively with a monoclonal IgG2a anti-fluorescein antibody with the same intrinsic affinity of 2-3 X 10(10) M-1. C1q binding efficiency to antibodies was significantly enhanced when the immunoglobulins interacted with immobilized multivalent antigen. Monoclonal IgM antibody bind identically to six F1-carrier protein conjugates independent of epitope (F1) density. In contrast, the C1q-antibody interaction binding was dependent upon epitope density. An average distance between F1 epitopes of 80 A was optimal for C1q binding by IgM. At low concn of IgM, when fluorescein was bound by antigen-binding sites on adjacent subunits of an intact pentamer, C1q appeared to bind IgM intramolecularly.

Purification and characterization of the 1q subcomponent of canine complement and its use in the 125I-C1q binding assay for detection of immune complexes.

The complement subcomponent, C1q, was isolated from serum obtained from clinically normal dogs, using a rapid 2-step process involving affinity chromatography. Yield of C1q ranged from 8 to 10 mg/L of serum. Hemolytically active C1q had 3 protein bands after sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and formed a single line of identity with rabbit anti-canine C1q. The amino acid composition of canine C1q was similar to that of human C1q and contained a high percentage of glycine. Isolated canine C1q was iodinated, and the fluid-phase binding assay was used to detect circulating immune complexes in dogs with systemic lupus erythematosus and rheumatoid arthritis.

Preliminary studies on the detection, isolation and partial characterization of Clq-containing IgM immune complexes in sera of patients with primary biliary cirrhosis.

The complement system is activated in primary biliary cirrhosis (PBC) and this activated state may be medicated by immunoreactive IgM. To identify and further characterize the relationship between the complement (Clq) and IgM in PBC sera, we developed an anti-Clq ELISA method which allowed detection of Clq-containing circulating immune-like complexes. Utilizing this technique, sera from 3 out of 5 patients with PBC revealed circulating immune-like complexes. Moreover, when serum samples were specifically examined for the presence of IgM containing Clq complexes, four of four samples examined were positive. Additional experiments indicated that these immune-like complexes could be removed from PBC sera by means of an anti-Clq immunoadsorbent. Upon subsequent isolation and characterization, these immune-like complexes demonstrated polypeptide chains corresponding to both human Clq and human IgM. Our experimental studies establish that Clq-containing IgM-like complexes can occur in the serum of patients with PBC, and provide additional support for the proposal that immunoreactive IgM can contribute to the activated complement system observed in PBC.

A rapid and efficient method for the purification of the complement subcomponents C1r and C1s in zymogen form using fast protein chromatography.

The purification of the subcomponents C1r and C1s of the first component of complement involves multiple steps and is time-consuming. This accounts for the frequently observed partial activation of the subcomponents. In this report we propose a simplified procedure of purification using a batch method and fast protein chromatography avoiding a shift of pH. The method provides C1r and C1s in a yield of 35 and 60% respectively. In addition, this study provides a simple and sensitive test to assess functional purity of C1r and C1s with respect to the other C1 subcomponents.

Activation of the classical complement pathway by BioRex-70.

The cation exchange resin BioRex-70 was able to activate the classical complement pathway in human serum at 37 degrees C over the resin concentration range 0-5% (v/v). Using zymosan-treated human serum, it was found that the activation proceeded as far as complement protein C3.

Isolation and characterization of macrophage-derived C1q and its similarities to serum C1q.

Recently, we have shown that the collagen-like, Fc-recognizing subcomponent C1q of the first complement component is synthesized by human, guinea pig and mouse peritoneal macrophages. To test whether macrophages may contribute to the serum pool of C1q, C1q was purified from guinea pig serum and from guinea pig peritoneal macrophage supernatants and compared for similarities. Both molecules had a similar sedimentation rate (macrophage C1q: 11.3 S, serum C1q: 11.2 S) and showed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions three identical bands with molecular weights of Mr, 29 000, Mr, 27 000 and Mr 23 000 for the A, B and C chains, respectively. Both C1q molecules migrated by immunoelectrophoresis in the gamma region and, in Ouchterlony analysis, showed complete antigenic identity with rabbit anti-serum C1q. These experiments demonstrate the antigenic and protein chemical similarities between serum C1q and C1q secreted by macrophages supporting the idea that macrophages have to be considered as one potential source of serum C1q. Furthermore, macrophage-derived C1q may be of importance in the local microenvironment at an inflammatory site involving macrophages.

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation.

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.