Isolation of human C1q as a by-product of routine fractionation.

Clq can be isolated as a by-product of large scale fractionation. The euglobulins of fraction III are submitted to chromatography on CM-cellulose in a batch-procedure. The active fraction being frequently contaminated by lysozyme the protein can be further purified by gel-filtration in the presence of carbohydrates. The purified Clq is suitable for the detection of immune complexes.     

Clq enhancement of IgG-dependent eosinophil-mediated killing of schistosomula in vitro

Antibody-dependent eosinophil-mediated cytotoxicity plays a role in host protection against metazoan parasite invasion. We examined a possible role for Clq in eosinophil-mediated cytotoxicity by using a Schistosoma mansoni schistosomula killing system in vitro. The addition of monomeric purified human Clq enhanced IgG-dependent human eosinophil-mediated killing from 1.4-fold to 2.3-fold (mean percent killing 12% +/- 4 vs 21% +/- 4, p less than 0.005) when the immune IgG concentration was low. In contrast, there was no significant enhancement of neutrophil-mediated killing. When the IgG concentration was increased fourfold Clq did not cause enhancement of eosinophil-mediated killing (35% +/- 9 vs 37% +/- 5). Preincubation of eosinophils with type 1 collagen abrogated Clq enhancement of killing, raising the possibility of a receptor-mediated process, which depends upon cellular binding of Clq via the collagenous portion of the molecule. Eosinophils and neutrophils were examined for the presence of Clq receptors by using 125I labeled Clq. Clq binding to both cell types was saturable, reversible, and specific, indicating that binding is through specific receptors. Type 1 collagen inhibited binding of Clq to cells, suggesting that Clq binding is via the collagenous stalk of Clq. The number of receptors was approximately twice as high for eosinophils as compared with neutrophils (1.9 X 10(7) vs 1.1 X 10(7), p less than 0.025). Affinity constants for the two cell types were similar (1.5 X 10(7) vs 1.3 X 10(7). These findings suggest that Clq and receptors for Clq on eosinophils may be important for eosinophil-mediated schistosomula killing.     

Complement-dependent induction of DNA synthesis and proliferation of human diploid fibroblasts

The effects of fresh human serum (FHS) and heat-inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq-depleted FHS to support enhanced DNA synthesis whereas Clq-depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS-mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement-dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20-fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody-independent role for complement in the activation of a subpopulation of human diploid fibroblasts.     

猪C1q的分离纯化与鉴定

猪Ｃ１ｑ由猪血清通过ＰＥＧ沉淀优球蛋白、低离子强度透析沉淀、ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４８亲和层析、ＳｅｐｈａｄｅｘＧ－２００凝胶层析等步骤分离纯化,每３００ｍｌ血清可制得９．８ｍｇＣ１ｑ,产率为４６．７％。纯化的猪Ｃ１ｑ在ＳＤＳ－ＰＡＧＥ上显示出三条染色带,分别在２９、２６、２２ｋＤ处,薄层扫描结果表明纯度达９１％;纯化的Ｃ１ｑ保持了较高活性,终浓度为４μｇ／ｍｌ时仍可使致敏的绵羊红细胞出现明显的凝集现象。猪Ｃ１ｑ－ＥＬＩＳＡ结果表明,动物Ｃ１ｑ代替人Ｃ１ｑ应用于临床检测是可行的。     

Use of Clq enzyme-linked immunosorbent assay to detect plant viruses and their serologically different strains

Clq was prepared from bovine serum using a simple method involving repeated dialysis at low ionic strength in the presence of chelating agents (yield c. 3 mg/100 ml serum). It was viable when stored at -18°C for up to 2 months, and at 4°C for at least 10 wk in a storage buffer containing 10% sucrose. When used in Clq ELISA this test was as sensitive as the direct double antibody sandwich form of ELISA (direct ELISA) in detecting purified potato virus Y (PVY), with a limit of detection in both methods of c. 15 ng/ml, and slightly more sensitive in detecting purified cocksfoot mild mosaic virus (CMMV), with limits of detection of c. 15 ng/ml and c. 15–60 ng/ml respectively. Using an antiserum to one strain of each virus, Clq ELISA readily detected strains of PVY, CMMV, Andean potato latent virus (APLV) and barley yellow dwarf virus (BYDV). This included detection of APLV-Hu by APLV-Caj antibodies and CMMV(G) by PMV(S) antibodies, neither of which system gives detection in direct ELISA. Clq ELISA was therefore less specific than direct ELISA in detecting serologically different virus strains. Virus detection by Clq ELISA was inhibited when sap of tobacco, Nicotiana clevelandii and Setaria italica was used at low dilution. Inhibition by N. clevelandii sap was alleviated by using increased concentrations of virus specific antibody to detect APLV and plum pox virus. Also, extracting APLV infective N. clevelandii or CMMV infective S. italica saps in a minimum of buffer, centrifuging at low speed and diluting the supernatant before testing, partially overcame the inhibition. The inhibitory substance(s) in sap may act by preventing the binding of Clq to virus-antibody aggregates. Sap of wheat, oat and barley did not appear to have an inhibitory effect and BYDV was readily detected in naturally infected field grown plants of these species.     

Cross--reactivity of the V3-speciflc antibodies with the human C1q

It has been previously shown that the sequence similarity between a portion of the envelope glycoprotein 120 (gp120) from the human immunodeficiency virus type-1 (HIV-1) and several types of human collagen and collagen-like molecules exists. That observation led to the suggestion that the antibodies against the third hypervariable region (V3) of HIV-1 gp120 (V3-specific antibodies) might have a role in the autoimmune phenomena observed in HIV-infected persons. In this study we have examined the cross-reactivity of the V3-specific antibodies purified from sera of HIV-infected individuals, sera obtained from the rheumatoid arthritis and systemic lupus crythematosus patients, as well as from the sera of healthy volunteers with the separate chains of a subcomponent of the first component of the human complement system, Clq. Our results show that the V3-specific antibodies are present in the sera of the HIV-infected individuals, patients suffering of the systemic autoimmune diseases as well as in the sera of healthy volunteers. Whereas these antibodies appeared in the HIV+-sera after antigen challenge, those present in the HIV- -sera probably represent the antibodies that are cross-reactive with the antigen. V3-reactive antibodies can be purified by affinity chromatography and they were highly specific for the V3-peptide. Additionally, they showed cross-reactivity with the separate chains of the human Clq as well as with the chicken collagen type VI. Possible physiological implications are discussed.     

Evidence for arginine residues in the immunoglobulin-binding sites of human Clq

The immune complex binding activity of human Clq was lost following treatment of the protein with the arginine-selective reagents cyclohexane 1,2-dione and phenylglyoxal. Both inactivations followed pseudo-first-order kinetics. The affinity of Clq for immune complexes was reduced 7-fold following cyclohexane-1,2-dione treatment, and could be substantially restored by treatment of the modified protein with hydroxylamine. Heat-aggregated IgG protected Clq against inactivation by both reagents. Incorporation of 25 molecules of [7-¹⁴C]phenylglyoxal per Clq molecule completely inactivated the protein. These data are consistent with the presence of arginyl residues in the immunoglobulin recognition sites of human Clq.     

Evidence for histidine residues in the immunoglobulin-binding site of human C1q

The immunoglobulin-binding activity of subcomponent Clq of human complement is lost following treatment with diethylpyrocarbonate; the inactivation showed first-order kinetics with respect to time and modifier concentration. Soluble IgG oligomers protected Clq against diethylpyrocarbonate modification. Treatment of modified Clq with hydroxylamine resulted in an 85% recovery of its ability to bind to aggregated immunoglobulin. The inactivation process was associated with modification of 12.1 +/- 0.7 histidine residues per Clq molecule. These data are consistent with the presence of histidine residues in the immunoglobulin-binding sites of Clq; these residues may participate in ionic interactions with the carboxyl groups known to be in the Clq binding site of IgG.     

Clq inhibition of the interaction of collagen with human platelets.

Human Clq, isolated in pure state after affinity chromatography on IgG-Sepharose, inhibited collagen-induced aggregation and release of 14C-Serotonin from prelabeled human platelets. Platelet aggregation induced by ADP or thrombin was not inhibited by Clq. Also, the adherence of platelets to glass surfaces was significantly diminished by Clq. In contrast, aggregated Clq mimicked the effect of collagen in causing platelet aggregation and release of serotonin. It appears that monomeric Clq, which has structural similarities to collagen competes with collagen for specific sites on the platelet surface.     

抗人Clq单克隆抗体轻链可变区基因的克隆及序列测定

 抗人Ｃｌｑ单克隆抗体轻链可变区基因的克隆及序列测定陈克敏,朱锡华（第三军医大学免疫学教研室,重庆６３００３８）目前国内外对基因工程抗体的研究较多,但尚未见补体基因工程的研究报道．一般的方法是经ＲＮＡ反转录合成ｃＤＮＡ,再以ＰＣＲ扩增,克隆Ｉｇ可变区基...     

Solvent effects on the structure of rabbit Clq, a subcomponent of the first component of complement.

The effect of solvent conditions on the conformation of rabbit Clq was studied by both spectroscopic and nonspectroscopic methods. The conformation of Clq in buffered saline solutions at pH 7.4 or 6.0 did not differ significantly from Clq at twice the saline concentration as determined with circular dichroism, difference spectroscopy, and tritium-hydrogen exchange techniques. Addition of calcium to the buffers had no structural effects in any of the conditions examined. Hydrogen exchange experiments performed at pH 7.4 were also unaffected by magnesium, manganese, or ethylenediaminetetraacetic acid. With all the methods used a pH effect was observable between 5.1 and 8.3. From solvent perturbation difference spectroscopy results it was calculated that the equivalent of 10 +/- 2 and 6 +/- 1 mol of tyrosine and tryptophan/mol of Clq, respectively, became exposed at the lower pH. A small positive CD band in the 231 to 235 nm region decreased in wavelength and increased in magnitude as a function of decreasing pH, indicating tyrosine exposure at the lower pH and possibly changes in the collagen-like structure of Clq. Hydrogen exchange experiments indicate a small, but significant, conformation transition occurring in the pH 5 region and a stabilization of conformation between pH 6 to 8. From these results the conformational pH dependence was interpreted as an acid expansion of Clq with a minor conformational transition occurring between pH 5 AND 6. These effects may in part be associated with decreased Clq-Ig interactions which have been observed at the lower pH.     

Solution structure of human and mouse immunoglobulin M by synchrotron X-ray scattering and molecular graphics modelling. A possible mechanism for complement activation

The pentameric 71-domain structure of human and mouse immunoglobulin M (IgM) was investigated by synchrotron X-ray solution scattering and molecular graphics modelling. The radii of gyration RG of human IgM Quaife and its Fc5, IgM-S, Fab'2 and Fab fragments were determined as 12.2 nm, 6.1 nm, 6.1 nm, 4.9 nm and 2.9 nm in that order. The RG values were similar for mouse IgM P8 and its Fab'2 and Fab fragments, despite the presence of an additional carbohydrate site. The IgM scattering curves, to a nominal resolution of 5 nm, were compared with molecular graphics models based on published crystallographic alpha-carbon co-ordinates for the Fab and Fc structures of IgG. Good curve fits for Fab were obtained based on the crystal structure of Fab from IgG. A good curve fit was obtained for Fab'2, if the two Fab arms were positioned close together at their contact with the C mu 2 domains. The addition of the Fc fragment close to the C mu 2 domains of this Fab'2 model, to give a planar structure, accounted for the scattering curve of IgM-S. The Fc5 fragment was best modelled by a ring of five Fc monomers, constrained by packing considerations and disulphide bridge formation. A position for the J chain between two C mu 4 domains rather than at the centre of Fc5 was preferred. The intact IgM structure was best modelled using a planar arrangement of these Fab'2 and Fc5 models, with the side-to-side displacement of the Fab'2 arms in the plane of the IgM structure. All these models were consistent with hydrodynamic simulations of sedimentation data. The solution structure of IgM can therefore be reproduced quantitatively in terms of crystallographic structures for the fragments of IgG. Putative Clq binding sites have been identified on the C mu 3 domain. These would become accessible for interaction with Clq when the Fab'2 arms move out of the plane of the Fc5 disc in IgM, that is, a steric mechanism exposing pre-existing Clq sites. Comparison with a solution structure for Clq by neutron scattering shows that two or more of the six globular Clq heads in the hexameric head-and-stalk structure are readily able to make contacts with the putative Clq sites in the C mu 3 domains of free IgM if if the Clq arm-axis angle in solution is reduced from 40 degrees-45 degrees to 28 degrees. This could be the trigger for Cl activation.     

Inhibition of the binding and activation of the first component of human complement. The effect of synthetic peptides, immunoglobulin fragments and various proteins

A study has been carried out on the inhibition of the subcomponent Clq binding to sensitized sheep erythrocytes (EA) by the following synthetic peptides mimicking the structure of a putative complement binding site of immunoglobulin G: Boc-Trp-Tyr, Boc-Tyr-Trp, Trp-Tyr, Boc-Trp-Phe, Boc-D-Trp-D-Tyr, Boc-D-Tyr-D-Trp, Boc-Leu-Leu, Ac-Phe-Tyr, and commercial Thr-Lys-Pro-Arg (tuftsin). Boc-Trp-Tyr was found to be the most potent inhibitor of Clq binding to EA (Ki 2.86 X 10(-4) M), tuftsin ranking second with Ki 6 X 10(-4) M. The D,D-dipeptides failed to inhibit the Clq binding at the investigated concentrations. Insoluble Z-Trp-Tyr-OMe activated a classical pathway of complement system, as monitored by consumption of C4, C2 and C3 components. Synthetic octapeptide Boc-Glu-Val-Asp-Leu-Leu-Lys-Asp-Glu-OMe (corresponding to the sequence 36-43 of beta 2-microglobulin) inhibited the Clq binding with Ki 4.7 X 10(-4) M, which gave grounds for localizing the complement binding site in beta 2-microglobulin. The finding in the Clq structure of the peptide sequence homologous to than of the pepsin active site, as well as the close similarity in the specificity of these proteins towards hydrophobic amino acid residues justified the assumption on the same structural bases of their specificity. The results of the present study, along with the literature data, underlie the hypothesis on the involvement in the complement binding of the following IgG residues: Trp277, Tyr278, Lys320, Lys322, Glu318 and Lys290. The enlisted residues are closely located in the three-dimensional structure of the CH2 domain of IgG. Lysozyme and lactalbumin having the sequences homologous to Trp277-Tyr278 of IgG inhibited Clq binding to EA with Ki 3 and 1.5 microM respectively.     

IgG binding to cytoskeletal intermediate filaments activates the complement cascade

The cellular plasma membrane becomes permeable to macromolecules during the cell injury process. This results in exposure of the interior of the cell to plasma proteins and to high-affinity binding of the Fc part of IgG to intermediate filaments (Hansson, G K, Starkebaum, G A, Benditt, E P & Schwartz, S M, Proc natl acad sci USA 81 (1984) 3103). Such IgG binding could be an early step in a process that serves to eliminate the injured cell. We have now identified its effect on the complement system. Intermediate filaments were reconstituted in vitro from purified vimentin, and incubated with plasma proteins. Cross-linker experiments showed binding of the heavy chain of IgG to vimentin, indicating that the vimentin protein carries an Fc-binding site. In contrast, no direct binding of complement factor Clq to vimentin could be detected. Binding of both IgG and Clq could, however, be detected by immunofluorescence when cytoskeletons of cultured endothelial cells were incubated with fresh serum. Therefore, IgG binding to filaments in the presence of serum is accompanied by Clq binding to IgG. This was in turn followed by fixation of C4 and C3 to intermediate filaments in a process that was dependent on both Ca2+, Mg2+ and Clq, indicating that it was part of a complement activation via the classical pathway. Exposure of fresh serum to intermediate filaments also resulted in production of the anaphylatoxic complement cleavage fragment. C3a, with a dose-response relationship between the amount of filaments present and the amount of C3a generated. Chemotactic activity towards granulocytes and monocytes was also generated by exposure of serum to intermediate filaments, and this activity was dependent on the presence of complement factor C5 and on the classical complement activation cascade, implying that it was due to the C5a peptide. Exposure of the interior of the cell to plasma proteins thus results in binding of IgG to intermediate filaments and activation of the complement cascade via the classical pathway. This, in turn generates bioactive mediators which may recruit leukocytes to the injured cell (C5a) and have profound effects on vascular permeability (C3a, C5a). We propose that this is part of a scavenger mechanism for the elimination of damaged cells.     

Use of glutaraldehyde-treated erythrocytes as indicator cells for the identification of the Clq component on the macrophage membrane by a rossette assay

Glutaraldehyde-treated sheep red blood cells (EGA) form rosettes with macrophages of various origin. The binding of EGA to cell surface is mediated by the membrane associated Clq as demonstrated by the inhibition of EGA rosette formation either after treatment of macrophages with anti-Clq serum or after treatment of EGA with Clq. The results clearly demonstrate that the EGA-binding receptor of macrophage is the Clq component.     

F(ab')2 antibody fragments against Trypanosoma cruzi calreticulin inhibit its interaction with the first component of human complement

Trypanosoma cruzi calreticulin (TcCRT), described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host/parasite equilibrium. In an in vitro correlate of this situation, we show that the Clq/TcCRT interaction is inhibited by F(ab')2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion Clq, and this Clq is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate Clq could be inhibited by F(ab')2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process.     

Fluorometric detection of low temperature thermal transitions in the Clq component of human complement

Fluorescence studies with the human complement component Clq were performed as a function of temperature and demonstrated the existence of low temperature, thermally induced structural transitions in the Clq molecule. Both intrinsic protein fluorescence and the fluorescence of the apolar probe 2-p-toluidinylnaphthalene-6-sulfonate independently showed thermal transitions at 15°C, 35°C and 48°C. Clq activity measurements indicated no loss of hemolytic activity at temperatures below 46°C. It is proposed that these structural transitions are a consequence of the internal flexibility of the native Clq molecule.     

Cytoplasmic origin of the so-called nuclear neutral histone protease.

1. We have investigated the origin of proteolytic activity which causes degradation of histones in chromatin isolated from Xenopus liver and the rat liver at neutral pH. Polyacrylamide disc gel electrophoresis was used for detection of proteolytic products of histones. 2. No proteolytic degradation of histones occurs in chromatin isolated from Xenopus erythrocytes and rat liver according to our procedure even after prolonged incubation at pH 8.0 and pH 5.0. However with chromatin isolated from Xenopus liver a high level of histone degradation is observed under similar conditions. 3. Mixing isolated nuclei from Xenopus erythrocytes with a crude cytoplasmic fraction from Xenopus liver causes histone proteolysis in isolated chromatin at pH 8.0. In similar experiments with corresponding fractions from rat liver histone proteolysis can be introduced only after repeated freezing and thawing of the cytoplasmic fraction. 4. A purified lysosomal preparation from rat liver causes a similar type of histone degradation upon incubation with chromatin from Xenopus erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal erythrocytes and rat liver. 5. The neutral proteolytic activity that can be introduced in isolated chromatin by a crude cytoplasmic fraction and by a purified lysosomal fraction from rat liver is inhibited by sodium bisulphite. 6. We conclude that the neutral proteolytic activity which causes degradation of histones in isolated chromatin is due to a contamination with neutral protease(s) originating from cytoplasmic organelles.     

Protein disulphide-isomerase of chick-embryo tendon.

Protein disulphide-isomerase can be partially purified from the high-speed-supernatant fraction of extensively disrupted chick-embryo tendon tissue. The catalytic properties of the preparation resemble those of the enzyme from mammalian liver. Gel electrophoresis and isoelectric focusing show the enzyme to be very acidic, with pI 4.4 +/- 0.3. Gel filtration indicates an Mr for the active enzyme of 140 000. The enzyme can be partially purified by preparative gel filtration or isoelectric focusing, but its limited stability has prevented purification to homogeneity; active fractions from both gel filtration and isoelectric focusing show two major polypeptide components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The major polypeptides present in partially purified preparations have Mr 45 000 and 55 000; the latter band co-distributes with the enzyme activity in fractionations by both gel filtration and isoelectric focusing. The subcellular location of the enzyme cannot be established from work on homogenates of whole tissue, which are extensively disrupted. In homogenates from isolated tendon cells, the enzyme is located in a vesicle fraction that is excluded from Sepharose 2B but is of low density and can only be sedimented at very high speeds. This fraction is identified as deriving from the endoplasmic reticulum on the grounds of marker-enzyme studies and electron microscopy.     

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.