Search PubMed⌕ Search

Biomedical subjects

M G Colomb

Publications and source records attributed to M G Colomb.

At least 73 records · Page 4Linked to original sources
PubMed1984

Activation of C1.

The first component of complement, C1, is a calcium-dependent complex of two loosely interacting subunits: C1q, responsible for the binding of activators to C1; C1r2-C1s2, which supports the autoactivation potential of C1, together with the proteolytic activity of activated C1- on its two substrates, C4 and C2. Isolated dimeric C1r2 is able to autoactivate through an intradimer cross-proteolysis; this capacity is lost when C1r2 is associated with two molecules of C1s inside the calcium-dependent C1r2-C1s2 subunit; this capacity is again observed in reconstituted C1. A model for reconstituted soluble C1 is proposed, based on electron microscopy, neutron diffraction, ultra-centrifugation, various biochemical findings, as well as functional properties of C1 or of its subcomponents. The flexible rod-like structure of C1r2-C1s2 is folded around two arms of C1q, with the catalytic domains of C1r and C1s inserted inside the cone defined by the C1q stalks. Activation of C1 which, in vivo, is controlled by C1 inhibitor, can be achieved by various activators, such as immune complexes; it appears to result from the suppression of a negative control and resides in a positive modulation of the intrinsic autocatalytic potential of C1r inside C1.

Amino Acid Sequence↗
PubMed1984

Diamine-induced dissociation of the first component of human complement, C1.

Lysine has been shown to inhibit spontaneous and antibody-dependent C1 activation. This paper demonstrates that lysine does not prevent autoactivation of purified C1r. 20 mM lysine, 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane or 1,5-diaminopentane are able to dissociate C1 into its two entities, C1q and the calcium-dependent C1r2-C1s2 complex. Ig-ovalbumin insoluble complexes bearing C1 are also dissociated by lysine and the above-mentioned diamines used at the same concentration: C1q remains bound to the complexes whereas the C1r2-C1s2 complex is partially solubilized. The effect of lysine or diamines is not due to a competition with calcium for calcium-binding sites, as increasing concentrations of calcium even slightly increase the dissociation due to the amines. The dissociative effect is dependent on the carbon chain length of the diamines, with an optimum for 1,3-diaminopropane. It is also dependent on the relative 'cis-position' of the amino groups in the diamines. Polyamines such as spermine and spermidine are also able to dissociate C1 with even a higher efficiency than lysine and putrescine. Thus, a diamine-induced 'structural inhibition' of C1 is demonstrated, of potential interest for a pharmacological control of complement activation.

Calcium↗
PubMed1984

Characterization of the C1q receptor on a human macrophage cell line, U937.

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.

Binding Sites↗
PubMed1984

Comparative study of the fluid-phase proteolytic cleavage of human complement subcomponents C4 and C2 by C1s and C1r2-C1s2.

The C3 convertase of the classical pathway of complement is composed of fragments C4b and C2a resulting from cleavage of C4 and C2 by activated C1. The limited proteolysis of these two different substrates by the same protease, C1s, has been studied in the fluid phase using purified proteins. The turnover numbers of C2 and C4 cleavage by C1s were affected to different extents, depending on whether C1s was alone or associated with C1r or with monoclonal antibodies to C1s. The binding of C2 to C4 favours the proteolysis of C2 by C1s, as revealed by the use of I2-treated C2.

Antibodies, Monoclonal↗
PubMed1983

[Role of immunoglobulins in the activation of complement].

Depending on their class and sub-class, immunoglobulins may activate the complement by the classical pathway or by the alternative pathway. This activating property depends on how immunoglobulins are associated. Activation by the classical pathway implies binding to, and activation of C1. Binding is essentially an ion type interaction between C1q and--in the case of human IgG's for instance--the C gamma 2 domain. It has recently been investigated in depth by various techniques, such as inhibition of the C1q--IgG interaction by chemical compounds or by peptides isolated from C gamma 2. Activation of C1 seems to involve some elements of C gamma 3. In addition to initiating the classical pathway, immunoglobulins may act as acceptors of the classical C3-convertase (C4b C2a); a structure accepting the newly formed C4b is present in the Fab portion of IgG. It would appear that optimal functioning of the complement system is achieved when the C1 activating site and the C3-convertase forming site are close together on the IgG molecule, since the newly born peptidic fragments have a very short life. Immunoglobulins may activate the alternative pathway via their Fab portion. This property is directly related to the ability of Ig's to accept nascent C3b, which probably includes that of accepting nascent C4b as described above. In the alternative pathway C3-convertase may form when structural conditions around IgG-bound C3b encourage binding of B to C3b rather than the intervention of I and H factors which degrade C3b. The Ig-C3b interaction responsible for activation of the alternative pathway may also be directly involved in solubilization of immune complexes during activation of complement.

Animals↗
PubMed1983

Biosynthesis in vitro of complement subcomponents C1q, C1s and C1 inhibitor by resting and stimulated human monocytes.

The capacity of cultured human monocytes to synthesize and to secrete the subcomponents of C1 and C1 inhibitor was examined. Non-stimulated monocytes secreted C1q and C1s from day 5 of culture. C1s reached a plateau immediately at its maximum level, whereas C1q secretion increased progressively until the end of the second week. Between day 12 and day 25, C1q secretion remained nearly constant (1-15 fmol/day per microgram of DNA, depending on the donor), whereas C1s secretion decreased and even in some cases stopped. C1r and C1 inhibitor were not secreted in detectable amounts by these resting cells. Stimulation of monocytes by yeasts, immunoglobulin G-opsonized sheep red blood cells or latex beads did not modify consistently C1q and C1s secretion. Activation by conditioned media from mitogen-, antigen- or allogeneic-stimulated lymphocyte cultures increased C1q production from 2 to 7 times and re-activated C1s secretion. Under the same conditions of activation, C1 inhibitor was secreted (up to 300 fmol/day per microgram of DNA) and C1r became detectable in culture supernatants. Isolated human monocytes are thus able to synthesize the whole C1 subcomponents; C1, if assembled, could be protected from non-immunological activation by locally produced C1 inhibitor. Activated monocytes appear to be a good tool for studying the assembly of C1 subcomponents and the role of C1 inhibitor in this process.

Cells, Cultured↗
PubMed1983

Autoactivation of human complement subcomponent C1r involves structural changes reflected in modifications of intrinsic fluorescence, circular dichroism and reactivity with monoclonal antibodies.

Autoactivation of C1r is closely correlated with an irreversible increase of its intrinsic fluorescence. The activation and the fluorescence increase of C1r are accelerated on addition of activated C1r. Ca2+, di-isopropyl phosphorofluoridate and C1 inhibitor, which all inhibit, although to different extents, C1r activation, inhibit in parallel the fluorescence increase. C1r activation is blocked at pH 4.0-5.0, whereas it is accelerated at pH 10.5; under the same conditions the fluorescence increase shows parallel effects. No such fluorescence increase is observed during C1s activation by trace amounts of C1r. Far-u.v. circular-dichroism spectra of C1r indicate 73 and 78% of unordered form in both the proenzyme and the activated species respectively. The slight changes observed on activation are not restricted to C1r, as comparable results are obtained for proenzyme and activated C1s. C1r activation appears thus to involve structural changes leading to an 'activated state' distinct from the 'proenzyme state'. Monoclonal antibody to activated C1r is poorly reactive with proenzyme C1r, a finding that also supports this hypothesis.

Antibodies, Monoclonal↗
PubMed1982

A study of a covalent-like interaction between soluble nascent C4b and C4-binding protein.

In the classical pathway of complement, the interaction between C4b and C4bp can be considered as a control of the C3 convertase formation. Purified C4-binding protein (C4bp) interacts with soluble nascent C4b to form covalent-like complexes; the interaction is also possible with nascent C4b-like C4, but not with C4, C4b or C4b-like C4. Formation of the complexes upon incubation of C4bp, C4 and C1s appears to involve a single link between a subunit of C4bp and the alpha' chain of C4b, as observed by SDS-polyacrylamide gel electrophoresis in reducing conditions (160 000 dalton band). In non-reducing conditions, a mixture of C4b-C4bp complexes is observed as a function of the C4b:C4bp molar ratio, with apparent molecular weights differing by a value of 210 000 and reflecting different C4b-C4bp associations. A maximum of five molecules of C4b are bound per molecule of C4bp, which appears to consist of 10 subunits of apparent molecular weight 72 000. The link between C4b and C4bp is partially destroyed by 1 M hydroxylamine at pH 9.0; its formation is strongly inhibited by 3.5 mM hydroxylamine or 60 mM methylamine at pH 9.0. These findings suggest an ester or amide bond between the activated carboxyl group of the thioester bridge in the alpha' or alpha chain of nascent C4b or C4b-like C4 and a hydroxyl or amino group of C4bp. Thus, C4bp might compete with other C4b acceptors such as membranes or IgG.

Carrier Proteins↗
PubMed1982

Structural features of the first component of human complement, C1, as revealed by surface iodination.

Lactoperoxidase-catalysed surface iodination and sucrose-gradient ultracentrifugation were used to investigate the structure of human complement component C1. 1. Proenzymic subcomponents C1r and C1s associated to form a trimeric C1r2-C1s complex (7.6 S) in the presence of EDTA, and a tetrameric Clr2-C1s2 complex (9.1 S) in the presence of Ca2+. Iodination of the 9.1 S complex led to a predominant labelling of C1r (70%) over C1s (30%), essentially located in the b-chain moiety of C1r and in the a-chain moiety of C1s. 2. Reconstruction of proenzymic soluble C1 (15.2 S) from C1q, C1r and C1s was partially inhibited when C1s labelled in its monomeric form was used and almost abolished when iodinated C1r was used. Reconstruction of fully activated C1 was not possible, whereas hybrid C1q-C1r2-C1s2 complex was obtained. 3. Iodination of proenzymic or activated C1 bound to IgG-ovalbumin aggregates led to an equal distribution of the radioactivity between C1q and C1r2-C1s2. With regard to C1q, the label distribution between the three chains was similar whether C1 was in its proenzymic or activated form. Label distribution in the C1r2-C1s2 moiety of C1 was the same as that obtained for isolated C1r2-C1s2, and this was also true for the corresponding activated components. However, two different labelling patterns were found, corresponding to the proenzyme and the activated states.

Centrifugation, Density Gradient↗
PubMed1982

Fluid phase activation of proenzymic C1r purified by affinity chromatography.

1. Proenzymic C1r was purified from human plasma in a two-step technique involving indirect affinity chromatography on Sepharose Ig anti-C1s. The capacity of C1r to monomerize at pH 5.0 and to redimerize at neutral pH was used for selective elution of C1r. The yield in purified C1r was 39% from plasma; no trace of contaminating serine proteases was detected from [3H]diisopropyl phosphorofluoridate labelling of C1r. 2. C14 was able to undergo a two-way autoactivation: an intramolecular catalytic process catalysed by proenzymic C1r itself and an intermolecular reaction catalysed by activated C1r formed in the process of the reaction. DFP (5mM) and C1 Inh at a C1 Inh/C1r ratio of 1:1 were effective on the solely intermolecular activation, leading to partial inhibition of the autoactivation from proenzymic C1r: C1r formed during the activation was titrated by the inhibitors. Calcium, high ionic strength or acid pH decreased C1r activation. The pH effect was characterized by a slowed-down reaction below pH 6.0 and no net influence at values as high as 10.5. The two types of activation developed similarly as a function of pH. 3. Peripheral iodination of C1r revealed differences in label distribution between proenzymic (A chain moiety 48%, B chain moiety 52%) and activated C1r (A chain 20%, B chain 80%). Two different conformational states of C1r were also suggested by 125I-labelling at different temperatures.

Calcium↗
PubMed1982

Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1.

Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.

Binding Sites↗
PubMed1980

A study on the structure and interactions of the C1 sub-components C1r and C1s in the fluid phase.

1. Both proenzyme and activated C1r, which are dimers at pH 7.4, dissociated into monomers at pH 5.0 (C1r) and 4.0 (C1r), as shown by the decrease of apparent molecular weight and of sedimentation coefficient, which was shifted from 7.1 S (dimer) to 5.0 S (monomer). 125I-labelling of C1r in the presence of lactoperoxidase occurred, for the dimer, 16-20% in the A chain and 80-84% in the B chain, whereas the distribution was 67.5% and 32.5%, respectively, for the monomer. It appears likely that the two monomers of C1r interact through their A chain and that the A and B chains are relatively independent from each other. 2. 125I-labelling of C1s in the presence of lactoperoxidase confirmed the calcium-dependent dimerization of this subcomponent. In the monomer, the B chain appears to be embedded in the A chain, as shown by the 125I- distribution in these chains, which was 5% and 95%, respectively. This changed after dimerization to 25% and 75%, respectively, which suggests that interactions occur through the A chain of each monomer and lead to an unfolding of the B chain. 3. C1r dimer and C1s monomer were found to interact in the absence of calcium to form a C1r2-C1s complex (7.7 S), whereas in the presence of calcium the two sub-components were associated into a C1r2-C1s2 complex (8.7S). It appears likely that the formation of this tetrameric complex involves both calcium-dependent, and calcium-independent binding forces, and that C1r and C1s interact through their respective A chain which, in the case of C1s, is hidden upon association.

Calcium↗
PubMed1980

Purified proenzyme C1r. Some characteristics of its activation and subsequent proteolytic cleavage.

1. Upon incubation for 1 h at 37 degrees C, proenzymic C1r was activated by a proteolytic cleavage comparable to that observed in vivo; after reduction and alkylation, two fragments of apparent molecular weights 57 000 and 35 000 were evident on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The activation kinetics were slightly sigmoidal and nearly independent of C1r concentration. They were characterized by a marked thermal dependence (activation energy = 45 kcal/mol). The reaction was inhibited by calcium and p-nitrophenyl-p'-guanidinobenzoate, but poorly sensitive to di-isopropyl phosphorofluoridate. The dependence of the activation rate on pH was unusual; it decreased progressively in the acid range (pH 4.5-6.5) which coincides with the dissociation of the C1r-C1r dimer. Above pH 6.5, the rate increased slightly and showed no clear maximum. These results are consistent with an intramolecular autocatalytic activation mechanism involving the pro-site of each subunit of the C1r-C1r dimer. 2. During a 5 h incubation period at 37 degrees C, C1r underwent two proteolytic cleavages which led to the successive removal of two fragments, alpha (35 000) and beta (7000-11 000) from each subunit, leaving a dimeric molecule of reduced size (Mr = 110 000; s20,w = 6.1 S). The proteolytic process was nearly independent of C1r concentration and characterized by a pH optimum at 8.5-9.0, and a high activation energy (36.8 kcal/mol). Calcium and p-nitrophenyl-p'-guanidinobenzoate, and also di-isopropyl phosphorofluoridate and benzamidine were inhibitors of this reaction. The product, C1r II, retained the original antigenic properties of C1r and a functional active site, but lost the capacity to bind C1s. These results are consistent with an autocatalytic intramolecular proteolysis mediated by the active site of each subunit of the C1r-C1r dimer.

Complement Activating Enzymes↗