C1 dissociation. Spontaneous generation in human serum of a trimer complex containing C1 inactivator, activated C1r, and zymogen C1s

Activation of the C1 complex in the presence of C1 inactivator (C1 IA) is known to result in the formation of tetramer C1 IA-C1r-C1s-C1 IA complexes that are dissociated from C1q. Both C1r and C1s of the tetramers are present in their activated forms. The present investigation concerned the generation of trimer complexes containing C1 IA, activated C1r, and zymogen C1s (C1 IA-C1r-C1s). C1 IA-C1r-C1s were released from C1q and were formed in high concentration during prolonged incubation (1 to 3 days) of normal serum at 37 degrees C without addition of activators. By contrast, dissociation of C1 with formation of C1 IA-C1r-C1s-C1 IA was complete within 30 min at 37 degrees C, when the serum was treated with heat-aggregated IgG (1 g/liter). On size exclusion chromatography (TSK-4000), C1 IA-C1r-C1s and C1 IA-C1r-C1s-C1 IA emerged with apparent m.w. of 320,000 and 460,000, respectively. The composition of the complexes was examined by absorption of serum with F(ab')2 anti-C1s- or anti-C1r-coated Sepharose beads. Eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting. Under nonreducing conditions, heat-aggregated IgG-treated serum showed high concentrations of C1 IA-C1r (m.w. 202,000) and C1 IA-C1s (m.w. 194,000), while serum incubated at 37 degrees C without activators showed high concentrations of C1 IA-C1r but no C1 IA-C1s. Under reducing conditions, heat-aggregated IgG-treated serum showed m.w. 120,000 and 110,000 complexes of C1 IA and the C1r and C1s light chains, respectively. Uncleaved C1s and the m.w. 120,000 complex was found in serum that was incubated at 37 degrees C without activators. Consistent with results obtained by size exclusion chromatography, analysis by crossed immunoelectrophoresis and by electroimmunoassay showed that C1s could be released from C1 IA-C1r-C1s in the presence of EDTA.     

Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca²⁺-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.     

The cleavage of two C1s subunits by a single active C1r reveals substantial flexibility of the C1s-C1r-C1r-C1s tetramer in the C1 complex

The activation of the C1s-C1r-C1r-C1s tetramer in the C1 complex, which involves the cleavage of an Arg-Ile bond in the catalytic domains of the subcomponents, is a two-step process. First, the autolytic activation of C1r takes place, then activated C1r cleaves zymogen C1s. The Arg463Gln mutant of C1r (C1rQI) is stabilized in the zymogen form. This mutant was used to form a C1q-(C1s-C1rQI-C1r-C1s) heteropentamer to study the relative position of the C1r and C1s subunits in the C1 complex. After triggering the C1 by IgG-Sepharose, both C1s subunits are cleaved by the single proteolytically active C1r subunit in the C1s-C1rQI-C1r-C1s tetramer. This finding indicates that the tetramer is flexible enough to adopt different conformations within the C1 complex during the activation process, enabling the single active C1r to cleave both C1s, the neighboring and the sequentially distant one.     

Mapping Surface Accessibility of the C1r/C1s Tetramer by Chemical Modification and Mass Spectrometry Provides New Insights into Assembly of the Human C1 Complex

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca²⁺-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340–19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB₁-EGF-CUB₂ interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.     

Identification of the C1q-binding Sites of Human C1r and C1s: A REFINED THREE-DIMENSIONAL MODEL OF THE C1 COMPLEX OF COMPLEMENT*

The C1 complex of complement is assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a Ca²⁺-dependent tetramer of two modular proteases C1r and C1s. Resolution of the x-ray structure of the N-terminal CUB₁-epidermal growth factor (EGF) C1s segment has led to a model of the C1q/C1s-C1r-C1r-C1s interaction where the C1q collagen stem binds at the C1r/C1s interface through ionic bonds involving acidic residues contributed by the C1r EGF module (Gregory, L. A., Thielens, N. M., Arlaud, G. J., Fontecilla-Camps, J. C., and Gaboriaud, C. (2003) J. Biol. Chem. 278, 32157–32164). To identify the C1q-binding sites of C1s-C1r-C1r-C1s, a series of C1r and C1s mutants was expressed, and the C1q binding ability of the resulting tetramer variants was assessed by surface plasmon resonance. Mutations targeting the Glu¹³⁷-Glu-Asp¹³⁹ stretch in the C1r EGF module had no effect on C1 assembly, ruling out our previous interaction model. Additional mutations targeting residues expected to participate in the Ca²⁺-binding sites of the C1r and C1s CUB modules provided evidence for high affinity C1q-binding sites contributed by the C1r CUB₁ and CUB₂ modules and lower affinity sites contributed by C1s CUB₁. All of the sites implicate acidic residues also contributing Ca²⁺ ligands. C1s-C1r-C1r-C1s thus contributes six C1q-binding sites, one per C1q stem. Based on the location of these sites and available structural information, we propose a refined model of C1 assembly where the CUB₁-EGF-CUB₂ interaction domains of C1r and C1s are entirely clustered inside C1q and interact through six binding sites with reactive lysines of the C1q stems. This mechanism is similar to that demonstrated for mannan-binding lectin (MBL)-MBL-associated serine protease and ficolin-MBL-associated serine protease complexes.The classical pathway of complement, a major component of innate immune defense against pathogens and altered self, is triggered by C1, a 790-kDa Ca²⁺-dependent complex assembled from a recognition protein C1q and C1s-C1r-C1r-C1s, a tetramer of two modular proteases, C1r and C1s, that respectively mediate activation and proteolytic activity of the complex (1–3). C1q has the overall shape of a bunch of tulips and comprises six heterotrimeric collagen-like triple helices that assemble through their N-terminal moieties to form a “stalk” and then diverge to form individual “stems,” each prolonged by a C-terminal globular recognition domain (4). C1r and C1s are homologous modular proteases each comprising, starting from the N-terminal end, a C1r/C1s, sea urchin EGF² (uEGF), bone morphogenetic protein (CUB) module (5), an EGF-like module (6), a second CUB module, two complement control protein modules (7), and a serine protease domain. This modular structure is shared by the mannan-binding lectin-associated serine proteases (MASPs), a group of enzymes that associate with mannan-binding lectin (MBL) and the ficolins and thereby trigger activation of the lectin pathway of complement (8).Assembly of the C1s-C1r-C1r-C1s tetramer involves Ca²⁺-dependent heterodimeric C1r-C1s interactions between the CUB₁-EGF segments of each protease (9–12). Similarly, MASP-1, MASP-2, MASP-3, and mannan-binding lectin-associated protein 19 (MAp19), an alternative splicing product of the MASP-2 gene comprising the N-terminal CUB₁-EGF segment of MASP-2, all associate as homodimers through their N-terminal CUB₁-EGF moieties (13–15). The structures of human C1s CUB₁-EGF, human MAp19, human MASP-1/3 CUB₁-EGF-CUB₂, and rat MASP-2 CUB₁-EGF-CUB₂ have been solved by x-ray crystallography (16–19), revealing that these domains all associate as head-to-tail homodimers through a highly conserved interface involving interactions between the CUB₁ module of one monomer and the EGF module of its counterpart. In addition, all CUB modules contained in these structures were found to contain a hitherto unrecognized Ca²⁺-binding site involving three conserved acidic residues (Glu⁴⁵, Asp⁵³, and Asp⁹⁸ in C1s), defining a novel CUB module subset diverging from the type originally described in the spermadhesins (20).Mutagenesis studies have recently established that assembly of the MBL- and ficolin-MASP complexes involves a major electrostatic interaction between two acidic Ca²⁺ ligands from the MASP CUB modules and a conserved lysine located in the collagen fibers of MBL and ficolins (16, 18, 21, 22). In the case of C1, a hypothetical model of the C1q/C1r/C1s interface, involving interaction between acidic residues mainly contributed by the C1r EGF module and unmodified lysine residues also located in the collagen-like stems of C1q, was derived from the x-ray structure of the C1s CUB₁-EGF interaction domain (16, 23). The aim of this work was to use site-directed mutagenesis to delineate the sites of C1r and C1s involved in the interaction between C1s-C1r-C1r-C1s and C1q. Our data rule out our previous interaction model and provide evidence that C1 assembly involves the same basic Ca²⁺-dependent mechanism as demonstrated in the case of MBL-MASP and ficolin-MASP complexes.     

Conformation and restricted segmental flexibility of C1, the first component of human complement

Seventy selected images of chemically crosslinked C1 are analyzed to illustrate structural details of the C1qC1r2C1s2 complex. From inspection of these images, the C1r2C1s2 tetramer can be seen to be located in the region of the C1q arms, cleanly separated from the C1q heads and from at least 90%, if not all, of the C1q stem. From measurements made upon 65 images, the semicone angles formed between the spreading arms and the symmetry axis passing through the stem of C1 may be calculated. Unlike C1q, for which a wide variety of angles is found, the C1 complex appears to possess a restricted range of angular flexibility with an average value of about 50 degrees. The volume inside the cone formed by the spreading arms of C1q is too small to contain the entire C1r2C1s2 tetramer; at least some of the tetramer must lie outside the cone when it is bound to C1q to form C1. From our knowledge of the sizes and structures of its subunits, and from symmetry considerations, a model is proposed for the configuration of the C1 complex in which the middle portion of the C1r2C1s2 tetramer is centrally located among the arms close to the stem of the C1q and with the two protruding ends of the tetramer wrapped around the outside of the cone. Functional implications of this more rigid structure are discussed with relevance to C1q-induced aggregation of latex beads and C1-induced disaggregation.     

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.     

X-ray structure of the Ca2+-binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement

C1, the complex that triggers the classical pathway of complement, is assembled from two modular proteases C1r and C1s and a recognition protein C1q. The N-terminal CUB1-EGF segments of C1r and C1s are key elements of the C1 architecture, because they mediate both Ca2+-dependent C1r-C1s association and interaction with C1q. The crystal structure of the interaction domain of C1s has been solved and refined to 1.5 A resolution. The structure reveals a head-to-tail homodimer involving interactions between the CUB1 module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca2+ ion is bound to each EGF module and stabilizes both the intra- and inter-monomer interfaces. Unexpectedly, a second Ca2+ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu45, Asp53, Asp98, and two water molecules. These acidic residues and Tyr17 are conserved in approximately two-thirds of the CUB repertoire and define a novel, Ca2+-binding CUB module subset. The C1s structure was used to build a model of the C1r-C1s CUB1-EGF heterodimer, which in C1 connects C1r to C1s and mediates interaction with C1q. A structural model of the C1q/C1r/C1s interface is proposed, where the rod-like collagen triple helix of C1q is accommodated into a groove along the transversal axis of the C1r-C1s heterodimer.     

C1 inhibitor-dependent dissociation of human complement component C1 bound to immune complexes.

The interaction of C1 inhibitor with complement component C1 bound to immune complexes was examined by using 125I-labelled C1 subcomponents. The inhibitor binds rapidly to subcomponent C1s, and more slowly to subcomponent C1r. Formation of the C1r-C1 inhibitor complex causes rapid dissociation of subcomponents C1r and C1s from the antibody-antigen-component C1 aggregate. The rate and extent of this release are proportional to C1 Inhibitor concentration and are also dependent on ionic strength. Results obtained with purified C1 Inhibitor, plasma or serum as source of C1 Inhibitor are all closely comparable. Only slight dissociation of subcomponent C1q is observed under the same range of conditions. The implications of the release phenomenon are discussed in relation to the structure of component C1 and the possibility of differential turnover of C1 subcomponents.     

Antibody-independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4.

The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 X 10(5) molecules per bacterium, with a Ka of 1.4 X 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 X 10(5) molecules per bacterium, and a Ka of 0.25 X 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.     

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.     

Characteristics of complement subcomponents C1r and C1s synthesized by Hep G2 cells.

The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.     

Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, and the MBL-associated protein MAp19

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.     

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.     

The unactivated form of the first component of human complement, C1.

The first component of complement, C1, was isolated unactivated from human serum by repeated additions of di-isopropyl phosphorofluoridate during isolation. The unactivated subcomponents were also isolated, and evidence is given that the three subcomponents C1q, C1r and C1s account wholly for the activity of component C1 in serum. No evidence could be found for a fourth subcomponent, C1t. The approximate molar proportions of the subcomponents in serum are C1q/C1r/C1s = 1:2:2. Optimum activity by haemolytic assay was found at approximate molar proportions C1q/C1r/C1s of 1:4:4. No activity was found when subcomponents were assayed singly or in pairs, except for subcomponents C1q and C1s, which in molar ratio 1:4 gave 15-20% of the activity of the mixture C1q + C1r + C1s. The proteolytic activity of the isolated subcomponent C1s varied according to the method of activation used. Subcomponents C1q + C1r + C1s and C1q + C1s in the presence of antibody-antigen aggregates were activated and inactivated simultaneously, showing a peak of activity and subsequent loss of activity. Both reactions are probably due to proteolysis, and analysis of the peptide bonds split will be necessary to distinguish these two phenomena.     

Molecular modelling of human complement subcomponent C1q and its complex with C1r2C1s2 derived from neutron-scattering curves and hydrodynamic properties.

Models for the structures of subcomponent C1q of first component C1 of human complement and its complex with subunit C1r2C1s2 are compared with experimental neutron-scattering curves. The length of the C1q collagenous arm is closer to 14.5 nm than to 11.5 nm proposed from electron microscopy, and this is consistent with the primary sequence of C1q. The mean C1q base-arm angle is 40-45 degrees and C1q is found to be flexible: the base-arm angle can vary up to 30 degrees from equilibrium at any moment. The complex of C1r2C1s2 and C1q requires a large shape change in C1r2C1s2. Ring-like models for C1r2C1s2 are not as successful at rationalizing the scattering data as are models that involve C1r2C1s2 binding to one side of C1q. Hydrodynamic calculations of the sedimentation coefficients for C1q and C1 are generally consistent with these neutron models.     

Chemical and functional characterization of a fragment of C1-s containing the epidermal growth factor homology region

C1-s, one of the three subcomponents of C1-, the first component of complement, is a serine protease comprising two disulfide-linked chains, the B chain, containing the catalytic site, and the A chain, involved in Ca2+ binding and Ca2(+)-dependent interaction(s) with the other C1- subcomponents. In an attempt to identify the regions responsible for the latter functions, C1-s was submitted to limited proteolysis with plasmin, a treatment that split the A chain into three major fragments, alpha 1, alpha 2, and gamma. Fragment alpha 2, which comprised the epidermal growth factor-like (EGF-like) region of C1-s, was heterogeneous, starting at serine 97 or phenylalanine 105 and ending at lysine 195. This fragment was reduced and alkylated and then digested with elastase, and three peptides covering positions 131-135, 131-139, and 131-140 were characterized by amino acid analysis, Edman degradation, and mass spectrometry, showing that position 134 of C1-s is occupied partly by an asparagine (47%) and partly by an erythro-beta-hydroxyasparagine, in contrast with the homologous position (150) of C1-r which only contains erythro-beta-hydroxyasparagine. As measured by equilibrium dialysis, native alpha 2, like the other plasmin-cleavage fragments, did not retain the ability of intact C1-s to bind Ca2+. In the same way, plasmin cleavage abolished the ability of C1-s to dimerize or to associate with C1-r in the presence of Ca2+. In contrast, both alpha 2 and the N-terminal alpha 1 fragment, starting at serine 24 of the A chain, were able to compete significantly with intact C1s for the formation of the Ca2(+)-dependent C1-s-C1r-C1-r-C1-s tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of C1q, C1s and C-1 Inh synthesized by stimulated human monocytes in vitro

C1q, C1s and C1 Inh synthesized and secreted by human monocytes were characterized by SDS-PAGE. C1q is formed of three chains A (Mr approximately 35 000), B (Mr approximately 33 000) and C (Mr approximately 25 000) which are associated in two subunits A-B and C-C. It appears identical to C1q purified from plasma. C1s is secreted as a non-activated, monocatenar protein of Mr approximately 87 000 identical to proenzymic C1s from plasma. Secreted C1 Inh (Mr approximately 100 000) has a slightly higher Mr than purified plasmatic C1 Inh. Monensin treatment of the cells favours the intracytoplasmic accumulation of products at various glycosylation stages.     

Measurement of macromolecular interactions between complement subcomponents C1q, C1r, C1s, and immunoglobulin IgM by sedimentation analysis using the analytical ultracentrifuge

The interactions between the complement components and with immunoglobulins are greatly enhanced by lowering the ionic strength and become readily measurable by physical techniques. Thus, the binding between C1q and IgM was previously shown to be appreciable (k = 1 x 10(6) M-1) at 0.084 M ionic strength (Poon, P.H., Phillips, M.L., and Schumaker, V.N. (1985) J. Biol. Chem. 260, 9357-9365). We have now found that, at 0.128 M ionic strength, the binding between human C1- (the activated first component of complement) and IgM was strong at physiological concentrations (k = 1 x 10(7) M-1), while under the same conditions binding between C1q and IgM was not observed. To explore the nature of the interactions responsible for this enhanced binding by C1- over C1q, mixtures of the various subcomponents of C1- were studied alone and with IgM. C1r2 did not bind to C1q, even when the ionic strength was reduced to 0.098 M, nor did the presence of C1r2 enhance the binding of C1q to IgM. In contrast, two C1s2 independently bound to C1q (k = 1 x 10(6) M-1), and caused a marked increase in its association with IgM (k = 5 x 10(6) M-1) at 0.098 M ionic strength. No detectable interaction was found between C1s2 and/or C1r2 and IgM in the absence of C1q. Moreover, there was no detectable interaction between the C1(-)-like complex formed between C1r2C1s2 and the collagenous C1q stalks (pepsin-digested C1q) and IgM. These data suggest that the binding of C1s2 to C1q, either alone or together with C1r2, induces a conformational change in C1q which results in additional C1q heads binding to complementary sites on IgM.