First Component Of Human Complement Research Articles

The first component of human complement (C1): activation and control.

The first component of human complement (C1) is a 750 000 dalton glycoprotein that requires calcium or other specific metal ions to maintain its native structure and function. Under physiologic conditions, C1 comprises two weakly interacting subunits, C1q and C1r2s2, with C1q containing the binding site(s) for activators and C1r2s2 possessing enzymatic potential. C1 circulates in a precursor state and only after "activation" does it acquire functional activity, manifested as enzymatic activity specific for its natural substrates C2 and C4. C1 activation, which is accompanied by limited proteolysis and conformational changes, can be induced by immune complexes or certain nonimmune substances. With C1 binding to an immune complex, the strength of interaction between C1q and C1r2s2 increases. C1 also spontaneously activates at 37 degrees C by an intramolecular autocatalytic mechanism although at a slower rate than that induced by activators. C1 functions are controlled by the serum glycoprotein C1-inhibitor (C1-In) which blocks the enzymatic activities of activated C1 (C1). Under physiologic conditions, C1 has a half-life of only 13 seconds in the presence of C1-In. C1 is efficiently disassembled by C1-In, thereby releasing two inactive C1rC1s(C1-In)2 complexes per C1 molecule, leaving C1q activator-bound with biologically reactive sites uncovered that are not expressed in macromolecular C1. The most recently recognized function of C1-In is that of controlling the C1 activation process itself. While having only limited effect on immune complex-induced C1 activation, C1-In effectively controls certain nonimmune-induced as well as spontaneous C1 activation. Thus C1-In plays an important role in regulating nonspecific complement activation. The latter observation is relevant for the understanding of the human disease hereditary angioedema. An overabundance of spontaneous C1 autoactivation, due to low C1-In levels, might underlie the abnormal activation of complement via the classical pathway detected in the sera of these patients. Finally, recent studies indicate that C1 may have other important biologic functions in addition to initiating the complement cascade.

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.

Neutron scattering studies of the isolated C1r2C1s2 subunit of first component of human complement in solution.

The subunit complex C1r2C1s2 of the first component of complement was investigated by small-angle neutron scattering in both the activated and unactivated forms. From these experiments, a molecular weight of 390,000 for C1r2C1s2 was found. The matchpoint was determined to be 43% 2H2O. Both results are consistent with composition data. The partial specific volume is 0.751 ml/mg. The radius of gyration at infinite contrast was found to be 17 nm for C1r2C1s2 and 1.1 nm for the cross section. Models for C1r2C1s2 were computed by the method of hard spheres, in which C1r2C1s2 was represented by spheres 0.87 nm diameter arranged in a straight rod of length 59 nm and a circular cross section of 3.2 nm. This rod can be bent at one or two places by up to 60 degrees without significant effect on the calculated radii of gyration. The model is in agreement with published ultracentrifugation and electron microscopy data.

Nature of the metal ion requirement for assembly and function of the first component of human complement.

The first component of human complement (C1) was reconstituted from equimolar concentrations of its purified subunits C1q, C1r, and C1s, in the presence of each of nine different metal ions for the purpose of studying the qualitative and quantitative nature of the metal ion requirement for C1 assembly and function. For C1 reconstituted with each metal ion, three assays characteristic of C1 were performed as follows: (1) spontaneous C1 activation in the absence of the regulatory protein C1-inhibitor was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis by simultaneously quantifying the specific proteolysis of the C1r and C1s subunits; (2) C1 activation induced by aggregated IgG in the presence of C1 inhibitor was similarly analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and (3) formation of 16 S macromolecular C1 was determined in the analytical ultracentrifuge. For all experiments, trace metal contaminants were removed from buffers and proteins. All divalent cations tested from the first transition period of the periodic table (i.e. Ca2+, Mn2+, Co2+, Ni2+, and Zn2+) effectively mediated the formation of functional macromolecular C1. Dose curves showed maximal C1 assembly and activation at ion concentrations of 30 to 50 microM for each of the above metal ions. However, when ion concentrations were increased above 50 microM, C1 assembly and activation became inhibited. The further to the right in the periodic table, the better inhibitor was the metal ion. Competition experiments indicated that the ion binding sites mediating inhibition are distinct from those promoting activation. Other metal ions that also effectively mediated C1 assembly and function were Cd2+ and Tb3+; however, Mg2+ and Ba2+ were ineffective. All metal ions that mediated C1 assembly and activation also promoted C2 consumption by C1 in normal human serum treated with aggregated IgG. In conclusion, the assembly and function of C1 can be mediated by numerous metal ions. In direct opposition to accepted theory, there is no specific requirement for calcium.

Electron microscopy of the first component of human complement.

Electron microscopy of the first component of human complement.

The binding and activation of the C1r-C1s subunit of the first component of human complement

The value of the functional affinity constant between 125I-labelled Clq and the Clr-Cls tetramer (when free in solution) in the formation of Cl was found to be 3.6 × 10 7 M −1. When Clq was bound to activating immune complexes, the value of K was about 10-fold higher before initiation of activation and there was a further two to three-fold rise as activation proceeded. The addition of an excess of unlabelled Clq increased the rate of activation of 125I-labelled Cl, suggesting an interaction between Clr-Cls and two neighbouring Clq molecules. It is suggested that the tetramer Clr-Cls may bind bivalently to Clq when free in solution, but on binding to activating complexes, one of the Clr-Cls binding sites is detached from Clq and becomes bound to a site on the complex. The resultant spatial rearrangement of the Clr molecules within the tetramer may be optimal for autocatalytic activation of Clr.

The dissociation properties of native C1

The first component of human complement (C1) readily dissociates under physiologic conditions into two subunits - C1q and C1r 2C1s 2. The equilibrium constant for this reaction has been determined for native C1 in fresh normal human serum by hemolytic titration. Standard technology was modified to simulate physiologic conditions. Furthermore, assays were carried out at numerous poncentrations of sensitized erythrocytes, thereby allowing the calculation of the percent of associated C1 at different total C1 concentration. Increased C1 dissociation was observed with dilution. From these data, an association constant of 4.5 × 10 8 M −1 was calculated for native C1. Thus in normal human serum approximately ten percent of the C1 is present as free C1q and C1r 2C1s 2.

Spontaneous activation of the first component of human complement (C1) by an intramolecular autocatalytic mechanism.

For biochemical characterization, the first component of human complement (C1) was reconstituted from physiologic concentrations of purified C1q, 125I C1r, and 131I C1s. Upon incubation at 37 degrees C, C1 spontaneously activated, as evidenced by the characteristic proteolysis of the C1r and C1s polypeptide chains as detected by SDS-PAGE analysis. This spontaneous C1 activation followed first-order kinetics (t 1/2 = 4 min and k = 0.173 min-1) with an activation energy of 19.1 kcal/mol. Spontaneous C1 activation was unaffected by the general protease inhibitor phenylmethylsulfonylfluoride (PMSF) but reversibly blocked by a known inhibitor of C1 activation, nitrophenylguanidinobenzoate (NPGB). Spontaneous C1 activation was measured at C1 concentrations ranging from 9 to 160 nM (i.e., 0.05 to 1.0 times physiologic concentrations). The data indicate that C1 spontaneously activates by an intramolecular autocatalytic mechanism, for first-order kinetics were observed over the entire concentration range with t 1/2 = 4 min at each concentration. However, the percentage of activable C1 decreased with dilution due to C1 dissociation (i.e., C1qr2s2 leads to C1q + C1r2s2). The observed concentration of C1 that spontaneously activated at each dilution equalled the concentration of C1 present as macromolecular C1. When reconstituted C1 was mixed with normal human serum (NHS) and then incubated at 37 degrees C, spontaneous C1 activation was completely inhibited. Pretreating NHS at 56 degrees C for 30 min destroyed its inhibitory activity. In conclusion, C1 spontaneously autoactivates at 37 degrees C by an intramolecular mechanism. This activation is suppressed in NHS.

Completion of the amino acid sequences of the A and B chains of subcomponent C1q of the first component of human complement.

The sequences of amino acid residues 109--224 of the A chain, and residues 109--22 of the B chain, of human subcomponent C1q are given. These results, along with previously published sequence data on the N-terminal, collagen-like, regions of the A and B chains [Reid (1979) Biochem. J. 179, 367--371] yield the complete amino acid sequences of the A and B chains of subcomponent C1q. The asparagine residue at position A-124 has been identified as the major site of asparagine-linked carbohydrate in subcomponent C1q. When the sequences of the C-terminal, 135-residue-long, 'globular' regions of A and B chains are compared they show 40% homology. The degree of homology over certain stretches of 15--20 residues, within the C-terminal regions, rises up to values of 73%, indicating the presence of strongly conserved structures. Structure prediction studies indicate that both the A and B chain C-terminal regions may adopt a predominantly beta-type structure with apparently little alpha-helical structure.

A new role for C-1-inhibitor in homeostasis: control of activation of the first component of human complement.

A new role for C-1-inhibitor in homeostasis: control of activation of the first component of human complement.

Formation of a conformationally changed C1r, a subcomponent of the first component of human complement, as an intermediate of its autoactivation reaction

Formation of a conformationally changed C1r, a subcomponent of the first component of human complement, as an intermediate of its autoactivation reaction

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.

Ultrastructure of the first component of human complement: electron microscopy of the crosslinked complex.

Electron micrographs are shown of the first component of human complement (C1) which has been crosslinked with a water-soluble carbodiimide to prevent dissociation into its C1q and C1r2C1s2 subunits. Two projections of the crosslinked molecule are seen in the electron micrographs, which are called "top" and "profile." In both views, the C1q heads are visible. From the top, the C1r2C1s2 tetrameric subunits appears to be located centrally on the C1q and folded to form a compact mass obscuring most of the arms and central bundle. In profile, the tetramer appears to be located in the region of the arms between the C1q heads and the central bundle. Both the heads and the rod-like central bundle appear to be free of C1r2C1s2 in these profile projections. Sometimes it is possible to count more than six domains in the region of the C1q heads, as though a portion of the tetramer had unfolded to protrude among the heads.

Stoichiometry and sedimentation properties of the complex formed between the C1q and C1r2C1s2 subcomponents of the first component of complement.

We have examined the functional and hydrodynamic properties of the first component of human complement, C1, and the activated first component, C1-, reassembled in the presence of Ca++ from C1q and either the C1r2C1s2 or the C1r-2C1s-2 tetramer. Reconstituted C1 has hemolytic activity similar to C1 in serum. As long as either tetramer is in excess and the total concentration is low, we find that only a 1:1 complex is formed between C1q and either the unactivated or activated tetramer. This complex sediments at 15.9 +/- 0.2 Svedbergs and has a complex sediments at 15.9 +/- 0.2 Svedbergs and has a m.w. of 739,000 +/- 37,000. The boundary shape of the sedimenting C1- preparation was broader than that of C1 suggesting the association constant between C1r2C1s2 and C1q may have decreased upon activation. At elevated concentrations, with more than a molar excess of C1q, C1 aggregated to form both 16S and 23S species.

The first component of human complement: on the mechanism of activation by some carbohydrates.

Four synthetic saccharides (mono-, di-, tri-, tetra-) linked to the carrier 8-methoxycarbonyloctanol were used in the presence or absence of a polysaccharide (PS) purified from ant venom to study the activation of C1 and the consumption of C4 in normal serum. The carrier was essential for the recorded activities of the saccharides. The alpha D mannose-O-carrier did not cause C1 activation and C4 consumption by itself, but it enhanced these activities when combined with the venom PS. The alpha L fuc(1 leads to 4)beta D glc-NAc-O-carrier caused C1 activation but no C4 consumption by itself at a high concentration (1.4 mg/ml), and enhanced C1 activation and C4 consumption in combination with the venom PS. The beta D gal(1 leads to 3)alpha L fuc(1 leads to 4)beta D glc-NAc-O-carrier caused both C1 activation and C4 consumption in normal human serum by itself. The alpha L fuc(1 leads to 2)beta D gal[alpha L fuc(1 leads to 4)]beta D glc-NAc-O-carrier neither caused C1 activation and C4 consumption by itself, nor did it enhance these activities when combined with the venom PS. Neither EAC1hu nor EAC1hu bound 3H-PS or 3H-beta D gal(1 leads to 3)alpha L fuc(1 leads to 4)beta D glc-NAc-O-carrier. Also, the precursor human C1 on EA (EAC1) was not activated by the venom PS. Thus, the overall evidence shows that human precursor C1 is activated by these carbohydrates solely by an interaction with C1q subunit of macromolecular C1.

Semi-flexible joint in the C1q subunit of the first component of human complement

C1q appears in electron micrographs in two different projections: lateral projections, in which the molecules resemble bunches of tulips; and top projections, in which the molecules are seen as six terminal subunits connected to a central portion. We have measured 39 particularly well-formed top views to determine the distribution of distances of terminal subunits from the central portion, from which may be calculated the distribution of the angles made by the connecting strands with an axis through the central portion. This distribution peaks sharply at a preferred angle of 50 °. A limited degree of flexibility must exist, however, for a few molecules are found with angles ranging from 20 ° to 80 °. Therefore, we suggest the existence of a semi-flexible joint at the point of interruption of the collagen-like amino acid sequence where the connecting strands join to the central portion of the C1q molecule.

Influence of ω-Aminoacids on the Activation of the First Component of Complement at Low Ionic Strength

Cl, is the first component of human complement, was activated to Cls by diluting human plasma in a solution of low ionic strength. The addition of lysine and its related aminoacids to plasma resulted in inhibition of Cl activation, alpha, omega-diaminoacids such as lysine and ornithine were most effective and omega-aminoacids such as 6 aminohexanoic acid were far less effective. Tranexamic acid which is most potent in inhibition of fibrinolysis among those aminoacids was least effective in the inhibition of Cl activation. omega-aminoacids or their analogues hardly inhibited Cls which had already been activated.

The conformation of subcomponent C1q of the first component of human complement.

The complement system in the blood of vertebrates1,2 has a vital role in the defence of the body against invading foreign material. Although it can be triggered before immunity has developed, it is usually activated following an initial intervention by antibodies, and about 20 serum proteins are involved. Some of these act as controlling inhibitors while others form nine components which, if following the classical activation pathway, all take part in bringing about membrane damage and cell lysis. The first component C1 consists of three subcomponents, C1q, C1r and C1s. C1q binds to antibody–antigen complexes as the first stage of fixation3 and as it shows no enzymatic activity by itself, conformational changes on binding have been proposed as the driving mechanism of the subsequent reactions. Since structural investigation by electron microscopy4–7 leaves doubt as to whether unnatural constraints were applied in the preparation and treatment of specimens, techniques such as X-ray or neutron small-angle scattering, which investigate the equilibrium state in dilute solution, provide valuable complementary evidence for both its findings and those of other physicochemical methods8. We have already carried out neutron work on the conformation of immunoglobulin and the influence of hapten binding (to be published) and are now studying the conformation of C1q as a preliminary to investigating the composite structures resulting from their mutual interaction. As evidence of the structure of this molecule has accumulated, the question has arisen of whether it maintains a compact closed conformation in its native state or a more open one in readiness for interaction with other proteins. The neutron small angle scattering curve is sensitive to conformational changes and although our measurements are not yet complete, model calculations suggest that the structural arrangement is indeed open.

Effect of C [formula omitted]-inhibitor, benzamidine, and heat treatment on the hydrolytic activity of a subcomponent of the first component of human complement, C [formula omitted

Inhibition of one subcomponent of the first component of human complement, C 1 s, ∗ ∗ The symbols for components used in this paper conform to the recommendations of the World Health Organization Committee on Complement Nomenclature, Immunochemistry (1970) 7, 137–142. Activated components were indicated by placing a bar over the numeral which refers to the active component or subcomponent. by C 1 -inhibitor, benzamidine, and heating at 52°C was investigated using various synthetic and natural substrates for C 1 s. The esterolytic activity of C 1 s toward acetyl-arginine methyl ester (AcArgOMe) was found to be more stable than that toward acetyl-glycyl-lysine methyl ester (AcGlyLysOMe) and acetyl-tyrosine ethyl ester (AcTyrOEt) against inhibition of C 1 INH and heating at 52°C. Also, the AcArgOMe hydrolytic activity of C 1 s was less susceptible to benzamidine than the AcGlyLysOMe and AcTyrOEt hydrolytic activity of C 1 s. The K j value calculated with AcArgOMe as the substrate for C 1 s was fond to be 7-fold larger than that calculated with AcGlyLysOMe and AcTyrOEt as substrates, suggesting that the catalytic site for AcArgOMe is different from that for AcGlyLysOMe and AcTyrOEt. The proteolytic activity of C 1 s towards C4 was found to be more stable than that towards C2 against inhibition by C 1 INH and heat-treatment. These results suggested that there are two C 1 s species; one is responsible for the hydrolysis of C2, AcGlyLysOMe, and AcTyrOEt, and another is for the hydrolysis of C4 and AcArgOMe. The former is more sensitive than the latter to inhibition by C 1 INH, benzamidine, and heat-treatment.

Active Disassembly of the First Complement Component, C1̄, by C1̄ Inactivator

Abstract In order to clarify its mechanism of disassembly, the first component of human complement, C1, was reconstituted from C1q, 125I-C1r, and 131I-C1s. This radiolabeled C1 bound tightly to aggregated IgG covalently linked to Sepharose 4B. The subsequent addition of C1̄ inactivator (C1̄-In) led to the rapid release of equimolar quantitites of 125I-C1r̅ and 131I-C1s̅ from the agg IgG. SDS-PAGE analyses of such eluates showed that one C1̄-In molecule was associated with each C1r̅ polypeptide chain and another C1̄-In molecule was bound to each C1s̅ polypeptide chain. The released C1r̅, C1s̅, and C1̄-In co-sedimented in sucrose density gradients with a rate of 9S and monospecific anti-C1s̅ pelleted all three proteins. Therefore, C1r̅, C1s̅, and C1̄-In, in a molar ratio of 1:1:2, respectively, are released from C1̄ as a complex. An analogous 9S complex containing C1r̅, C1s̅, and C1̄-In was generated in normal human serum after incubation with activators of the classical complement pathway. The C1r̅C1s̅(C1̄-In) complexes generated in both serum and the purified system were stable in the presence of EDTA. A diffusion coefficient of 2.3 × 10-7 cm2/sec was determined for the C1r̅C1s̅(C1̄-In) complex from its behavior on gel filtration. An m.w. of 330,000 was then calculated from its sedimentation and diffusion coefficients. This m.w. is consistent with the value of 382,000 for a molecule of composition C1r̅C1s̅(C1̄-In)2 obtained by summing the weights of the subunits. These results indicate that C1-In efficiently disassembles C1̄, thereby releasing two C1r̅C1s̅(C1̄-In)2 complexes per C1̄ molecule.