Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement.

The reaction of [14C]methylamine with native human C3 led to the stoichiometric incorporation of methylamine, loss of hemolytic activity, and the concomitant exposure of a sulfhydryl group that could be labeled with [14C]iodoacetamide. Both labeled sites were located in the C3d portion of the alpha-chain, which is known to contain the metastable binding of C3b. The methylamine-modified C3 [C3(CH3NH2)] was shown to exhibit many of the functional properties of C3b, although the C3a portion of the molecule remained covalently attached. C3(CH3NH2) bound Factor B and beta 1H, and could be cleaved by C3b inactivator in the presence of beta 1H. C3(CH3NH2) added to human serum caused activation of the alternative pathway and consumption of C3. In presence of Factors B and D and Mg++, C3(CH2NH2) formed a C3 convertase. The convertase-forming material could be removed from solution by anti-C3a Sepharose and the preformed convertase was completely inhibited by purified antibody to C3a. This antibody did not affect the function of the C3 convertase that contained C3b. Similar functional properties were exhibited by C3 exposed for short periods of time to relatively low concentrations of chaotropic reagents, such as KSCN or guanidine. These results suggest that the initial C3 convertase of the alternative pathway may be formed from native C3, without proteolysis, by the attack of a variety of nucleophiles including water. The C3 convertase formed from this altered C3 then generates by proteolytic cleavage the initial metastable C3b that is capable of attaching to receptive surfaces. Conversion of C3 to C3b exposes one sulfhydryl residue as does modification of C3 with methylamine. When the C3d portion of C3b bound to zymosan particles via the metastable binding site was treated with radiolabeled methylamine, the fragment was released from the particles in radiolabeled form. These findings are consistent with the concept that native C3 contains an active carbonyl group, probably in the form of a thioester, which can either react with water to form functionally C3b-l;ike C3 or, upon enzymatic conversion of C3 to C3b, allows C3b to form an ester bond with hydroxyl groups on the target surface.

Deficiency of the fifth component of complement in mice with an inherited complement defect.

The inherited complement deficiency of certain inbred strains of mice was shown to be due to an isolated lack of the fifth component of complement. The protein MuB1 (or hc'), which is present in normal mouse serum but absent from the serum of complement-deficient mice, was shown to be immunochemically related to the fifth component of human complement (C'5). C'5 hemolytic activity was specifically inhibited in human serum by mouse anti-MuB1 and in normal mouse serum by mouse antiserum to human C'5. Highly purified human C'5 reconstituted the hemolytic activity of complement-deficient mouse serum. It was, therefore, concluded that he', or MuB1, constitutes the murine analogue of the fifth component of human complement. The MuB1 concentration in normal mouse serum was found to be subject to a sex-related variation. By quantitative precipitin analysis it was demonstrated that serum from male mice contains twice as much MuB1 as that of female mice. This difference in C'5 concentration was also detected by hemolytic assay. In addition, C'6 and C'7 were also found to be subject to sex-related variations.

The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement.

Anaphylatoxin activity was derived from both human C'5 and C'3 molecules. This was achieved in the case of C'5 by interaction with trypsin or with EAC'4, (oxy)2a, 3. The smooth muscle-contracting material obtained from the treated C'5 was found to be a fragment of approximately 9,000-11,000 molecular weight. Its action was inhibited with antihistamine. The trypsinized C'5 also increased vascular permeability in guinea pig skin. When human C'3 was incubated with C'3 inactivator complex, which consists of a cobra venom protein and a beta-globulin of human serum, anaphylatoxin activity was observed. The activity was associated with a fragment cleaved from the C'3 molecule, having a molecular weight of between 6,000 and 15,000 as determined by gel filtration techniques. Similar activity was derived from C'3 by the C'3-converting enzyme in free or in cell-bound form. The C'5 anaphylatoxin failed to cross-desensitize guinea pig ileum to the contracting capacities of C'3 and guinea pig anaphylatoxin and vice versa. Anaphylatoxin prepared from C'3 by all methods mentioned above caused cross-desensitization to the other C'3 derivatives, but failed to desensitize to guinea pig anaphylatoxin.

Enharncement of the hemolytic activity of the second component of human complement by oxidation.

A method has been described for the chemical modification of human C'2 which results in a pronounced enhancement of its hemolytic activity and a marked increase in the stability of the intermediate complex EAC'1a,4,2a prepared with the modified C'2. Both effects are fully explained by the observed increase in activity and stability of the C'3 converting enzyme, C'4,2a, following its generation with modified C'2. Evidence has been presented in support of the hypothesis that the modification resulting from treatment of C'2 with a critical concentration of iodine consists of oxidation of one or more sulfhydryl group within the molecule.

The second component of human complement: its isolation, fragmentation by C'1 esterase, and incorporation into C'3 convertase.

A method has been described for the purification and isolation of the second component of complement (C'2) from human serum. The protein is a beta(1)-globulin with an approximate molecular weight of 117,000. Immunochemical analysis using a variety of specific antisera, including a monospecific antiserum to the isolated protein, indicate that the C'2 protein represents a heretofore unrecognized human serum constituent. Isolated C'2 contained 2 x 10(9) "effective molecules" per microgram and 1000 hemolytically active C'2 molecules were required to produce a single hemolytically effective C'2 site on erythrocytes undergoing immune cytolysis. C'1 esterase treatment of C'2 resulted in reduction of both its electrophoretic mobility and its molecular size, the latter observation indicating fragmentation of the molecule. Direct evidence was presented for the physical presence of C'2 as an integral part of the enzyme C'3 convertase.

Mechanism of action of factor D of the alternative complement pathway.

Factor D (C3 proactivator convertase) of human serum has been shown to be absolutely necessary for alternative pathway function, for activation of the C3/C5 convertase of that pathway and not to be a subunit of this enzyme. Factor D was found to be present in human plasma in active form only, at a concentration of 2 microgram/ml, and not to be controlled by plasma protease inhibitors or by spontaneous decay. Unlike trypsin, factor D cleaves and activates factor B only when it is in Mg++-dependent complex with C3b, has no esterolytic activity, and is unable to cleave the B chain of insulin. The alleged functional and antigenic relationship of factor D to alpha-thrombin could not be verified. The results of this study led to the description of the mechanism of action of factor D in terms of the cryptic site hypothesis.

The c3-activator system: an alternate pathway of complement activation.

Evidence has accumulated indicating the existence of a second complement activation mechanism which is functionally analogous to C1, C2, and C4. The noncomplement protein C3PA, previously recognized through its ability to form a complex enzyme with a protein from cobra venom, appears to be the precursor of the C4,2 analogue. It is a thermolabile beta-globulin with a molecular weight of 80,000. When serum is treated with naturally occurring plant or bacterial polysaccharides, the C3PA is cleaved into at least two fragments, one having the electrophoretic mobility of a gamma-globulin and a molecular weight of 60,000, and the other being an acidic peptide with a molecular weight of 20,000. The larger fragment has the ability to cleave C3 into C3a and C3b and is therefore called C3 activator. It arises from the action of an as yet unidentified serum enzyme upon the C3PA, which is tentatively called C3PA convertase. In addition to endotoxins, yeast cell walls, inulin, and agar, aggregates of immunoglobulins were found to be activating substances, including human IgA, guinea pig gamma1, and duck antibody. Serum depleted of C3PA had reduced E. coli bactericidal and increased hemolytic activity. The relationship of the C3-activator system to experimental and clinical observations has been discussed.

The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex.

Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.

The role of properdin in the alternate pathway of complement activation.

Properdin (P), a highly basic euglobulin, was purified from human serum to molecular homogeneity without the use of zymosan. Isolated P was found to efficiently initiate activation of the alternate pathway of complement activation (C3 activator or properdin system) and to be an essential component during its early reaction stages. The activity of isolated P did not require the presence of an activating polysaccharide. It was therefore concluded that purified P had been obtained in an activated form (P). In an isolated reaction system containing purified C3, C3 proactivator (C3PA), and C3 proactivator convertase (C3PAse), P was able to mediate the activation of C3PAse which in turn activated C3PA to cleave C3. This activation of C3PAse was found to depend on the presence of native C3. These results allowed the formulation of a concept in which P is envisaged to act as a modulator of native C3 enabling it to activate C3PAse. Activation of C3 was efficiently mediated by P in the fluid phase. Efficient activation of C5, however, required the participation of an insoluble polysaccharide (zymosan). The possibility is raised therefore that P might also be an integral part of the multimolecular C5 convertase of the alternate pathway of complement activation.

Fourth component of human complement: description of a three polypeptide chain structure.

The fourth component of human complement (C4) was shown to be composed of three distinct polypeptide chains linked by disulfide bonds and noncovalent forces. The sum of the molecular weights of the chains equalled that of the intact molecule. The mol wt of the alpha-, beta-, and gamma-chains were respectively, 93,000, 78,000, and 33,000 daltons. Action of C1s on C4 affected only the alpha-chain, reducing its mol wt to 87,000 daltons. The size of the activation peptide. C4a, is therefore estimated to be 6,000 and that of the major fragment C4b, 198,000 daltons. Periodic acid-Schiff-stained SDS polyacrylamide gels of reduced C4 revealed carbohydrate to be associated with all three chains. A modification of the original method of isolation of C4 is presented.

The eighth component of human complement (C8): isolation, characterization, and hemolytic efficiency.

A method was described allowing the isolation of the eighth component of complement, C8, from human serum. C8 is an immunochemically definable, heretofore unrecognized serum protein with a molecular weight of approximately 150,000 and an electrophoretic mobility of a gamma(1)-globulin. It may be obtained with 5% yield as a 5000-fold purified, physicochemically and immunochemically homogeneous protein. Under optimal experimental conditions, a few molecules of C8 were found sufficient for lysis of a cell.

The reaction mechanism of human C5 in immune hemolysis.

The data presented here indicate that the C5 reaction step may proceed via the specific attachment of C5 to EAC1,4,2,3 and the formation of a hemolytically active C5 intermediate complex. During this process only a minor proportion (less than 4%) of C5 offered to EAC1,4,2,3 becomes bound, although the remaining C5 also participates in the reaction as evidenced by its inactivation in the fluid phase. Once bound, C5 is exceptionally efficient in producing hemolysis, requiring less than seven specifically bound molecules per cell for the production of a hemolytic lesion. The extent of formation of the C5 intermediate complex is primarily dependent on the number of molecules of C4, 2 and C3 present on the cells employed for its generation. In these respects, the mode of action of C5 is completely analogous to that of the other components of complement thus far investigated. The C5 step differs, however, in other aspects. The binding of C5 is influenced by C6 and C7, components which are thought to act subsequent to it in the complement sequence. In addition, the hemolytic activity of the isolated C5 intermediate complex is exceedingly labile, having an average half-life at 30 degrees C of only 9 min. This characteristic distinguishes the C5 step, along with the C2 step, as potentially rate-limiting in the complement reaction. However, unlike C2, C5 remains firmly cell-bound during the decay process and apparently undergoes an alteration in situ which renders it hemolytically unreactive. Finally, C5 is unique in that it readily adsorbs in native form to unsensitized erythrocytes. This nonspecifically bound C5 remains firmly attached, although it may be specifically utilized as a source of C5 by an ongoing complement reaction. The significance of the marked affinity of native C5 for cell-surface receptors remains to be determined.

Lysis of erythrocytes by complement in the absence of antibody.

A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement.

Blood coagulation initiation by a complement-mediated pathway.

A variety of complement-activating substances, including inulin, immunoglobulin aggregates, bacterial endotoxins, and staphylococcal protein A, were found to initiate blood coagulation through a complement-mediated pathway. These substances markedly accelerated blood coagulation in normal rabbit blood. That this clot-promoting activity requires an intact complement system was demonstrated by an almost total lack of effect on blood from rabbits with an inherited deficiency of the sixth component of complement (C6). Small amounts of isolated C6 conferred to C6-deficient blood the ability to respond with accelerated coagulation upon activation of the complement system. In addition, it was determined that activation of complement through the previously described C3 activator system resulted in the initiation of blood coagulation. The participation of C1, C2, and C4 was not necessary.

Formation of C3a and C5a anaphylatoxins in whole human serum after inhibition of the anaphylatoxin inactivator.

Two biologically and chemically distinct anaphylatoxins (ATs) could be generated in whole human serum after removal of the AT inactivator (AI) by immune-absorption or after inhibition of AI with 1 M epsilon-aminocaproic acid (EACA). Both human ATs could be generated by treatment of serum with antigen-antibody complexes, which activate the classical complement pathway, and with inulin or yeast, both of which trigger the alternate pathway. The ATs were isolated from serum in active form and characterized as C3a and C5a. Although human C3a had been characterized previously, C5a had not. The molecular weight of human C5a AT was 17,500; its electrophoretic mobility at pH 8.5 was -1.7 x 10(-5) cm(2) V(-1) s(-1). The minimal effective concentration in vitro was 7.5 x 10(-10) M. The minimal effective doses of human C5a in producing a wheal and erythema in the human skin was 1 x 10(-15) mol. The results strongly suggest a biological function for both ATs and indicate that the expression of their activity is controlled by the AI of normal blood plasma.

C3 proactivator convertase and its mode of action.

The activity in human serum which is responsible for conversion of C3 proactivator (C3PA) to C3 activator was shown to reside in a 3S alpha-globulin. The factor, called C3PA convertase (C3PAse), was obtained in partially purified form. For conversion of C3PA, C3PAse required participation of metal ions and of a C3 fragment, which in physicochemical and antigenic properties resembled C3b. Isolated, native C3 failed to substitute for the fragment, but did restore the impaired C3 activator system in hydrazine-treated serum. Unlike native C3, the C3 fragment initiated conversion of C3PA in whole serum. A hypothetical concept which envisions the C3 fragment as effector of C3PAse has been proposed.

The membrane attack mechanism of complement. Verification of a stable C5-9 complex in free solution.

The membrane attack mechanism of complement, C5 to C9, has previously been postulated to associate on the target cell surface to a stable decamolecular complex with a calculated mol wt of 995,000. A soluble and stable complex consisting of C5, C6, C7, C8, and C9 has now been demonstrated to arise as a consequence of complement activation by the classical or alternate pathway. It has a sedimentation coefficient of 22.5S and a mol wt of 1 million daltons, and it migrates on electrophoresis at pH 8.6 as an alpha-globulin. The stable and soluble C5b-9 complex cannot bind to erythrocytes and has no demonstrable cytolytic activity. However, due to partially unsaturated binding sites for C9, it can bind additional C9 and thus function as an inhibitor of lysis of EAC1-8 by C9. These results support the concept according to which the membrane-bound attack system of complement represents a stable, decamolecular assembly of C5b-9. Unlike its analogue in free solution, the membrane-bound complex is cytolytically active.

Cytolytic lymphocytic cells with complement receptor in human blood. Induction of cytolysis by IgG antibody but not by target cell-bound C3.

Human blood lymphocytes were fractionated on glass bead columns charged with sheep erythrocyte (Es) membranes-bearing human C3b (7,000-10,000 molecules/Es). In the passaged cells the proportion of C receptor lymphocytes was strongly reduced, in parallel with the capacity to lyse chicken erythrocytes (Ec) in the presence of IgG-rabbit anti-Ec antibody. In other experiments, lymphocytes forming rosettes with Es bearing activated rabbit complement [C(ra)] from C6-deficient rabbits were removed by centrifugation through human serum albumin-gelatine mixtures. This procedure also depleted the lymphocyte preparations of antibody-dependent cytolytic effector cells. The results suggest that rations of antibody-dependent cytolytic effector cells. The result suggest that such effector cells have receptors for human C as well as for C(ra). Lymphocytes were not able to lyse erythrocytes bearing either human C3b (similar to 30,000 molecules/Ec) or activated C(ra) in the absence if IgG antierythrocyte antibodies. Under the same experimental conditions these target cells were efficiently lysed in the presence of small amounts of IgG antitarget cell antibodies. This suggests that the interaction between the cellular Fcreceptors and the Fc part of the inducing antibodies is of special significance for the triggering of the cell-mediated lytic reaction. However, although target cell-bound C did not trigger cytolysis, it seemed to potentiate antibody-dependent cytolysis, probably by enhancing effector cell-target cell contacts.

The membrane attack mechanism of complement: the three polypeptide chain structure of the eigth component (C8).

The purification of human C8 in milligram quantities from outdated human serum was achieved by ammonium sulfate precipitation (37.5-50% saturation) and ion exchange column chromatography employing CM-32 cellulose and QAE-Sephadex. The yield of C8 activity ranged from 2-9%, and the average purification was 1,700-fold. Fully reduced C8 was shown by SDS polyacrylamide gel electrophoresis to have three polypeptide chains which were present in equimolor ratios: alpha, 77,000 daltons; beta, 63,000 daltons; and gamma, 13,700 daltons. C8 denaturation by SDS and urea in the absence of reducing agents revealed two noncovalently linked subunits: alpha-gamma, 99,000 daltons, and beta, 75,000 daltons.

Assembly of the cytolytic alternative pathway of complement from 11 isolated plasma proteins.

The known cytolytic function of the alternative pathway in serum was quantitatively reproduced by combining 11 isolated plasma proteins at their respective serum concentrations. These proteins are: C3, Factor B, Factor D, C3b inactivator, beta1H, native properdin, C5, C6, C7, C8, and C9. In absence of activators of the alternative pathway, this mixture was stable at 37 degrees C as evidenced by lack of consumption of Factor B, C3, and C5. Upon addition of either rabbit erythrocytes or neuraminidase-treated sheep erythrocytes, cell lysis ensued and the extent of lysis was dependent on dose of the component mixture. The dose-response curves obtained with the isolated component mixture and with C4-depleted serum were virtually indistinguishable. Nonactivator erythrocytes (untreated sheep erythrocytes) were not lysed by the component mixture. Deletion of properdin resulted only in a twofold diminution of the hemolytic activity of the component mixture. No immunoglobulin requirement was apparent. These results indicate that the cytolytic systems studied are internally sufficient and capable of coupling the initiation and amplification sequence with the cytolytic membrane attack sequence.