Human complement component C4. Structural studies on the fragments derived from C4b by cleavage with C3b inactivator.

1. One of the activation products of C4, C4b, was prepared, and the reactive thiol group on the alpha'-chain was radioactively labelled with iodo[2-14C]acetic acid. The alpha'-chain was isolated and the N-terminal amino acid sequence of the first 13 residues was determined. 2. C4b was cleaved by C3bINA in the presence of C4b-binding protein and C4d and C4c isolated. The radioactive label and therefore the reactive thiol group were located to C4d. 3. C4c was reduced and alkylated and the two alpha'-chain fragments of C4c were separated. 3. The molecular weights, amino acid analyses and carbohydrate content of the three alpha'-chain fragments were determined. C4d has a mol.wt. of 44500 and a carbohydrate content of 6%. The two alpha'-chain fragments of C4c have mol.wts. of 25000 (alpha 3) and 12000 (alpha 4) and carbohydrate contents of 10 and 22% respectively. 4. The N-terminal amino acid sequences of C4d, the alpha 3 and the alpha 4 fragments were determined for 18, 24 and 11 residues respectively and, by comparison with the N-terminal sequence of the C4b alpha'-chain, the 25000-mol.wt. fragment (alpha 3) was shown to be derived from the N-terminal part of the alpha'-chain. 5. C-Terminal analyses were done on the alpha'-chain and its three fragments. Arginine was found to be the C-terminal residue of C4d and of the alpha 3 fragment. The C-terminal residue of the alpha'-chain and of the alpha 4 fragment could not be identified. The order of the three fragments of the alpha'-chain is therefore: alpha 3(25000)--C4d(44500)--alpha 4(12000). The specificity of C3bINA is for an Arg--Xaa peptide bond.     

Purification of the human complement control protein C3b inactivator.

An alternative method of isolation from human plasma is described for C3b inactivator, C3bINA, the proteinase that in conjunction with either beta 1H or C4b-binding protein will hydrolyse respectively C3b or C4b, the activation products of the third, C3 and fourth, C4, components of complement. The purification is by chromatography of plasma on columns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500ml of plasma) is severalfold higher than in previously described methods. The sensitivity of the assay for C3bINA has been increased by including optimal amounts of beta 1H, and it was observed that beta 1H was essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution or bound to a cell surface. Native C3 is not hydrolysed by C3bINA + beta 1H, but the haemolytically inactive form that appears on prolonged storage at 4 degrees C or on freezing and thawing is hydrolysed and gives fragments of the alpha-chain of 75000 and 43000 apparent mol.wt. As the alpha'-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67000 and 43000 apparent mol.wt. when incubated with C3bINA + beta 1H, this suggests that the larger fragment is N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clear classification of the type of proteinase to which this enzyme belongs has been obtained.     

Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3d,g (alpha 2D), and C3g

The fragments that result from the inactivation of C3b have not been completely characterized. Initial inactivation is catalyzed by the protease factor I, which, in the presence of its cofactor (factor H), cleaves two peptide bonds in the alpha'-chain of C3b. This results in the release of a small peptide (C3f, Mr 3000) from iC3b, which consists of the C3 beta chain covalently bonded to two alpha'-chain-derived peptides (Mr 68,000 and Mr 43,000). Surface-bound iC3b is cleaved at a third site by factor I to produce C3c and C3d,g (or alpha 2D). The factor I cofactor for this cleavage is the C3b receptor that is present on erythrocyte and leukocyte membranes. This report describes the isolation and initial structural characterization of C3c and C3d,g generated in whole blood after complement activation with cobra venom factor. These fragments were compared with the C3 fragments isolated from the serum and plasma of a patient with complement activation in vivo. The fragments were isolated with two solid phase monoclonal antibodies, one of which recognizes a determinant on C3g (clone 9) and one of which recognizes a determinant on C3c (clone 4). C3c isolated from normal blood showed three polypeptides that had apparent m.w. of 75,000, 43,000, and 27,000. The C3d,g consisted of a single polypeptide chain with a m.w. of 40,000. Amino terminal sequence analysis showed that the Mr 27,000 peptide from C3c is derived from the amino terminal portion of the alpha'-chain of C3b, whereas the Mr 43,000 peptide is derived from the carboxy terminus of the same chain. Amino terminal sequence analysis showed also that C3g is derived from the amino terminus of C3d,g. The C3 fragments isolated from a patient with partial lipodystrophy, nephritic factor activity, low serum C3 levels, and circulating C3 cleavage products showed a more complicated pattern on SDS-PAGE. The fragment isolated with clone 9 had an apparent m.w. of 40,000, identical to C3d,g generated in vitro, and it had the same amino terminal sequence as C3d,g generated in vitro. The eluate from insolubilized clone 4, however, showed prominent bands with Mr of 75,000, 56,000, 43,000, and 27,000, together with a triple-banded pattern at 68,000 and a minor band at 80,000. This eluate thus appears to contain C3c, and iC3b or an iC3b-like product. The origin of the Mr 56,000 and Mr 80,000 peptides have not yet been determined. These studies, with previous data, definitively order the C3c and C3d,g peptides in the alpha-chain of C3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation and deregulation of the fluid-phase classical pathway C3 convertase

Three mechanisms that regulate the formation and function of the fluid-phase classical pathway C3 convertase (C4b2a) have been elucidated: a) a temperature-mediated intrinsic decay of the enzyme; b) an extrinsic accelerated decay mediated by the effect of the serum protein C4b-binding protein (C4-bp); and c) the inactivation of C4b in the C4b-C4b-p complex by the proteolytic action of C3b/C4b inactivator (I), which cleaves the alpha 1-chain of C4b yielding C4d (alpha 2-chain), and C4c (alpha 3-, alpha 4-, beta-, gamma-chains). A fourth mechanism is described based on the observation that the IgG fraction of the serum of certain patients with glomerulonephritis contains a protein that prevents the intrinsic and C4-bp-mediated decay of surface-bound C4b2a. This protein prolongs the half-life of fluid-phase C4b2a from 10 min to more than 5 hr, increasing the utilization of C3. It also inhibits the decay mediated by C4-bp by preventing the dissociation of C2a from the C4b, 2a complex. In addition, I alone or in the presence of C4-bp fails to cleave the alpha 1-chain of C4b in the stabilized C4b, 2a complex. This protective property of the stabilizing factor (NFc) requires the presence of C2a because C4b was not protected unless it was bound to C2a. Therefore, NFc provides a mechanism by which the serum regulatory proteins are bypassed.     

Complement receptor binding of C3b-coated cells treated with C3b inactivator, beta 1H globulin and trypsin.

Treatment of 125I-C3b bound to EAC1423b with C3b inactivator (C3bINA) and beta 1H globulin (beta 1H) cleaved the alpha-chain of C3b into 65,000- and 42,000-dalton fragments, both of which remained disulfide-bonded to the intact beta-chain (C3bi). Subsequent treatment with trypsin (0.1 microgram/ml) released 125I into the supernatant and yielded cells coated with a 33,000-dalton fragment of alpha-chain, presumably C3d. These results are in agreement with those obtained by others using fluid phase C3b. C3b-coated cells (EAC1423b) adhered to complement (C) receptors on human erythrocytes, glomeruli, and monocytes. C3bi-coated cells adhered to the receptors on glomeruli and monocytes, but not to those on human erythrocytes. C3d-coated cells adhered only to the monocyte receptors. The findings suggest that the glomerular C receptor recognizes portions of the C3 molecule different from those recognized by either the erythrocyte or monocyte receptors.     

Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.     

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.     

Phylogeny of C4b-C3b cleaving activity: similar fragmentation patterns of human C4b and C3b produced by lower animals

Functional and structural studies of the activated proteins of the complement system C4b and C3b have led to the identification of cleavage products resulting from the effect of the regulatory proteins, factor I, H, and C4b binding protein (bp). In this paper we report the results of studies that investigated the capacity of plasma or serum from a wide range of phylogenetic species to yield similar cleavage products. Sera and plasma from mammals, reptiles, amphibia, and fishes are capable of cleaving fluid phase human C4b and C3b, generating apparently the same fragments as observed using normal human serum: alpha 2, alpha 3, alpha 4 from the alpha' chain of C4b: and alpha-68, alpha-46, alpha-43, and alpha-30 from the alpha' chain of C3b. When C3b bound to a cell membrane is used C3c and C3dg are generated. The generation of these fragments from C3bi is a dose-dependent reaction. There is no correlation between the evolution of the species and the quantitative capability to degrade the substrates. Birds possess only a limited capability to degrade the alpha' chain of C4b and have no cleaving activity for C3b, whereas sera from more primitive vertebrate species (chondrichthyes and agnatha) fail to participate in the reaction. Contrary to other species, the proteins in fish serum or plasma responsible for the degradation of C4b and C3b show a unique requirement for Ca2+ ions. Magnesium and barium are less effective, and in their presence a 65,000 dalton intermediate product is observed. These results demonstrate that protein(s) displaying proteolytic activity for products of complement activation, probably related to I, H, and C4bp, are present in plasma of species whose evolution have preceded humans by 300 million years. Moreover, the recognition of human substrates and the generation of fragments identical to those produced by human serum suggests that human C4b and C3b share structural characteristics with their evolutionary ancestors in the serum or plasma of the species studied.     

Cleavage of human C5 by trypsin: characterization of the digestion products by gel electrophoresis.

Human C5 is composed of two nonidentical polypeptide chains, alpha and beta (m.w. 130,000 and 80,000, respectively) linked together by disulfide bonds and noncovalent forces. Cleavage of C5 by trypsin fragments with increased anodic mobilities. Limited digestion of C5 by trypsin (substrate to enzyme ratio 10:1 w/w at 37 degrees C for 1 min) resulted in the release of a small terminal alpha-chain peptide (alpha1, m.w. 15,000) probably analogous to C5a, from a large fragment, C5b (m.w. 195,000) composed of an intact beta-chain disulfide linked to an alpha-chain that has a lower m.w. (alpha' 115,000). Further digestion (37 degrees C, 5 min) resulted in cleavage of the alpha-chain at multiple sites with the production of three peptides from the alpha'-chain (alpha2I, 23,500; alpha2II 15,700 and alpha2III 10,200) and a residual fragment, C5c (m.w. 144,000). The alpha1 and alpha2 peptides are not covalently linked to the beta-chain nor to one another. The C5c fragment on the other hand is composed of small peptides of the alpha'c chain (alpha3 14,000; alpha4I 9,000; ALPHA 4II 11,000; alpha 5 23,000 to 30,000) which are linked to the beta-chain and also probably to one another by covalent bonds. Secondary cleavage occurred upon prolonged digestion with trypsin (37 degrees C, 20 min), and this resulted in the progressive erosion of the alpha'c peptides and the conversion of C5c to smaller C5c-like species.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.     

Phylogeny of regulatory proteins of the complement system. Isolation and characterization of a C4b/C3b inhibitor and a cofactor from sand bass plasma

We have previously demonstrated that the alpha'-chain of human activated form of the fourth (C4b) and third (C3b) component of C are cleaved by plasma or serum from vertebrate species spanning through 300,000,000 yr of evolution yielding fragments identical with those obtained with human plasma. In this study, we investigated the molecular basis of this reaction. We chose barred sand bass plasma because this is the most primitive species analyzed possessing these activities. Barred sand bass plasma proteins were separated on a Sephadex G-200 column and the eluted samples analyzed for C4b and C3b cleavage. Individual fractions were inactive, but degradation was obtained when proteins of 380 and 155 kDa were combined. In contrast to the human regulatory proteins, the sand bass proteins require Ca2+ ions. K76COOH, an inhibitor of human factor I, inhibited the function of the 155-kDa but not of the 380 kDa-fraction. Thus it appears that the 155-kDa fraction functions as the C4b/C3b cleaving enzyme (I) and the 380-kDa material as its cofactor. Further purification of the 380-kDa fraction yielded a protein that by SDS-PAGE consisted of two noncovalently linked subunits of 110 and 42 kDa at a molecular ratio of 2:1. These two chains were antigenically distinct, and constitute domains of the same protein. The 110-kDa peptide binds C4b and not C3b but it fully expresses the cofactor function for the 155-kDa fraction on the cleavage of both C4b and C3b. Limited tryptic digestion of the 110-kDa domain demonstrated C4b binding activity in fragments of 34, 25, and 23 kDa. The activity of the 34-kDa fragment was the same as that of the undigested protein. Comparison of the amino acid composition of the barred sand bass cofactor and of human C4bp shows similar high content of cysteine and proline but not of tryptophan. It differs from human factor H in cysteine, serine, proline, and tryptophan. These studies indicate that regulatory proteins for the C4b and C3b C fragments may have appeared very early phylogenetically.     

Membrane receptors of mouse leukocytes. I. Two types of complement receptors for different regions of C3

Mouse leukocytes were studied for membrane receptors for the third C component by rosette formation with C coated erythrocytes (EAC). Methods were devised for the preparation of EAC complexes containing either mouse C3b or mouse C3d. EAC 1-3dmo were prepared from EA treated with whole mouse serum while EAC 1-3bmo were produced from EAC 142hu treated with whole mouse serum containing sodium suramin. The specificity of the EAC complexes for mouse leukocytes was confirmed by inhibition experiments using fluid phase human C3d. Low concentrations of fluid phase human C3d inhibited EAC1-3dmo rosettes but failed to inhibit EAC 1-3bmo rosettes. Eight-fold higher concentrations of fluid phase C3d caused partial inhibition of EAC1-3bmo rosette formation with lymphocytes, but not with other types of murine leukocytes. Thus mouse leukocytes apparently contain the same two types of C receptors as do human and guinea pig leukocytes. Mouse CR1 is specific for a non-C3d region of C3b, (possibly analogous to human C3c) whereas mouse CR2 is specific for both C3d and the C3d region of C3b.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

Formation and structure of the C5b-7 complex of the lytic pathway of complement.

The formation and structure of the complement cytolytic intermediary complex, C5b-7, were studied with the aim of determining the interactive regions of C5, C6, and C7. The structure of human complement component C5 was elucidated by the application of limited proteolysis which generated well characterized major polypeptide fragments of this molecule. Plasmin, thrombin, and kallikrein cleave C5b with greater facility than C5. The most useful cleavage of C5b was effected by plasmin because the fragmentation pattern was similar to the processing of C3b by factors H, I, and kallikrein. Plasmin hydrolyzes peptide bonds within the alpha'-chain of C5b, resulting in a four-chain fragment, C5c (M(r) = 142,000), and a single chain fragment, C5d (M(r) = 43,000). Circular dichroism spectroscopic analyses indicated that C5d is substantially richer in alpha-helical content than is C5c (27 versus 9%). Polyclonal antibodies directed against C5c blocked the interaction of C5b-6 with C7, whereas antibodies directed against C5d inhibited the binding of C5 with C3b. Chemical cross-linking using a cleavable radioiodinated photoreactive reagent revealed that both C6 and C7 associate preferentially with the alpha'-chain of C5b. The reversible interactions of C5 with C6, C7, and major polypeptide fragments derived from these were investigated with solid phase binding assays. The results indicate that the carboxyl-terminal domains of C6 and C7, which have cysteine-rich modules homologous to those found in factors H and I, have the capacity to link specifically with C5.     

Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46).

Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.     

Two clusters of acidic amino acids near the NH2 terminus of complement component C4 alpha'-chain are important for C2 binding

Previous work has indicated a role for the NH2-terminal segment of the C3 alpha'-chain in the binding interactions of C3b with a number of its protein ligands. In particular, we have identified two clusters of acidic residues, namely, E736 and E737 and to a lesser extent D730 and E731, as being important in the binding of C3b to factor B and complement receptor 1 and the binding of iC3b to complement receptor 3. Whereas human C3 and C4 have an overall sequence identity of 29%, over a segment near the NH2 termini of their respective alpha'-chains the sequence identity is 56% (70% chemical similarity). Given the functional similarity between the C4b-C2 and C3b-B interactions in the respective formation of the classical and alternative pathway C3 convertases, as well as the sequence conservation of two acidic clusters, we hypothesized that residues 744EED and 749DEDD within the NH2-terminal segment of the C4 alpha'-chain would mediate in part the binding of C2 to C4b. We tested this hypothesis using three independent approaches. Site-directed mutagenesis experiments revealed that replacing subsets of the charged residues by their isosteric amides within either acidic cluster resulted in molecules having reduced C2 binding activity. Moreover, a synthetic peptide (C4 residues 740-756) encompassing the two acidic clusters was a specific inhibitor of the binding of C2 to red cell-associated C4b. Finally, Ab raised against the above peptide was able to block the interaction between red cell-associated C4b and fluid phase C2. Taken together, these results strongly suggest that the NH2-terminal acidic residue-rich segment of C4 alpha'-chain contributes importantly to the interaction of C4b with C2.     

Characterization of functional properties of C4-binding protein by monoclonal antibodies

We prepared mouse monoclonal antibodies to human C4-binding protein (C4-bp) by fusing spleen cells from mice immunized with purified C4-bp to the mouse myeloma line P3U1. Of four monoclonal antibodies that reacted with intact C4-bp, two were specific for a 48K fragment, one of the chymotryptic cleavage products of C4-bp, and one was specific for another fragment (160K). The fourth monoclonal antibody did not react with either fragment. One of the monoclonals that reacted with the 48K fragment blocked the binding of C4-bp to cell-bound C4b. This monoclonal antibody (TK3) also inhibited two other functions of C4-bp, serving as an essential cofactor for C3b/C4b inactivator (I) in the cleavage of fluid-phase C4b and accelerating the decay of C2a from the C4b,2a complex. The other monoclonals had little or no effect on these activities of C4-bp. In addition, we found that the 48K fragment lost the binding affinity for C4b. However, it can function as a cofactor for I and as a decay-accelerator, although its activities were about 200 times weaker than intact C4-bp on a molar basis. The monoclonal antibody TK3 completely inhibited these activities of the 48K fragment. On the basis of these findings, we conclude that the functionally active site of C4-bp is located on the 48K fragment. Probably, the cofactor and decay-accelerating activities of C4-bp result from the binding of C4-bp to C4b.     

Isolation of an iC3b forming enzyme from peritoneal polymorphonuclear leukocytes of guinea pigs

We have investigated fluid phase cleavage of C3b by peritoneal polymorphonuclear leukocytes of guinea pigs and found that polymorphonuclear leukocytes expressed an iC3b forming enzyme as well as C3b receptor with maturation in peritoneal cavity. The iC3b forming enzyme was found to be distinct from C3bINA, a physiological iC3b forming enzyme in plasma, since the activity was inhibited by monoiodoacetic acid and did not require a cofactor plasma protein, beta 1H, for the cleavage of C3b into iC3b. The iC3b forming enzyme is gradually released upon incubation of PMN at 37 degrees C. The molecular weight of the iC3b forming enzyme was estimated to be 48,000 from gel filtration on Sephadex G-200.     

Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.