Evidence for a C4b binding site on the C2b domain of C2

We raised murine mAb against human C protein C2. The representative mAb 3A3.3 (IgG1 kappa) recognized an epitope on the C2b domain of C2, as determined by binding and inhibition of binding radioassays. The hemolytic activity of purified human C2 and of C2 in normal human serum was inhibited by the mAb. The rate of decay of the C3-convertase at 30 degrees C was not affected by the mAb. C2 binding to EAC4b was inhibited by intact IgG and the Fab fragment of the mAb; 50% inhibition required 1 microgram/ml of either. The data suggest the presence of a C4b-binding site on the C2b domain of C2 and that the mAb recognizes an epitope at, or adjacent to, this site. The C2b portion of the C2 molecule may be important in assembly of the classical pathway C3-convertase.     

Mouse C3b/C4b inactivator: purification and properties

Mouse C3b/C4b inactivator (C3b/C4bINA) was purified approximately 400 times from mouse serum. It is a beta-globulin and consists of 2 disulfide bonded chains of m.w. 60,000 and 35,000. Under nonreducing conditions, its m.w. is 95,000. It cleaves the alpha'-chain of cell-bound C4b into 3 fragments: alpha 2, alpha 3, alpha 4. The alpha 2 fragments remain bound to the cell surface (C4d), and the rest of the molecule (C4c) is released into the fluid phase. In fluid phase, C3b/C4bINA cleaves the alpha'-chain of C4b in a similar manner but only in the presence of mouse or human C4-binding protein (C4-bp). Mouse C4-bp and human C3b/C4bINA do not cleave human C4b, although mouse C4-bp binds to human C4b. This incompatibility suggests that C4-bp and C3b/C4bINA must interact to cleave fluid phase C4b. Mouse C3b/C4bINA also cleaves the alpha'-chain of human C3b in solution into 2 fragments in the presence of human beta 1H. Therefore, it is likely that mouse and human C3b/C4bINA are homologous proteins. A monospecific antiserum to mouse C3b/C4bINA has been prepared in rabbits. By crossed immunoelectrophoresis, this antiserum detects, in addition to the protein described above, a fast beta-globulin with a m.w. of approximately 200,000 and antigenically identical to C3b/C4bINA but enzymatically inactive. This protein could represent a precursor of C3b/C4bINA.     

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.     

Control of C1 activation by nascent C3b and C4b: a mechanism of feedback inhibition

We have demonstrated that immune complexes turn over C1, i.e., limiting quantities of immune complexes activate an excess of C1. This was readily apparent in a system of purified C1 and C1-inhibitor (C1-In) but not in normal human serum (NHS). The following results indicate that C3 and C4 are the serum factors responsible for the inhibition of C1 turnover by immune complexes. 1) In a purified protein system composed of C1 and C1-In at pH 7.5, ionic strength 0.14 M, doses of immune complexes that activated all the C1 in 60 min at 37 degrees C yielded no detectable C1 activation when C2, C3, and C4 were also present. All proteins were at their physiologic concentrations. Activation was quantified by SDS-PAGE analysis and hemolytic titration 2) In order to inactivate C3 and C4, NHS was treated with 50 mM methylamine (MeAm) for 15 min at 37 degrees C, after which the MeAm was removed by dialysis. The activities of C1, C2, and C1-In were unaffected by this treatment. Doses of immune complexes that consumed no C1 in NHS, consumed all the C1 in MeAm-treated NHS (MeAm-NHS). 3) Reconstitution of MeAm-NHS with physiologic concentrations of C3 and C4 rendered the serum again resistant to excessive C1 consumption by immune complexes. Immune complexes used in these studies included EA-IgG, EA-IgM, tetanus-human anti-tetanus, and aggregated human IgG. There appeared to be specificity to the inhibition reaction since C4 by itself could inhibit C1 consumption by EA-IgM, whereas the presence of C3 was also required to control EA-IgG. Finally, N-acetyl-L-tyrosine was added to NHS at a final concentration of 30 mM. This nucleophile did not interact with native C3 or C4, nor did it directly activate C1. However, upon the addition of low doses of immune complexes, acetyl tyrosine did yield uncontrolled C1 activation, presumably by binding nascent C3b and C4b and thereby blocking their attachment to the immune complexes. We conclude that in NHS there is a mechanism of feedback inhibition by which nascent C3b and C4b inhibit C1 turnover by immune complexes. This mechanism of control might be physiologically important in that it prevents excessive complement activation by low concentrations of immune complexes.     

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.     

Intracellular degradation of the complement C3b/C4b receptor in the absence of ligand

Human neutrophils (PMN) respond to various soluble stimuli by translocating intracellular complement C3b/C4b receptors (CR1) to the cell surface. Ligand-independent internalization of surface CR1 has been demonstrated previously, but the fate of total cellular CR1 during PMN stimulation has not been determined. In order to study the fate of CR1 during neutrophil activation, we have employed a unique approach for the quantitative analysis of intracellular antigens which allows simultaneous measurement of total cellular and surface membrane antigen pools. Stimulation of isolated PMN with N-formyl-Met-Leu-Phe or ionomycin resulted in a mean 7-fold increase in surface CR1 expression within 15 min. Total cellular CR1 decreased by as much as 45% within 15 min, with loss continuing for up to 1 h. Inclusion of NH4Cl during PMN stimulation inhibited the loss of total CR1 without affecting surface CR1 expression. Addition of phenylmethylsulfonyl fluoride inhibited loss of total CR1 and enhanced the stimulus-induced increases in surface CR1. These data suggest that intracellular degradation of CR1 occurs during stimulation of PMN and may involve proteolysis in an acidic intracellular compartment. Since our experiments were done with isolated PMN in the absence of serum and complement components, this degradation occurred in the absence of C3b, the ligand for CR1. To our knowledge, ligand-independent degradation of a cell surface receptor has not been previously detected.     

The superfamily of C3b/C4b-binding proteins

The determination of primary structures by amino acid and nucleotide sequencing for the C3b-and/or C4b-binding proteins H, C4BP, CR1, B, and C2 has revealed the presence of a common structural element. This element is approximately 60 amino acids long and is repeated in a tandem fashion, commencing at the amino-terminal end of each molecule. Two other complement components, C1r and C1s, have two of these repeating units in the carboxy-terminal region of their noncatalytic A chains. Three noncomplement proteins, beta 2-glycoprotein I (beta 2I), the interleukin 2 receptor (IL 2 receptor), and the b chain of factor XIII, have 4, 2 and 10 of these repeating units, respectively. These proteins obviously belong to the above family, although there is no evidence that they interact with C3b and/or C4b. Human haptoglobin and rat leukocyte common antigen also contain two and three repeating units, respectively, which have more limited homology with the repetitive regions in this family. All available data indicate that multiple gene duplications and exon shuffling have been important features in the divergence of this family of proteins with the 60-amino-acid repeat.     

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.     

Endogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes

Decay-accelerating factor (DAF) is a membrane glycoprotein found on various cells that are in contact with complement. It inhibits the formation of the C3 convertases of the complement system, both the classic (C4b2a) and alternative (C3bBb) pathways. In this investigation, we used a homobifunctional cross-linking reagent to search for a DAF ligand on the surface of cells subjected to complement attack. We found that DAF forms complexes with C4b and C3b deposited on the same erythrocytes, but not with the physiologic degradation products of these complement fragments, that is, C4d or C3dg. Taken together with prior observations that DAF action is reversible, and DAF does not affect the structure of C4b or C3b, these findings suggest that DAF functions by competitively inhibiting the uptake of C2 or factor B, and preventing the assembly of the C3 convertases.     

The surface protease PgtE of Salmonella enterica affects complement activity by proteolytically cleaving C3b, C4b and C5

Complement activity in mammalian serum is fundamentally based on three homologous components C3b, C4b and C5. During systemic infection, the gastrointestinal pathogen Salmonella enterica disseminates within host phagocytic cells but also extracellularly. Consequently, systemic Salmonella transiently confronts the complement system. We show here that the surface protease PgtE of S. enterica proteolytically cleaves C3b, C4b and C5 and that the expression of PgtE enhances bacterial resistance to human serum. Degradation of C3b was further enhanced by PgtE-mediated plasminogen activation.     

Polymorphism of the C3b/C4b receptor (CR1): characterization of a fourth allele

The receptor for C3b/C4b (C3bR or CR1) has an unusual polymorphism in which three codominant alleles determine variants with a large difference in Mr (160,000, 190,000, or 220,000). We found an individual who has, in addition to the common 190,000 Mr molecule, a C3bR whose Mr is 250,000. In this proband and in some members of his family, this novel heterozygous phenotype can be isolated from 125I surface-labeled cells by iC3 or iC4 affinity chromatography or by immunoprecipitation with the use of polyclonal or monoclonal anti-C3bR. Relative to the 190,000 Mr C3bR, E from individuals in this family have 20 to 30% of the total receptor counts in the 250,000 Mr C3bR. However, on C3bR-bearing leukocytes there is a much larger amount of the 250,000 Mr C3bR (approximately 60%) relative to the 190,000 Mr C3bR. Similar to the other three C3bR variants, the Mr is 5,000 greater on polymorphonuclear cells than on E, and treatment of this new C3bR with endoglycosidase F decreases its Mr by approximately 10,000. Therefore, because this variant is inherited and has structural and functional similarities to the other three C3bR, we conclude that this 250,000 Mr CR1 probably represents a fourth allele.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.     

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.     

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.     

Translation of the human C3b/C4b receptor mRNA in a cell-free system and by Xenopus oocytes

The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190,000, 220,000, 250,000, and 280,000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220,000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CR1 and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CR1 monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.     

Characterization of a soluble form of the C3b/C4b receptor (CR1) in human plasma

A radioimmunoassay with the use of soluble 125I-Fab monoclonal anti-CR1 and rabbit IgG anti-CR1 bound to Staphylococcus aureus particles was employed to detect and quantitate CR1 antigen in human plasma. Among 16 normal individuals the concentration of soluble CR1 in plasma ranged from 13 to 81 ng/ml, and a similar range of concentration was found in plasma from 15 patients having systemic lupus erythematosus (SLE). The amount of plasma CR1 in normal donors, but not in SLE patients, significantly correlated with the number of CR1 sites on erythrocytes (r = 0.90, p less than 0.001), and was 7.1% of the amount of receptor that was present on erythrocytes in blood. The concentration of soluble CR1 was not diminished by ultracentrifugation or ultrafiltration of plasma, was not affected by various modes of anti-coagulation or even by clotting of blood, and did not change during incubation of blood at 4 degrees C for up to 4 hr. On sucrose density gradient ultracentrifugation of plasma the CR1 was distributed as a broad peak that overlapped the plasma protein profile. The Mr of plasma CR1 was identical to that of erythrocyte CR1 when assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunoblotting. In addition, the plasma form of CR1 exhibited the same structural phenotype as did receptor from erythrocytes of the same individual. CR1 antigen purified from plasma was as active as CR1 from erythrocytes in promoting the cleavage by factor I of C3b to iC3b, C3c, and C3dg. Therefore, a functionally and structurally intact form of soluble CR1 resides in plasma.     

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.