A new apoptotic pathway for the complement factor B-derived fragment Bb

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.     

Absence of induction of IL-1 production in human monocytes by complement fragments

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.     

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Expression and function of an early activation marker restricted to human B cells

A B cell-specific monoclonal antibody (anti-Ba) was prepared. In two-color FACS analysis the anti-Ba reacted with a subpopulation of Ig+ or B1+ cells obtained from tonsils, but did not react with most B1+ cells derived from PBL. Activation of B cells from PBL with TPA or anti-mu induced Ba expression and the addition of PHA-conditioned supernatant with anti-mu-enhanced Ba expression. Other B cell activators, such as Staphylococcus aureus Cowan I (Staph-A) or PWM plus T cells, could induce Ba expression. Ba expression was observed 6 hr after stimulation and reached a peak level at 72 hr. Ba expression was strictly restricted to B cells. H-7, a specific inhibitor of protein kinase C (C-kinase), displayed a dose-dependent inhibitory effect on Ba expression, showing dependency on C-kinase for Ba expression. Anti-Ba inhibited B cell proliferation induced by anti-mu and B-BCGF distinct from BSF-1. The results presented in this study suggest that the Ba antigen on B cells may be comparable to the Tac antigen on T cells.     

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.     

Hypolipidemic therapy modulates expression of apolipoprotein B epitopes on low density lipoproteins. Studies in mild to moderate hypertriglyceridemic patients

Low density lipoproteins (LDL) of untreated moderate to severe hypertriglyceridemic patients (HTG-LDL) are smaller in size and are relatively enriched in triglycerides and proteins compared with normal LDL (N-LDL). HTG-LDL also manifest defective binding to the LDL receptors of normal cultured human fibroblasts. These structural and functional defects are reversible by effective hypolipidemic therapy. The aims of the present study were to confirm the reversibility of the structural and functional defects in mild to moderate hypertriglyceridemic patients and also to test the hypothesis that therapy improved the binding of HTG-LDL to cells by modulating epitopes of apolipoprotein B (apoB-100) on the surfaces of LDL particles. Fasting plasma samples were obtained from five mild to moderate hypertriglyceridemic patients before and 3 weeks after bezafibrate therapy when mean triglyceride levels were 436 and 157 mg/dl (P less than 0.01), respectively. LDL particles were isolated by zonal ultracentrifugation, characterized chemically, and assayed for cell association and proteolytic degradation in-up regulated normal human skin fibroblasts. LDL immunoreactivity was tested in solid phase competitive binding radioimmunoassays (RIA) using three monoclonal antiLDL antibodies (Mab). Mab 464B1B3 and Mab 465B6C3 react against epitopes in the COOH-terminal (T2/K4) fragment of apoB-100. Mab D7.1 reacts with an epitope in the midportion (T3/K3) fragment. Mab 464B1B3 inhibits the binding of LDL to the LDL receptor. Hypolipidemic treatment altered the composition of LDL. Mean LDL triglycerides fell from 9.4 to 5.8% of LDL mass (P less than 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Two monoclonal antibodies to actin: one muscle selective and one generally reactive

Two IgG1, kappa monoclonal antibodies (Mab) against actin have been obtained from a fusion in which chicken gizzard actin was used as the immunogen. One Mab, designated B4, shows a preferential reactivity toward enteric smooth muscle actin but also cross-reacts with skeletal, cardiac, and aorta actins on the basis of immunoblots, ELISA assays, and indirect immunofluorescence. However, this antibody does not react with either cytoplasmic actin in any of these assay systems. A second Mab, designated C4, reacts with all six known vertebrate isoactins as well as Dictyostelium discoideum and Physarum polycephalum actins. Thus B4 Mab appears to react with an epitope that is at least partially shared among the muscle actins but not found in cytoplasmic actins, while C4 Mab binds to an antigenic determinant that has been highly conserved among the actins. The binding sites of both Mabs on skeletal actin overlap that of pancreatic DNase I. Both antibodies bind a SV8 proteolytic product comprising the amino-terminal two-thirds of the actin molecule, and their epitopes appear to overlap since C4 can compete for the binding of B4 to skeletal actin. Neither antibody is able to prevent actin polymerization.     

Monoclonal antibodies to protein kinase C gamma. Functional relationship between epitopes and cofactor binding sites

Four monoclonal antibodies (mAbs), derived from high-responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype gamma 1, kappa and 15G4 is of isotype gamma 2b, kappa. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific for the bovine gamma isoform of protein kinase C and cross-react with protein kinase C gamma from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase C gamma cofactor-dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor-independent catalytic activity of protein kinase C gamma. Competition analysis between these mAbs and phosphatidylserine, 12-O-tetradecanoylphorbol 13-acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase C gamma. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase C gamma.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.     

Analyses of epididymal sperm surface antigens by monoclonal antibodies against hamster spermatozoa

BALB/c mice were immunized with spermatozoa from cauda epididymides of hamsters and the immune spleen cells were fused with mouse myeloma cells (P3U1). Seven hybridomas (GHS-1,-2,-3,-4,-5,-6, and -7) that produced monoclonal antibodies (Mabs) binding to the epididymal spermatozoa were established. Three Mabs (GHS-3,-4, and -6) were IgM and the other four were IgG1. All Mabs reacted to hamster spermatozoa from cauda epididymides but none of the Mabs except GHS-5 and -7 reacted to spermatozoa in testis. GHS-5 and -7 Mabs bound to the acrosome region of spermatozoa in both testis and epididymis. The antigens corresponding to GHS-2, -4, and -6 Mabs appeared to be excreted from epithelial cells of caput epididymis, while those to GHS-1 and -3 Mabs seemed to be produced in cauda epididymis. Both groups of the antigens bound to the surface of spermatozoa during their epididymal transit. Immunoblotting analyses of epididymal fluid showed that the antigen epitopes corresponding to GHS-1,-2,-3,-4, and -6 Mabs were distributed to multiple components with different molecular weights ranging from over 100 to 25 kd. The distribution patterns of the epitopes corresponding to GHS-1 and -3 Mabs and GHS-2,-4, and -6 Mabs were very similar, respectively, but each group pattern was quite different from each other. GHS-5 Mab reacted to a component of sperm extract with a molecular weight of around 94 kd, while GHS-7 Mab failed to recognize any components transblotted.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Role of the interleukin 2 receptor in differentiation of a clone of Ly-1+ B cells

CH12.LX, an in vitro subclone of a murine B cell lymphoma that makes IgM reactive with sheep erythrocytes (SRBC), has cell surface receptors for the lymphokine interleukin 2 (IL 2). The binding of recombinant murine IL 2 to these receptors did not stimulate CH12.LX cells to differentiate and secrete antibody. However, the binding of either of two monoclonal antibodies (Mab) specific for the IL 2 receptor increased the proportion of CH12.LX cells that secrete hemolytic IgM. The effect did not require the presence of antigen. One of the Mab, 3C7, is known to block the binding of IL2 to its receptor on T cells, whereas the other, 7D4, which also reacts with the IL 2 receptor, does not block the binding of IL 2. The differentiation of CH12.LX induced by 3C7, but not that induced by 7D4, was inhibited by recombinant IL 2. Neither IL 2 (up to 200 U/ml) nor 3C7 (up to 10 micrograms/ml) had any significant influence on incorporation of [3H]thymidine; 7D4 at 10 micrograms/ml decreased thymidine incorporation by about 60%. Mitomycin C and hydroxyurea, which both inhibit the incorporation of [3H]thymidine into CH12.LX cells, also both induce antibody secretion. In both cases, the concentration necessary to cause differentiation is substantially lower than that needed to cause detectable inhibition of thymidine uptake. We conclude that the IL 2 receptor on CH12.LX cells is a functional signal transducing molecule, and we discuss the possible inverse relationship between proliferation and differentiation.     

Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.     

The C4A and C4B isotypic forms of human complement fragment C4b have the same intrinsic affinity for complement receptor 1 (CR1/CD35)

Several previous reports concluded that the C4b fragment of human C4A (C4Ab) binds with higher affinity to CR1 than does C4Bb. Because the isotypic residues, (1101)PCPVLD and (1101)LSPVIH in C4A and C4B, respectively, are located within the C4d region, one may have expected a direct binding contribution of C4d to the interaction with CR1. However, using surface plasmon resonance as our analytical tool, with soluble rCR1 immobilized on the biosensor chip, we failed to detect significant binding of C4d of either isotype. By contrast, binding of C4c was readily detectable. C4A and C4B, purified from plasma lacking one of the isotypes, were Cs converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers and higher oligomers by sequential chromatographic steps. The binding sensorgrams of C4Ab and C4Bb monomers as analytes reached steady state plateaus, and these equilibrium data yielded essentially superimposable saturation curves that were well fit by a one-site binding model. Although a two-site model was required to fit the equilibrium-binding data for the dimeric forms of C4b, once again there was little difference in the K(D) values obtained for each isotype. Independent verification of our surface plasmon resonance studies came from ELISA-based inhibition experiments in which monomers of C4Ab and C4Bb were equipotent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are consistent with recent C4Ad structural data that raised serious doubts about there being a conformational basis for the previously reported isotypic differences in the C4b-CR1 interaction.     

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.     

Differences between the binding sites of the complement regulatory proteins DAF, CR1, and factor H on C3 convertases

Binding studies using purified decay-accelerating factor (DAF), CR1, and Factor H indicate that the primary interaction of DAF with C3 convertases is with the Bb or C2a subunits, whereas CR1 and Factor H interact primarily with the C3b or C4b subunits. The ability of soluble DAF, CR1, or Factor H to decay C3b,Bb bound to zymosan was inhibited by various concentrations of fluid-phase competitors (C3b, Bb, C3b,Bb, C3b,B, C4b, or C4b,C2a) in 0.1% NP-40 at 22 degrees C. The apparent association constants (appKa) for DAF were 0.045, 0.067, 0.91, 0.71, 0.00045, and 0.53 microM-1, respectively. The appKa for CR1 were 0.50, 0.0040, 1, 1, 1, and 1.1 microM-1, respectively. The appKa for Factor H were 4.3, 0.0005, 2.9, 6.3, 0.27, and 0.29 microM-1, respectively. Thus, C3b binds to DAF with a 10-fold lower affinity than to CR1 and a 100-fold lower affinity than to Factor H. The appKa of C3b,Bb for the three proteins were more similar: DAF (0.91 microM-1), CR1 (1 microM-1), and Factor H (2.9 microM-1). DAF binds to Bb with a 50% higher affinity than to C3b, and to C4b,C2a with a 1000-fold higher affinity than to C4b alone. In contrast, CR1 and Factor H bind almost equally well to the C3 convertases and to their noncatalytic subunits. The affinity of DAF for CVF,Bb was similar to its affinity for Bb alone, suggesting that DAF does not recognize conformational determinants unique to Bb in C3 convertases.     

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.