Deficiencies in regulator proteins. 2. Factor I and H.
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Afibrinogenemia↗
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Results for “Complement C3b Inactivator Proteins”
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The modulation of the alternative pathway of complement in C2-deficient human serum by changes in concentration of the component and control proteins.
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Animals↗
Synthesis and regulation of complement protein factor H in human skin fibroblasts.
The alternative pathway of C activation is Ag-independent and forms a first line of defense against infection before immune response. The C3 convertase, C3bBb, formed during activation of the alternative pathway is tightly regulated, with destabilization produced by factor H. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrated that human skin fibroblasts synthesized and secreted factor H protein. Two forms of the protein were identified, the approximately 160-kDa form seen more prominently in serum and a 45-kDa form that has also been identified in serum. The cells contained two forms of factor H mRNA, 4.4 and 1.8 kb. IFN-gamma increased factor H protein synthesis and mRNA content. No effect was observed with LPS. Neither HepG2 cells or human peripheral blood monocytes synthesized factor H protein or contained factor H mRNA.
Cell Line↗
Purification and structural studies on the complement-system control protein beta 1H (Factor H).
An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.
Amino Acid Sequence↗
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.
Binding Sites↗
Murine protein H is comprised of 20 repeating units, 61 amino acids in length.
A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized with this cDNA probe. Ten positives were colony-purified, and the largest plasmid cDNA insert, MH8 (4.4 kb), was sequenced by the dideoxy chain termination method. MH8 contained the complete coding sequence for the precursor of murine complement protein factor H (3702 bp), 100 bp of 5'-untranslated sequence, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length. Similar repetitive units are present in the C4b binding protein, the C3b-receptor (CR1), complement factor B and C2, and in beta 2-glycoprotein I and the interleukin 2 receptor. This finding suggests a common evolutionary origin for regions of these proteins.
Amino Acid Sequence↗
Role of human factor I and C3b receptor in the cleavage of surface-bound C3bi molecules.
Control of functions mediated by the third component of complement (C3) depends on the rate of generation and degradation of biologically active C3 fragments. To evaluate the mechanisms of degradation of active C3 fragments, the role of the control protein C3b/C4b inactivator (factor I) was investigated under conditions approximating those found in vivo, i.e. in the presence of plasma. The breakdown of human erythrocyte-bound C3bi molecules in serum or plasma was mediated only by factor I, since factor I-deficient or -depleted plasma was inactive until reconstituted with highly purified factor I. The rate of cleavage of C3bi bound to human erythrocytes by purified factor I was not affected by the presence or absence of beta 1H (factor H). The released breakdown product of C3bi has been shown to be C3c antigenically and on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two different monospecific antibodies to the human C3b receptor totally abrogated factor I-mediated cleavage of cell-bound C3bi, suggesting that the C3b receptor (but not factor H) is required as an obligate cofactor. The rate of this C3b receptor-dependent, factor I-mediated cleavage of bound C3bi is strongly regulated by the surface to which C3bi is bound. Whereas C3bi bound to particulate nonactivators of the alternative complement pathway such as human erythrocytes is rapidly degraded by this mechanism, the rate of cleavage of C3bi bound to activators is significantly slower. These data suggest a physiologic role of C3b receptors in the degradation of biologically active C3 fragments deposited on host tissues. They also suggest that C3bi molecules on restricted surfaces are relatively stable and can thereby interact with complement C3 receptors in vivo.
Animals↗
Cleavage of C4b by C3b inactivator: production of a nicked form of C4b, C4b', as an intermediate cleavage product of C4b by C3b inactivator.
We have investigated the mechanism of cleavage of C4b into C4c and C4d by the C3b inactivator (C3bINA) and revealed the formation of a nicked form of C4b as an intermediate cleavage product. The cleavage of C4b by the C3bINA was a two-step reaction. The first cleavage occurred on the alpha-chain (89,000 daltons) yielding two fragments, 73,000 daltons and 16,000 daltons. These fragments were bound to each other or to the beta or gamma chain through disulfide linkages. Therefore, an altered form of C4b, C4b', consisting of four disulfide-linked polypeptide chains with the same m.w. as C4b, was produced as an intermediate cleavage product. Subsequently, the second cleavage occurred on the alpha-chain fragment of 73,000 daltons to produce the two generally recognized fragments, C4c and C4d. In both cleavage reactions, a high m.w. cofactor protein, C4bC3bINACo, which is the same protein described as the C4 binding protein, was required, suggesting that both of the proteolytic processes are catalyzed by the C3bINA.
Complement C3b Inactivator Proteins↗
Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.
Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.
Complement C3↗
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.
Animals↗
Purification and characterization of a novel protein that is required for degradation of N-alpha-acetylated proteins by the ubiquitin system.
Anion exchange chromatography of reticulocyte lysates revealed that the ubiquitin cell-free system can be resolved into two essential fractions: unadsorbed material (Fraction I) that contains ubiquitin and a high salt eluate (Fraction II) that contains the conjugating enzymes and the conjugate-degrading protease. Many proteins with exposed NH2 termini are degraded in a ubiquitin-supplemented Fraction II. However, this partially purified and reconstituted system does not degrade N-alpha-acetylated proteins. These proteins are degraded in whole lysates in a ubiquitin-dependent manner (Mayer, A. Siegel, N. R., Schwartz, A. L., and Ciechanover, A. (1989) Science 244, 1480-1483). It appears that a protein factor which is specifically required for the degradation of N-alpha-acetylated proteins is removed or inactivated during the fractionation of the lysate. Here we report the purification and characterization of a novel protein that is required along with the protease for the degradation of ubiquitin conjugates of histone H2A, an N-alpha-acetylated protein. The protein is not required for the degradation of ubiquitin conjugates of proteins with free NH2 termini. The protein, which is found in crude Fraction I, was purified approximately 200-fold by (NH4)2SO4 precipitation, Sephadex G-100 gel-filtration chromatography, Mono Q anion exchange chromatography, and an additional Sephadex G-100 gel filtration chromatography step. The protein is removed from Fraction I during the purification of ubiquitin and has not been previously recognized since the majority of the protein substrates evaluated in the cell-free system have free NH2 termini. The protein has an apparent molecular mass of approximately 92 kDa. It is a homodimer that is composed of two identical 46-kDa subunits. Initial analysis of the mechanism of action of this protein revealed that it must interact with the conjugates in order to allow proteolysis to occur. We designated the protein Factor H (Factor Hedva).
Acetylation↗
IL-1 and tumor necrosis factor. Similarities and differences in stimulation of expression of alternative pathway of complement and IFN-beta 2/IL-6 genes in human fibroblasts.
IL-1 and TNF induced concentration-related increases in the synthesis of factor B, C3, and IFN-beta 2/IL-6 in human skin fibroblasts. Effects of both stimuli were apparent with concentrations as low as 0.1 ng/ml and maximal responses were observed between 1 and 10 ng/ml; only for IL-1 induction of IFN-beta 2/IL-6 was there a further increase in response up to 100 ng/ml. For factor B and C3, maximal increases induced by IL-1 and TNF were similar: 119- and 109-fold for factor B and 15-fold and 11-fold for C3, respectively. Although both IL-1 and TNF increase synthesis of factor B and C3 in hepatocytes, the increases observed in fibroblasts were approximately 50- and 8-fold more for factor B and C3, respectively. Neither protein synthesis nor mRNA for IFN-beta 2/IL-6 was present in HepG2 cells either before or after stimulation with IL-1 or TNF. In contrast to the similarities between the effects of IL-1 and TNF on synthesis of factor B, C3, and IFN-beta 2/IL-6, only TNF increased synthesis of factor H. Because TNF induces membrane IL-1 in fibroblasts, it is possible to speculate that the effects of TNF on fibroblasts are due to induction of IL-1. An autocrine action of TNF through IL-1 is possible for TNF-induced synthesis of IFN-beta 2/IL-6, but the effects of TNF on synthesis of factor B, C3, and factor H indicated that TNF has effects on fibroblasts separate from IL-1. The effects of IL-1 and TNF on the synthesis of factor B and C3 in fibroblasts may be a part of an acute phase response occurring at a local level. However, the large responses in synthesis of factor B and C3 to IL-1 and TNF may suggest that factor B and C3 have a role, as yet undescribed, in tissues in addition to the role these proteins are known to play in inflammation.
Adult↗
A monomeric human C4b-binding protein (C4bp) more efficiently inactivates C3b than natural C4bp: participation of C-terminal domains in factor I-cofactor activity.
We designed a cDNA construct encoding an artificial membrane molecule consisting of all 8 short consensus repeats (SCRs) of human monomeric C4b-binding protein (C4bp) followed by DAF's GPI anchor, named mC4bp, and expressed the protein on swine endothelial cells (SEC). At the same level of expression, mC4bp protected host cells as effectively as DAF, the most potent complement (C) regulator on the membrane. This result was unexpected from the reported functional properties of natural multimeric C4bp. Here, we investigated the mechanism whereby mC4bp has potent cell-protective activity. Our results were as follows: (1) mC4bp serves more efficiently as a methylamine-treated C3 (C3ma)-inactivating factor I-cofactor than natural C4bp and as efficiently as MCP as a methylamine-treated (C4ma)-inactivating cofactor by fluid-phase cofactor assay: (2) the potency of C3ma inactivation by mC4bp and factor I is quite high compared to those of other cofactors: (3)blocking studies using mAbs against C4bp suggested that both the 48 kDa N-terminal fragment and the C-terminal domain near the portion responsible for bundle formation participate in the high C3ma-inactivating capacity of mC4bp. Thus, acquiring high C3ma-inactivating capacity secondary to monomeric alteration leads to high C regulatory activity of mC4bp. These results infer that mC4bp differs from C4bp in its potent factor I-cofactor activity and is a good candidate as a safeguard against hyperacute rejection of xenografts.
Complement C3b Inactivator Proteins↗
Hypocomplementemia in Reye syndrome: relationship to disease stage, circulating immune complexes, and C3b amplification loop protein synthesis.
Measurement of C1q, C2, C4, C5, C6, factor B, properdin, beta1H, and C3bINA were made in acute sera from 31 patients with Reye syndrome. Abnormalities were found in 18 patients. The magnitude of the complement component depression correlated with disease severity. Sera from patients with stage IV illness had significantly lower complement levels than did sera from patients with state I (P less than 0.001), II (P less than 0.05), and III (P less than 0.05) disease. Circulating immune complex measurements were performed on all 31 acute sera and were present in six (19%). However, from the results of the present study, it would appear that in the majority of the patients circulating immune complexes are not the cause of the lowered levels of, at least, C3 and factor B. Rather, these low levels could be explained as secondary to reductions in the levels of the C3b amplification loop control proteins beta1H and C3bINA.
Antigen-Antibody Complex↗
Enzymatic treatment transforms trypomastigotes of Trypanosoma cruzi into activators of alternative complement pathway and potentiates their uptake by macrophages.
In the absence of bound antibody, trypomastigote bloodstream forms of Trypanosoma cruzi fail to activate the alternative complement pathway. We now demonstrate that treatment with trypsin and, to a lesser extent, with sialidase converts these protozoa into activators of the pathway, as judged by their lysis in normal sera or sera genetically deficient in fourth or second component of complement (C4 or C2) and their Mg2+-dependent consumption of C3 as measured by crossed immunoelectrophoresis. In addition, after pretreatment with enzyme and incubation in C5-deficient serum, trypomastigotes were shown to possess both C3 and properdin factor B (B) on their surface as judged by immunofluorescence. Requirement for the late components C5-C9 was suggested by the failure of C5-deficient sera to lyse trypsin-treated parasites. The inability to activate the alternative complement pathway was regained by these organisms after incubation in vitro. This restoration of insusceptibility was inhibited when puromycin was included in the culture medium. Treatment of the trypomastigotes with trypsin also potentiated their uptake by mouse peritoneal macrophages without apparent interference with their capacity to differentiate and multiply inside the cell. These findings suggest that untreated trypomastigotes normally escape recognition by the alternative pathway in vivo because of the presence on their surface of trypsin- and sialidase-sensitive regulatory molecules, the expression of which is dependent on protein synthesis.
Animals↗
The complement system in bullous pemphigoid: VII. Fixation of the regulatory protein beta 1H globulin by pemphigoid antibody.
Using in vitro complement immunofluorescent staining methods, serum samples from 5 active cases of bullous pemphigoid, with pemphigoid antibody titers of 320 or greater, were tested for their ability to fix the regulatory protein beta 1H globulin in addition to C4 and C3. All 5 samples yielded positive C3, C4 and beta 1H staining reactions in a linear fashion along the basement membrane zone. Heat inactivation or treatment of the complement source (fresh normal human serum) with EDTA, Mg2-EGTA abolished all 3 staining reactions. Substitution of C2-deficient serum as the source of complement inhibited both C3 and beta 1H staining but had no effect on C4 staining. Use of serum devoid of beta 1H (R beta 1H) minimally enhanced C3 staining while no beta 1H staining was observed. The addition of beta 1H to R beta 1H restored positive beta 1H staining. Skin biopsies of perilesional skin from 6 patients with bullous pemphigoid demonstrated heavy in vivo deposition of beta 1H in addition to C3. These studies suggest that pemphigoid antibodies will fix the regulatory protein beta 1H in addition to other complement components, a phenomenon which requires activation of the classical complement pathway and generation of the C3b amplification convertase.
Antibodies↗
Activation of the alternative pathway of complement by cellulosic hemodialysis membranes.
Compared to cellulose acetate, hemodialysis with cuprophan membranes is associated with greater activation of the alternative pathway of complement. Previous studies have shown that this difference is not due to a greater number of potential covalent binding sites for activated C3 on cuprophan. To investigate further the factors that influence complement activation by hemodialysis membranes, proteins were eluted from serum-treated cuprophan and cellulose acetate membranes with hydroxylamine at alkaline pH and analyzed by SDS-PAGE and Western blot. Approximately 23 times more total protein was removed from cellulose acetate. Virtually all the C3 in the cellulose acetate eluate was in the form of inactive fragments C3c and C3dg. In contrast, the functionally active form of C3 (C3b) was a prominent constituent of the cuprophan eluate. The binding of factor B (precursor of the catalytic subunit of the C3 convertase) and factor H (regulatory protein of C3 activation) to serum-treated membranes was also analyzed. By Scatchard's method, the affinity constant at equilibrium for factor B binding (KB) to the two types of membranes was not significantly different; however, there were approximately four times more factor B binding sites on the cuprophan than on the cellulose acetate. For cuprophan, the number of factor B binding sites was 1.6 times greater than the number of factor H binding sites. These studies demonstrate that a portion of the C3b molecules that bind to cuprophan are protected from degradation, and suggest that the complement activating capacity of hemodialysis membranes is determined by biochemical properties that modulate both the binding of serum proteins to the membrane and the interactions of the endogenous regulatory proteins with membrane-associated C3b.
Cellulose↗
Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CR1) and comparison with H, C4b-binding protein (C4bp), and decay accelerating factor (DAF).
Four CR1 variants have been found in the normal population and are designated CR1-A (190,000 daltons), CR1-B (220,000 daltons), CR1-C (160,000 daltons), and CR1-D (250,000 daltons). In the present study, we first developed an improved chromatographic purification scheme for CR1 that does not employ a C3b affinity step. CR1 variants (A, B, and C) were then isolated, and their individual functional activity was assessed. Each possessed similar co-factor activity for I-mediated cleavage of C3(H2O), as well as for the inhibitory activity for fluid phase C3 convertases. These results indicate that, despite relatively large Mr differences, in the purified state these three CR1 variants have similar functional activities. The functional activity of CR1 was also compared with C4bp, H, and decay accelerating factor (DAF) in fluid phase assays designed to assess the inhibition of the C3 convertases and co-factor activity. On a molar basis, CR1 had approximately the same inhibitory activity as C4bp for the classical pathway convertase, and had the same as H for the alternative pathway convertase. These results indicate that CR1 encompasses the functional capabilities of both proteins. They also raise a number of interesting genetic and structural questions in regard to these complement regulatory proteins, because C4bp is thought to have multiple C4b binding domains, whereas H is reported to bind one C3b. DAF was an approximately fourfold better inhibitor of the alternative pathway convertase than CR1 or H, but was a fourfold less efficient inhibitor of the classical pathway convertase than CR1 or C4bp. The effective inhibitory capacity of DAF in these fluid phase assay systems suggests that the DAF substrate specificity is for the convertases. Fluid phase CR1 was twofold less efficient than H in serving as a co-factor for the first cleavage of fluid phase C3b, and hardly mediated the second cleavage. These data are in contrast to the co-factor activity of CR1 on a cell membrane, and provide additional evidence for the local environment being a critical modulator of the function of proteins that regulate the activation of C3.
Complement Activating Enzymes↗