[Compliment system proteins: properties and biological activity (Lecture)].
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Animals↗
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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↗
Synthesis and regulation of C1 inhibitor in human skin fibroblasts.
Proteins of the C1 complex, C1q, C1r, and C1s, of the classical pathway of complement activation are known to be synthesized in human skin fibroblasts. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrate that human skin fibroblasts synthesize and secrete C1 inhibitor with an apparent molecular mass of 78 kDa in the cell lysate and 102 kDa in the extracellular medium. This C1 inhibitor had the capacity to bind activated C1s. Fibroblasts synthesized 30- to 50-fold more C1 inhibitor than was synthesized in monocytes. As previously reported, fibroblasts also synthesized C1r and C1s. IFN-gamma, IFN-beta 1, and TNF had significant, but distinct, effects on synthesis of C1 inhibitor, C1r, and C1s. Incubation of the cells with IFN-gamma, 1000 U/ml, for 24 h induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 4.2-, 1.9- and 1.6-fold, respectively. IFN-beta 1 had effects similar to IFN-gamma, although smaller in magnitude. TNF, 12.5 ng/ml, induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 1.5-, 1.4- and 2.6-fold. IL-1, IFN-beta 2 (IL-6), and LPS did not affect synthesis of C1 inhibitor, C1r, or C1s. Fibroblasts are present in large amounts in most tissues. Synthesis of C1 inhibitor, C1r, and C1s by these cells could provide a source of these important proteins in body tissues. In addition, fibroblasts should be a good model for the in vitro study of genetic diseases involving the synthesis of these proteins.
Adult↗
The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b.
Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.
Amino Acid Sequence↗
Possible role for increased C4b-binding-protein level in acquired protein S deficiency in type I diabetes.
In this study, total protein S (PS) immunological levels, free-PS and C4b-binding-protein (C4bBP) concentrations, and PS functional activity were investigated in insulin-dependent (type I) diabetic patients and compared with nondiabetic subjects. Mean total PS antigen concentration was not different between diabetic patients and nondiabetic subjects, whereas free-PS levels and PS functional activity were significantly reduced in diabetic patients. C4bBP was increased in diabetic patients and correlated with HbA1 levels. This study shows that type I diabetic patients have depressed free PS and PS activity despite the presence of normal total PS concentration and suggests that this phenomenon is probably linked to the increase of circulating C4bBP.
Adult↗
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↗
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↗
[Protein chemistry of the complement system].
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Amino Acid Sequence↗
Human C4BP binds to the hypervariable N-terminal region of many members in the streptococcal M protein family.
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Amino Acid Sequence↗
Kinetics of interaction of C1 inhibitor with complement C1s.
The kinetics of inhibition of the complement serine protease, C1s, by its only known inhibitor, C1 inhibitor, have been measured by a variety of methods. One method continuously monitors the loss of esterolytic activity with a synthetic substrate coupled to a chromogen while another monitors the formation of a stable (covalent) complex by high-pressure size-exclusion chromatography under dissociating conditions. Additional methods employ fluorescence probes to follow the formation of bimolecular complexes but are not expected to distinguish between covalent product and noncovalent (reversible) intermediates. There was good agreement between rate constants obtained by the various methods over a broad range of inhibitor concentrations, suggesting that noncovalent intermediates do not accumulate to a significant extent. The reaction appears to be pure second order with a bimolecular rate constant of 6.0 X 10(4) M-1 s-1 at 30 degrees C, independent of Ca2+, and an activation energy of 11.0 kcal/mol. The rate increases up to 35-fold in the presence of heparin which was shown to bind to all three components (enzyme, inhibitor, and complex) with similar affinity (Kd = 2.0-3.3 microM). The fluorescent probe 1,1'-bis(anilino)-4-,4'-bi(naphthalene)-8,8'-disulfonate [bis(ANS)] bound to the complex with Kd = 0.26 microM under conditions where the individual components had little affinity for the dye, consistent with the generation of one or more hydrophobic binding sites on the protein surface during complex formation.
Complement Activating Enzymes↗
A new case of 'type II' inherited protein S deficiency.
Protein S inherited deficiency is associated with high risk of recurrent venous thrombotic disease (Broekmans et al, 1985a, b). Protein S exists as two forms in plasma, either free and functionally active or complexed with C4b-binding protein (C4b BP) and inactive (Dahlbäck & Stenflo, 1981). We report here the case of a 26-year-old woman and her brother, 28 years old, both suffering from recurrent venous thrombosis since the age of 20, diagnosed as severe protein S deficiency according to the following data: free protein S: 2.5-3% by ELISA, undetectable by electroimmunodiffusion (EID); total protein S: 13-16% by ELISA, 21-18% by EID, C4b BP: normal levels. Crossed immunoelectrophoresis using anti-protein S antibodies revealed only traces of protein S associated with C4b BP and no free protein S. All these assays were performed in the absence of any anticoagulant therapy. Among the investigated relatives, less severe protein S deficiency was observed in three children of the propositus: total protein S levels ranging from 41% to 50% (EID), 40-53% (ELISA); free protein S levels ranging from 16% to 18% (EID), 10-12% (ELISA); normal C4b BP levels. Crossed immunoelectrophoresis revealed traces of free protein S but a significant amount of protein S associated with C4b BP. From these results, we consider, according to Comp's classification (Comp et al, 1986a), that the propositus and her brother are the second case of protein S deficiency type II to be reported in the literature while her children belong to the type I category.
Adult↗
Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins.
Many strains of Streptococcus pyogenes bind C4b-binding protein (C4BP), an inhibitor of complement activation. The binding is mediated by surface M proteins in a fashion that has been suggested to mimic the binding of C4b. We have previously shown that a positively charged cluster at the interface between complement control protein domains 1 and 2 of C4BP alpha-chain is crucial for the C4b-C4BP interaction. To extend this observation, and to investigate the interaction with M proteins, we constructed and characterized a total of nine mutants of C4BP. We identified a key recognition surface for M proteins that overlaps with the C4b binding site because substitution of R64 and H67 by Gln dramatically reduces binding to both ligands. However, the analysis of all mutants indicates that the binding sites for C4b and M proteins are only overlapping, but not identical. Furthermore, M proteins were able to displace C4BP from immobilized C4b, whereas C4b only weakly affected binding of C4BP to immobilized M proteins. We found that the molecular mechanisms involved in these two interactions differ because the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b binding. In addition, six mAbs directed against the alpha-chain interfered with C4b-C4BP interaction, whereas only two of them efficiently inhibited binding of C4BP to M proteins. Collectively, our results suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional noncovalent forces cause tight binding of C4BP to streptococcal M proteins.
Amino Acid Substitution↗
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↗
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↗
Derivation of the sequence of the signal peptide in human C4b-binding protein and interspecies cross-hybridisation of the C4bp cDNA sequence.
A 5' cDNA clone coding for human C4b-binding protein (C4bp) was isolated, characterised and sequenced to complete the cDNA sequence coding for residues 1-32 thus confirming the protein sequence data of Chung et al. [(1985) Biochem. J. 230, 133-141]. The sequence extended to allow derivation of the putative leader peptide sequence which was 32 residues in length and showed a high of hydrophobicity typical of other documented leader sequences. Cross hybridisation was detected between the human C4bp cDNA probes and genomic DNA isolated from various species on Southern blots suggesting that genomic sequence homologous to that coding for C4bp has been conserved during evolution.
Amino Acid Sequence↗
A case report of deficiency in an inhibitor of calcium-dependent association of protein S with C4B-binding protein suggested by a modified crossed immunoelectrophoresis.
We have experienced a coagulation factor VIII-deficient patient whose plasma has normal protein S (PS) activity and masses of free PS and its bound form in complex with C4b-binding protein (C4BP). Although the patient's plasma showed a normal ratio of free PS to PS-C4BP complex in the presence of 5 mM EDTA, the plasma gave an abnormally retarding major C4BP peak together with a major PS peak in the crossed immunoelectrophoresis (CIE) in the presence of 2 mM CaCl2. It was revealed that the major peak was formed by a mixture of PS-C4BP complex and free form. The addition of normal human plasma (NHP) to the patient's plasma inhibited the retardation of the major PS-C4BP complex. These suggest that the patient's plasma lacks some component(s) to inhibit Ca(2+)-dependent association of PS with C4BP.
Adult↗
Human recombinant macrophage colony-stimulating factor (M-CSF) increases Cl-esterase inhibitor (Cl-INH) synthesis by human monocytes.
The ability of macrophage colony-stimulating factor (M-CSF) to influence production of complement proteins by cultured human monocytes was studied. Three-day-old cultures of human monocytes were treated with 250-1000 U/ml recombinant human M-CSF (Cetus Corporation, Emeryville, CA). After 3 days the M-CSF containing medium was replaced by the same medium without M-CSF, and the cells were cultured for 3 more days. Samples taken at the removal of M-CSF and 3 days later were tested for the concentration of factor B and Cl-esterase inhibitor (Cl-INH) using ELISA methods, and C2 by using an immunohaemolytic assay. M-CSF induced a marked dose-dependent increase in the synthesis of Cl-INH, but did not significantly change Bf and C2 production. The findings suggest that M-CSF is able to influence selectively the complement protein-producing ability of cultured human monocytes.
Cells, Cultured↗
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↗