Third Component Of Human Complement Research Articles

Hepatitis C Virus Proteins Inhibit C3 Complement Production

The third component of human complement (C3) plays a central role in innate immune function as its activation is required to trigger classical as well as alternative complement pathways. In this study, we have observed that sera from patients chronically infected with hepatitis C virus (HCV) displayed significantly lower C3 levels than sera from healthy individuals. Liver biopsy specimens from the same patients also exhibited lower C3 mRNA expression than liver tissues from healthy donors. C3 mRNA level was reduced in hepatocytes upon infection with cell culture-grown HCV genotype 1a or 2a in vitro. Further analysis suggested that HCV core protein displayed a weak repression of C3 promoter activity by downregulating the transcription factor farnesoid X receptor (FXR). On the other hand, HCV NS5A protein strongly downregulated C3 promoter activity at the basal level or in the presence of interleukin-1β (IL-1β) as an inducer. In addition, the expression of the transcription factor CAAT/enhancer binding protein beta (C/EBP-β), which binds to the IL-1/IL-6 response element in the C3 promoter, was inhibited in liver biopsy specimens. Furthermore, expression of C/EBP-β was reduced in hepatocytes infected with cell culture-grown HCV, as well as in hepatocytes transfected with the NS5A genomic region of HCV. Together, these results underscore the role of HCV NS5A protein in impairing innate immune function.

Human genetic variation in the Sierra de Gredos mountain (central Spain): study of several polymorphisms.

The variation in the third component of human complement (C3) and orosomucoid (ORM1) serum proteins, and 6-phosphogluconate dehydrogenase (6PGD), adenosine deaminase (ADA), esterase D (ESD), and acid phosphatase (ACP1) red cell enzymes was examined in two samples from autochthonous populations living on either side of the Sierra de Gredos range in Central Spain. These results support only a moderate differentiation between Gredos subpopulations, which is discussed in relation to other Iberian Peninsula groups. Allele frequencies in Gredos show a remarkable heterogeneity as contrasted with other Iberian populations for most of the markers examined; relatively high C3*S, ORM1*F, ADA*1, ESD*2, and ACP1*C gene frequencies are characteristics of Gredos samples. This differentiation was more marked for the Northern Gredos population and could be related to the geographical peculiarities of this region.

Polymorphism of the Third Component of Human Complement (C3) in Haryana, India

Using high veldge electrophoresis, C3 component of the human complement has been studied in a total of 95 planed malpes from the people of Haryana, a North Indian State.

Structural features of the human C3 gene: Intron/exon organization, transcriptional start site, and promoter region sequence

The third component of human complement (C3) is a key molecule in the activation of the complement cascade. C3 cDNA fragments were used to identify seven cosmid clones that covered all but 1 kilobase pair (kb) of the C3 gene. The remainder of the gene was cloned by using the polymerase chain reaction. These clones were used to identify the intron/exon boundaries and to map the gene. The C3 gene is 42 kb in length and comprises 41 exons ranging in size from 52 to 213 base pairs (bp). The transcription start site was identified by primer extension, and approximately 1 kb of DNA upstream of this site was sequenced. Putative TATA and CAAT boxes were identified along with a number of regions that shared homology with known regulatory sequences. These include responsive elements for interferon-gamma, interleukin-6, nuclear factor kappa B, estrogen, glucocorticoids and thyroid hormone. Several of these agents have been shown to affect C3 synthesis and mRNA levels. The sizes of the exons in C3 were compared to those of C4 and alpha 2-macroglobulin (alpha 2M). Thirty-nine of 41 exons in C4 were found to be of similar size to the analogous ones in C3, and two-thirds of those in alpha 2M were also similarly sized, supporting the hypothesis that these genes arose from a common ancestor.

Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC)

C3(H 2O), an inactive form of C3 present to a variable extent in most C3 preparations, has been isolated in 40 min from previously purified C3 using FPLC ion exchange chromatography on a Mono Q column. As many as six peaks were obtained from some C3 preparations, corresponding to different molecular forms of the protein. One of these peaks consisted of a molecular form of C3 with intact α and β chains, a free sulfhydryl group but no hemolytic activity and was identified as C3(H 2O). C3(H 2O) eluted as a homogeneous peak well resolved from native C3, C3b, high molecular weight aggregates and small degradation fragments. The same C3(H 2O) peak was generated from native C3 by repeated freeze-thaw cycles or NH 2OH treatment. C3(H 2O) α chain appeared as a doublet about 2 kDa heavier than native C3 α chain in low cross-linked gels. Two forms of C3b could be separated on the Mono S column, both able to form the C3 convertase. The present report described a very fast method to resolve and isolate to homogeneity C3(H 2O) and native C3 from C3 preparations. Both molecular forms of C3 are very suitable for studies of the initial and amplification C3 convertases of the alternative pathway of complement.

Complete Intron/Exon Organization of DNA encoding the α' Chain of Human C3

The third component of human complement (C3), a central molecule in both the classical and alternative pathways of complement, is comprised of two polypeptides, termed the alpha and beta chains. Activation of C3 cleaves the alpha chain into two fragments, C3a, an inflammatory peptide, and the alpha' chain which remains covalently linked to the beta chain. Proteolytic fragments derived from the alpha' chain during activation and regulation of complement play a significant role in host defense and regulation of the immune response. Two cosmid clones covering the alpha' chain region were used to characterize the structure of this portion of the C3 gene. The alpha' chain is encoded by 24 exons, which range in size from 52 to 213 base pairs (bp) with an average size of 115 bp. The splice donor sequence at the beginning of intron 12 has a rare sequence variant of GC instead of the usual GT sequence. Ten introns have been completely sequenced and were surprisingly short, ranging in size from 85 to 242 bp with an average of 140 bp. Other introns range in size from 250 bp to over 4 kilobases in length. The gene size for this portion of C3 is estimated to be 23-24 kilobases. Comparison of exon structure with protein domains and with peptide mapping studies demonstrates that several binding sites on C3 are encoded by single exons. These data support the hypothesis that individual exons can code for functional protein domains.

Evidence that SE is distal to LU on chromosome 19q.

Analysis of a family informative for chromosome 19 loci establishes that the Lutheran blood group locus (LU) lies between the third component of human complement (C3) and the secretor loci (SE). Previously published lod scores for C3:LU are increased from 2.94 to 3.80. The linkage relationships (zeta and theta) between C3, LU, and SE are examined, and the proposed order and approximate genetic distances are determined to be: pter--C3-10.6cM-cen-7.4cM-LU-9cM-SE--qter.

Storage of Platelets in Earth Orbit. Effect of Gravity and the Formulation of Plastic Storage Containers on Platelet IgG and C3

We investigated the effects of gravitational force and three different plastic formulations of the storage bags on the quality of stored platelets by measuring the changes in platelet-associated immunoglobulin G (IgG) and two antigenic markers of the third component of human complement (C3), C3d and C3c. Pooled platelets were stored in parallel at microgravity (MG) and on the ground (lg) using three different plastic polymers: (1) polyvinylchloride (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP); (2) PVC plasticized with trioctyl trimellitate (TOTM), and (3) unplasticized polyolefin (PO). The IgG and C3 were quantified by an automated antiglobulin consumption test in freeze/thaw disrupted platelets (total IgG or C3). The baseline values for platelet associated IgG and C3, measured after 2.5 days of storage at 1g just prior to the launch, fell within the normal range. Following an additional 6.5 day storage, platelets stored at MG had accumulated significantly less C3d than those stored at lg, suggesting that MG storage was beneficial. Specific plastic formulations also exerted a significant effect on the accumulation of these immunoproteins, the effect being particularly pronounced at MG. The smallest increases of IgG and C3 were seen in platelets stored in TOTM and the largest in those stored in DEHP. It is possible that further studies at MG would permit a clear characterization of the effects of other independent storage variables.

Heat-induced thiol-disulfide interchange reaction on the third component of human complement, C3.

The third component of human complement, C3 is composed of two disulfide-bridged polypeptide chains of Mr 120,000 (alpha chain) and Mr 70,000 (beta chain). C3 has a thioester bond that serves as a binding site for targets when C3 is activated. Heat treatment of C3 induces autolytic peptide bond cleavage at the thioester site in the alpha chain as well as rupture of the thioester bond. The alpha chain fragments are linked to each other and beta chain via disulfide bonds. This study, however, documented that prolonged heating gave rise to liberation of several fragments including beta and the larger fragment of alpha chain. Using a fluorescent thiol reagent and [14C]iodoacetamide, we analyzed thiol residues present on each fragment, and elucidated that the thiol residue exposed by rupture of the thioester bond shifts in turn to another fragment resulting in the liberation of the fragments. The results were compatible with those on C4, and suggested that the generated thiol residue induces thiol-disulfide interchange reaction. On heating of plasma, fragments of C3 were not released, while the cleavage of the alpha chain occurred more effectively. The heated C3 (56 degrees C, 15 min) became insusceptible to C3b inactivator (I) and factor H, suggesting that additional conformational change is accompanied with cleavage of the thioester bond.

Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose 5 N-acetylglucosamine 2-PA∼mannose 9 N-acetylglucosamine 2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.

Localization and functional significance of a polymorphic determinant in the third component of human complement

A polymorphic epitope in the third component of human complement was studied. This allotypic system is distinct from the electrophoretically determined C3 S/F polymorphism and is denned by the recognition of one allotype by a monoclonal antibody. Allotypic protein variants, C3 F+ (reactive with this antibody) and C3 s− (non-reactive with the antibody), were purified. Deglycosylation studies and N-terminal sequencing of CNBr fragments, reactive with the antibody, revealed that the polymorphic epitope was present in a β chain fragment of mol. wt 20,000. In the intact C3 molecule, this fragment is situated with N-terminus at residue No. 202, using the numbering of the cDNA derived amino acid sequence of human prepro C3. Addition of Fab fragments from the alloselective antibody preferentially inhibited the activity of C3 F+ in a haemolytic assay which is selective for the C3 activity in the alternative complement pathway.

A fluorimetric assay for native C3: The hemolytically active form of the third component of human complement

The content of native C3 in samples of purified C3 may be accurately determined using the fluorescent probe ANS (8-anilino-1-napthalene sulfonate). The assay is based on the 11.5-fold increase in fluorescence intensity of ANS which accompanies proteolytic conversion of native C3 to C3b. The assay may be performed in the presence of hemolytically inactive derivatives of C3 such as C3b and C3(H2O). It exhibits the unique feature of being independent of protein concentration and it does not require a C3 standard, other purified complement components, C3 depleted serum, cells or cell-bound intermediate complexes, such as EAC142. A method utilizing cation exchange chromatography (Mono S, Pharmacia) is also described for the rapid (30 min) analytical or preparative separation of native C3 from inactive forms of C3 and from C3 fragments.

Mechanisms of stable serum resistance of Neisseria gonorrhoeae.

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab')2 isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab')2 prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab')2. Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab')2 before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.

Biochemistry of C3 and related thiolester proteins in infection and inflammation.

The characterization of the reactive thiolester bond in the third component of human complement has led to the identification of homologous sites in a number of proteins. In addition to the participation of the C3 thiolester in the opsonic acylation of surface components of microorganisms, new evidence is emerging to implicate thiolester disruption by physiologic nucleophiles, such as ammonia, as a potent mediator of local inflammation in the lung, the kidney, and at endothelial surfaces. Manipulation of the thiolester bonds in these related proteins should permit us to understand, and ultimately to direct, the molecular mechanisms of inflammation.

Immunoblotting analysis of the peptide chain structure of the physiological breakdown products of the third component of human complement

AbstractImmunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified third component of human complement (C3) and its fragments and of unpurified C3 and its degradation fragments in serum or plasma has been examined. The pattern of C3 degradation observed corresponded well with previous observations made with purified proteins. The immunoblotting technique demonstrated a high resolution, and proved to be capable of providing a full analysis of mixtures of C3 fragments in serum with the use of chain‐fragment‐specific antibody preparations. Additional heterogeneity in C3 and its breakdown products were observed, and covalent adducts between activated C3 and other proteins were detected directly in serum and plasma without any purification of the components. The results further indicated that antibodies raised against denatured C3 reacted quantitatively and qualitatively better with the nitrocellulose‐bound C3 than those antibodies raised to native C3, probably reflecting the paratially denatured state of molecules after separation by polyacrylamide gel electrophoresis and blotting onto the nitrocellulose.

Proton nuclear magnetic resonance study of the third component of complement: solution conformation of the carboxyl-terminal segment of C3a fragment

A proton nuclear magnetic resonance (NMR) study is reported of des-Arg-C3a, which is a 76-residue fragment obtained from the N-terminal portion of the alpha chain of the third component of human complement. A method of carboxypeptidase digestion/difference spectroscopy [Endo, S., & Arata, Y. (1985) Biochemistry 24, 1561-1568] was used for the spectral assignments for Ala-76, Leu-75, Gly-74, His-72, His-67, and Ala-48. On the basis of the NMR results obtained for these residues, we conclude that in aqueous solution (1) the C-terminal segment Leu-73-Ala-76 is free from interactions with the rest of the C3a molecule and (2) the major part of the C-terminal segment takes an ordered conformation. We also suggest that the presence of a core, which is formed by segment Tyr-15-Tyr-59 [Huber, R., Scholze, H., Paques, E. P., & Deisenhofer, J. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1389-1399], is essential for the C-terminal segment in maintaining the ordered structure in aqueous solution. 1H NMR spectral data were also obtained for the intact C3 from human and porcine sources. The resonances for the C2-H protons of His-67 and His-72, which exist in the C3a part of the human C3 molecule, were assigned. Comparisons of the results obtained with those for des-Arg-C3a demonstrate that upon cleavage of C3a very little change, if any, is induced in microenvironments of His-67 and His-72 and a piece of segment that contains His-72 is exposed to solvent and highly flexible.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of action of blocking immunoglobulin G for Neisseria gonorrhoeae.

Blocking immunoglobulin G (IgG) inhibits complement-mediated killing of serum-resistant Neisseria gonorrhoeae (GC) in immune human serum. We examined the mechanism of action of blocking IgG. Presensitization of GC with increasing concentrations of blocking IgG or F(ab')2 before incubation with bactericidal antibody and absorbed pooled normal human serum increased consumption and deposition of the third component of human complement (C3) and the ninth component of human complement (C9) but inhibited killing in dose-related fashion. We next showed that blocking IgG or F(ab')2 partially inhibited binding of bactericidal IgG to GC. Also, binding of a monoclonal antibody recognizing GC outer membrane protein PIII was almost completely inhibited by blocking F(ab')2, confirming other work (Rice, P. A., M. R. Tam, and M. S. Blake, manuscript submitted for publication) showing that PIII is a target for blocking antibody. Studies of the C3 deposition site showed that one quarter of the C3 deposited on GC in the presence of blocking IgG bound covalently to the antibody molecule. Finally, 125I-GC constituents with covalently bound C3 were affinity purified on Sepharose bearing antibodies to C3 and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. C3 deposition on a 40,000-mol wt surface protein was enhanced six- to ninefold by blocking IgG, which indicates that the site of complement deposition was altered by blocking antibody. These studies show that blocking IgG competes with bactericidal antibody for binding to GC, but enhances rather than blocks complement activation, and leads to complement deposition at new sites that do not result in serum killing.

Fibrinogen binding inhibits the fixation of the third component of human complement on surface of groups A, B, C, and G streptococci.

Effects of fibrinogen binding to M protein-positive and -negative streptococci on fixation of the third component of human complement (C3) were determined. In all test cultures of serological groups A, B, C, and G fixation of C3 was observed in normal human serum as revealed by quantitative fluorescent immunoassay. Fibrinogen binding inhibited the fixation of C3 on streptococci. The degree of inhibition was proportional to the extent of fibrinogen binding. Thus, inhibition of C3 fixation was most pronounced in strongly fibrinogen-positive streptococci of groups A, C, and G and not demonstrable in fibrinogen-negative cultures of groups C and G. Trypsinization of the streptococci destroyed their capacity to bind fibrinogen and consequently the inhibitory effects on C3 fixation. The carboxymethylated alpha and beta chains of fibrinogen moderately inhibited C3 fixation whereas gamma chain had no influence. These studies may indicate that fibrinogen binding structures other than M protein could also be involved in streptococcal pathogenicity.

Structures of sugar chains of the third component of human complement.

Human C3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acetylated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of alpha- and beta-chains of C3 indicated that the alpha-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the beta-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.

Relationship between the third component of human complement (C3) bound to stored preserved erythrocytes and their viability in vivo.

In this study we evaluated whether the 4 degrees C storage-induced coating of red blood cells (RBC) with molecules of the third component of human complement, C3, affects the viability of the preserved RBC. To this end, we determined whether the amount of C3 bound to preserved RBC correlated with the 24-hour survival value. The % anti-C3c-induced agglutination of stored RBC provided an estimate of the amount of RBC-bound C3. In some cases, the number of RBC-bound C3c-containing molecules was also quantitated. The 24-hour survival of autologous RBC was measured in 114 cases. All units were initially stored at 4 degrees C as RBC concentrates followed in 21 cases by frozen storage and in 75 cases by biochemical rejuvenation and frozen storage. The data showed a significant correlation between % anti-C3c-induced agglutination of the preserved RBC and the length of 4 degrees C storage of the RBC concentrates. Neither freezing nor rejuvenation cleaved the C3c fragment from stored RBC. The 24-hour survival was significantly and negatively correlated with both the storage length of RBC concentrates at 4 degrees C and with the amount of RBC-bound C3 but not with RBC ATP level. These data suggest that the RBC-bound C3 either contributes to or is a marker for the extent of the preservation injury of RBC.