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PubMed1981

A monoclonal antibody against human complement component C3: the production of C3 by human cells in vitro.

A monoclonal antibody (WM1) against the third component of human complement (C3) was produced by fusing P3-X63-Ag8 mouse myeloma cells with spleen cells from BALB/c mice immunized with purified C3. The specificity of WM1 antibody against C3 was established by its capacity to inhibit a standard C3 hemolytic assay and to immunoprecipitate C3 from human serum in the presence of S. aureus bacteria. Indirect binding assays indicate that the antibody is directed the C3c portion of C3. The immunoprecipitation technique was used to screen for the presence of C3 in the culture supernatants of various cell lines. By this means, C3 was identified as a secreted product of human primary fibroblasts, transformed fibroblasts and D98/AH-2, a HeLa derivative, but not of other human cell lines. WM1 was unable to immunoprecipitate C3 from rabbit or mouse fibroblast culture supernatants.

Animals↗
PubMed2002

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection.

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.

Animals↗
PubMed1980

Experimental bovine trypanosomiasis (Trypanosoma vivax and T. congolense). II. Serum levels of total protein, albumin, hemolytic complement, and complement component C3.

Serum levels of total protein, albumin, activity of hemolytic complement, and complement component C3 were decreased in cattle infected with either T. congolense or T. vivax. The hemolytic complement activity was reduced most, i.e. to 20% (T. congolense) or 5% (T. vivax) of control levels. The development of hypocomplementemia was closely associated with the first peak of parasitemia.

Animals↗
PubMed1997

Phosphorylation of complement component C3 after synthesis in U937 cells by a putative protein kinase, casein kinase 2, which is regulated by CD11b: evidence that membrane-bound proteases preferentially cleave phosphorylated C3.

It was our aim in this study to investigate the possibility that the third component of complement (C3) is phosphorylated during synthesis and secretion in U937 cells. Labelling of U937 cells with [32P]Pi, followed by immunoprecipitation of C3 from cell lysates and culture supernatants at different time points, showed that C3 was phosphorylated intracellularly immediately before release into the medium, which initiated cleavage of the protein into an iC3b-like fragment. Stimulation of CD11b/CD18 increased phosphorylation 7-fold, from a basal level of 2%. The phosphorylation sites in C3 did not resemble those described previously for casein kinase (CK) 1, cAMP-dependent protein kinase A or calcium- and phospholipid-dependent protein kinase C. Instead, protein kinase CK2 was suggested inasmuch as: (1) CK2 was detected both on the cell surface and on shed microparticles; (2) phosphorylation of purified C3 by microparticles was abolished by a monoclonal antibody, anti-CK2; (3) the [32P]Pi tag of both phosphorylated C3 (secreted from U937 cells) and of microparticle-phosphorylated C3, which was cleaved either by membrane proteases or by leucocyte elastase, was found in a 40 and a 70 kDa polypeptide; (4) both secreted C3 and C3 phosphorylated in vitro were much more susceptible to cleavage by proteases. Generation of C3 fragments provides a means by which U937 cells can stimulate nearby cells which are expressing complement receptors. The present study demonstrates that the cleavage of C3 is controlled by an intracellular phosphorylation event regulated by CD11b/CD18.

Casein Kinase II↗
PubMed1993

Macrophage colony-stimulating factor (M-CSF) enhances complement component C3 production by human monocytes/macrophages.

The third complement component, C3, is an important factor in the host defense mechanism in which monocytes/macrophages participate as the primary phagocytes. Monocytes/macrophages are the principal extrahepatic producers of C3, and this C3 production is thought to be regulated by several cytokines. In the present study, we demonstrated that human macrophage colony-stimulating factor (M-CSF) enhanced C3 production by human peripheral monocytes in serum-free culture. Analytical immunoblot and ELISA showed that the presence of M-CSF increased the production of intracellular pro-C3 and extracellular C3 for 24 h in a dose-dependent manner. To confirm the rapid effect of M-CSF on C3 production, we performed metabolic labeling of C3 with [35S]methionine. The production of [35S]C3 for the first 6 h in the presence of M-CSF was also increased as compared to that in the absence of M-CSF. In addition to the previously reported effects of M-CSF on monocytes/macrophages, such as the enhancement of C3 receptor expression and C3 receptor-mediated phagocytosis, we consider that the effects of M-CSF demonstrated in this study are of importance in the local immune system organization of C3 and monocytes/macrophages.

Complement C3↗
PubMed2005

Complement activation in acute coronary syndromes.

The complement system is part of the host defence response. However, considerable evidence suggests that complement plays an important role in the pathophysiology of ischemic heart disease. The aim of this study was to evaluate complement activation in patients with all forms of acute coronary syndromes (ACS) and to examine the relationship between the degree of complement activation and myocardial injury. The study population included 152 subjects (26 females): 82 with ACS (35 acute myocardial infarction (AMI), 22 non-Q wave MI (NQMI), 25 unstable angina (UAP)) (Group A), 35 stable angina (SA) (Group B), and 35 healty control subjects (Group C). Complement 3 (C3), Complement 4 (C4), C-reactive protein (CRP), troponin I (TnI) as well as creatine kinase MB (CK-MB) were evaluated. Patients' blood samples were taken on admission (day 1) and after 2, 3 and 7 days in group A. However, only one measurement was performed in the groups B and C. Plasma C3 and C4 peak levels were significantly higher in patients with AMI (141+/-29 and 35+/-11 mg/dl) and NQMI (136+/-13 and 35+/-7 mg/dl) than in patients with SA (128+/-14 and 27+/-10 mg/dl) and the control subjects (114+/-22 and 22+/-7 mg/dl) (p<0.03). Also, C3 and C4 serum levels in patients with SA and UAP (126+/-16 and 31+/-7 mg/dl) were significantly higher than those in control subjects (p<0.01, p<0.03, respectively). At 1-week follow-up, there were no significant differences between the plasma levels of C3 and C4 in patients with UAP (p>0.05). However, plasma levels of C3 and C4 were significantly different between days in patients with AMI and NQMI (p<0.0001). Plasma C3 and C4 levels in ACS showed a relationship with peak CK-MB and Tn I levels (p<0.01). Plasma CRP level in ACS showed positive correlation with C3 (p<0.01) and C4 (p<0.001). In this study, we determined that plasma C3 and C4 levels were elevated in ACS and SA. Although C3 and C4 were higher in ACS and SA, the systemic levels of inflammatory markers in patients with SA and UAP were lower than those found in the AMI and NQMI groups. The relationship between C3, C4 levels and ACS further suggests that the complement activation is related to necrosis within the myocardium.

Acute Disease↗
PubMed1976

Genetic polymorphism of C3 and serum levels of immunoglobulins, C3, C4 components of complement and C3-proactivator in four different populations of Afghanistan.

The C3 phenotype distribution was studied in 4 different populations from Afghanistan. The gene frequencies of C3S allele were: Tajiks (0,8547), Pushtoons (0.8812), Hazaras (0.9036) and Osbeks (0.8530). These values were significantly higher than in European populations studied previously. No significant differences were found between the mean serum levels of C3, C4 and C3-proactivator among 4 population groups. A higher concentration of IgG, IgA and IgM was observed in Afghanistan sera than reported for Europeans.

Afghanistan↗
PubMed2003

The spotted wolffish (Anarhichas minor Olafsen) complement component C3: isolation, characterisation and tissue distribution.

The complement component C3 was isolated from spotted wolffish (Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, M(r)65 and 115kDa, respectively. The high molecular weight alpha-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the beta-alpha junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3.

Amino Acid Sequence↗
PubMed2001

Structure and biology of complement protein C3, a connecting link between innate and acquired immunity.

Complement protein C3 is a central molecule in the complement system whose activation is essential for all the important functions performed by this system. After four decades of research it is now well established that C3 functions like a double-edged sword: on the one hand it promotes phagocytosis, supports local inflammatory responses against pathogens, and instructs the adaptive immune response to select the appropriate antigens for a humoral response; on the other hand its unregulated activation leads to host cell damage. In addition, its interactions with the proteins of foreign pathogens may provide a mechanism by which these microorganisms evade complement attack. Therefore, a clear knowledge of the molecule and its interactions at the molecular level not only may allow the rational design of molecular adjuvants but may also lead to the development of complement inhibitors and new therapeutic agents against infectious diseases.

Amides↗
PubMed1984

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3.

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1998

Active sites in complement component C3 mapped by mutations at indels.

Engineered mutants of human complement component C3 were used to test the idea that sites of length polymorphisms in protein families (indels) can guide a search for protein:protein interaction sites. Sequence changes were introduced at each of the 27 indels in the C3/4/5 protein family, and mutants at 26 indels were expressed by transiently transfected COS cells. Expressed proteins were assayed 1) for concentration, by ELISA and by autoradiography of radiolabeled protein; 2) for classical pathway hemolytic activity; 3) for susceptibility to proteolytic activation by the alternative pathway and cobra venom factor C3 convertases; and 4) for susceptibility to complement factor I in the presence of factor H. Most of the mutations did not appreciably alter expression or activity relative to wild-type C3, consistent with the idea that most indels occur at the protein surface. Mutations at four indels severely damaged C3 functional activity, but did not affect the stability or structure of the protein, as assessed by their effects on expression by COS cells and on susceptibility to cleavage by C3 convertases and factor I. These indels are therefore near functionally important amino acid residues; they represent good candidates for sites of protein:protein interactions. Mutation of the sequence at a fifth indel altered the equilibrium between the latent and reacted C3 conformations, and mutations at 4 other indels substantially decreased both protein activity and expression. The mutants provided an overview of the structural and functional roles played by different parts of C3.

Amino Acid Sequence↗
PubMed1997

Cleavage of the complement system C3 component by HIV-1 proteinase.

The C3 factor of the complement system and its C3b fragment are cleaved in vitro by the proteinase of the human immunodeficiency virus, type 1 (HIV PR). The cleavage occurs in the alpha-chain of both substrates at multiple sites yielding a 100 kDa fragment of the C3 alpha-chain and multiple fragments of the C3b alpha-chain. The scissile bonds are: Ala86-Glu87, Leu310-Leu311, His641-Trp642 and Arg649-Ser650. The resulting fragments resemble the physiologically occurring inactive fragments of C3: C3c and C3d, suggesting a possible biological role of the HIV-proteinase in the complement inactivation process.

Amino Acids↗
PubMed1991

Localization of the human complement component C3 binding site on the IgG heavy chain.

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.

Amino Acid Sequence↗
PubMed1999

Simple quantification of complement factors C3 and C3b using separation by isotachophoresis.

The separation of complement factors C3 and C3b by isotachophoresis in 1% agarose gel followed by immunoprecipitation and quantification is presented. Glycine was used as spacer in a nonequilibrium isotachophoresis (Acevedo, F., J. Chromatogr. 1991, 545, 391-396). Tricine, beta-alanine and Tris were the leading ion, terminating ion and counter ion, respectively. After electrophoresis the gel was incubated in rabbit anti-human complement factor C3c. The amounts of C3 and C3b in the sample were measured by optical densitometry of the Coomassie Brilliant blue-stained immunoprecipitates in the agarose gel. The correlation coefficient obtained for the logarithm of the integrated densitometric measurement vs. the logarithm of the amount of applied C3 was higher than 0.98 in calibration experiments. The extent of complement factor C3 activation is calculated as the ratio between the amount of C3b and the amount of C3b plus C3 and expressed as percent. The progress of complement activation from human blood plasma samples induced by Mg2+ and zymosan are presented as examples.

Animals↗
PubMed1983

The incidence of sperm-associated immunoglobulin and C3, the third component of complement, in infertile men.

One hundred four consecutive men who underwent a semen analysis as part of their infertility evaluation were assayed for sperm-associated immunoglobulin (Ig) and the third component of complement (C3). Sixteen (15%) of the 104 men had increased IgG, and 10 (12%) of 86 men tested had increased IgA on their sperm; however, only 4 (5%) of the 87 men tested were positive for plasma IgG antisperm antibody. Only 2 (13%) of the 16 men with increased sperm-associated IgG had detectable IgG antisperm antibodies in their seminal plasma. Five (10%) of 50 men tested had increased sperm-associated C3; none had seminal plasma that deposited C3 on sperm. These data suggest that of the three compartments that can be tested--circulating plasma, seminal plasma, and the sperm surface--the direct measurement of sperm surface Ig may be most important in the evaluation of infertile men. The presence of sperm-associated IgG or IgA was not associated with semen abnormality. In contrast, the presence of sperm-associated IgG was associated with decreased mucus penetration at the time of postcoital testing despite normal-appearing mucus.

Complement C3↗
PubMed2001

Serine 132 is the C3 covalent attachment point on the CH1 domain of human IgG1.

The covalent binding of C3 (complement component C3) to antigen-antibody complexes (Ag.Ab; immune complexes (ICs)) is a key event in the uptake, transport, presentation, and elimination of Ag in the form of Ag.Ab.C3b (IC.C3b). Upon interaction of C3 with IgG.IC, C3b.C3b.IgG covalent complexes are formed that are detected on SDS-polyacrylamide gel electrophoresis by two bands corresponding to C3b.C3b (band A) and C3b.IgG (band B) covalent complexes. This allows one to evaluate the covalent binding of C3b to IgG antibodies. It has been described that C3b can attach to both the Fab (on the CH1 domain) and the Fc regions of IgG. Here the covalent interaction of C3b to the CH1 domain, a region previously described spanning residues 125-147, has been studied. This region of the CH1 domain is exposed to solvent and contains a cluster of six potential acceptor sites for ester bond formation with C3b (four Ser and two Thr). A set of 10 mutant Abs were generated with the putative acceptor residues substituted by Ala, and we studied their covalent interaction with C3b. Single (Ser-131, Ser-132, Ser-134, Thr-135, Ser-136, and Thr-139), double (positions 131-132), and multiple (positions 134-135-136, 131-132-134-135-136, and 131-132-134-135-136-139) mutants were produced. None of the mutants (single, double, or multiple) abolished completely the ability of IgG to bind C3b, indicating the presence of C3b binding regions other than in the CH1 domain. However, all mutant Abs, in which serine at position 132 was replaced by Ala, showed a significant decrease in the ability to form C3b.IgG covalent complexes, whereas the remaining mutants had normal activity. In addition we examined ICs using the F(ab')2 fragment of the mutant Abs, and only those containing Ala at position 132 (instead of Ser) failed to bind C3b. Thus Ser-132 is the binding site for C3b on the CH1 domain of the heavy chain, in the Fab region of human IgG.

Amino Acid Sequence↗
PubMed1998

Purification from human plasma of a hexapeptide that potentiates the sulfation and mitogenic activities of insulin-like growth factors.

The human plasma contains small peptide molecules known as low molecular weight growth factors synergistically increasing certain biological actions of insulin-like growth factors. In the present work we isolated and characterized a hexapeptide with HWESAS as structure. This purified peptide was absolutely necessary for the sulfation activity of insulin-like growth factor-I on chick embryo pelvic cartilages and improved the mitogenic activity of both insulin-like growth factors. The effects of this hexapeptide were confirmed by using the homologous synthetic peptide, that exhibited similar biological effects. Other synthetic peptides with structure derived from hexapeptide were shown to be active: the pentapeptide HWESA appeared more potent than the tripeptide HWE, which is about 170 to 200 times less active than the hexapeptide. The sequence of hexapeptide HWESAS is identified in only one human protein that is C3f, a fragment of C3 complement.

Animals↗