The purification and characterization of bovine C4, the fourth component of complement.
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ABSTRACT:
SUBMITTER: Booth NA
PROVIDER: S-EPMC1186463 | biostudies-other | 1979 Mar
REPOSITORIES: biostudies-other
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Project description:The fourth component of complement, C4, was isolated from human serum in good yield, and in confirmation of previous reports was shown to be formed from three peptide chains, alpha, beta and gamma, with apparent mol.wts. 90 000, 80 000 and 30 000 respectively. Preparative methods are described for the isolation of the three peptide chains and their amino acid analyses reported. Component C4 contains 7.0% carbohydrate, alpha-chain 8.6% and the beta-chain 5.6%. The N-terminal amino acid sequences are given for 12 residues of the alpha-chain, eight of the beta-chain and 19 of the gamma-chain.
| S-EPMC1164925 | biostudies-other
Project description:The fourth component of bovine complement, C4, was cleaved specifically by subcomponent C1s to produce two fragments, C4a and C4b. The smaller, C4a, was isolated in pure form and is a peptide of 9500 mol.wt. containing approx. 84 amino acids and no detectable carbohydrate. C4a has an amino acid composition that is comparable with the anaphylatoxins C3a and C5a, containing six cysteine residues/mol and a high proportion of basic residues. The amino acid sequence of the first thirteen residues shows four identities with the porcine C3a sequence. There is almost complete identity between the C4a sequence and that of the alpha-chain of human C4, indicating that this region is highly conserved. This evidence also clearly establishes that C4a is cleaved from the N-terminal of the alpha-chain of C4.
| S-EPMC1161638 | biostudies-other
Project description:Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.
| S-EPMC1186403 | biostudies-other
Project description:Human complement component C4 is coded by tandem genes located in the HLA class III region. The products of the two genes, C4A and C4B, are different in their activity. This difference is due to a degree of 'substrate' specificity in the covalent binding reactions of the two isotypes. Mouse also has a duplicated locus, but only one gene produces active C4, while the other codes for the closely related sex-limited protein (Slp). In order to gain some insight into the evolutionary history of the duplicated C4 locus, we have purified C4 from a number of other mammalian species, and tested their binding specificities. Like man, chimpanzee and rhesus monkey appear to produce two C4 types with reactivities similar to C4A and C4B. Rat, guinea pig, whale, rabbit, dog and pig each expresses C4 with a single binding specificity, which is C4B-like. Sheep and cattle express two C4 types, one C4B-like, the other C4A-like, in their binding properties. These results suggest that more than one locus may be present in these species. If this is so, then the duplication of the C4 locus is either very ancient, having occurred before the divergence of the modern mammals, or there have been three separate duplication events in the lines leading to the primates, rodents and ungulates.
| S-EPMC1136911 | biostudies-other
Project description:Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.
| S-EPMC1161639 | biostudies-other
Project description:Genetic factors have been estimated to account for about 25% of the variation in an adult's life span. The complement component C4 with the isotypes C4A and C4B is an effector protein of the immune system, and differences in the overall C4 copy number or gene size (long C4L; short C4S) may influence the strength of the immune response and disease susceptibilities. Previously, an association between C4B copy number and life span was reported for Hungarians and Icelanders, where the C4B*Q0 genotype, which is defined by C4B gene deficiency, showed a decrease in frequency with age. Additionally, one of the studies indicated that a low C4B copy number might be a genetic trait that is manifested only in the presence of the environmental risk factor "smoking". These observations prompted us to investigate the role of the C4 alleles in our large German longevity sample (? 700 cases; 94-110 years and ? 900 younger controls). No significant differences in the number of C4A, C4B and C4S were detected. Besides, the C4B*Q0 carrier state did not decrease with age, irrespective of smoking as an interacting variable. However, for C4L*Q0 a significantly different carrier frequency was observed in the cases compared with controls (cases: 5.08%; controls: 9.12%; p = 0.003). In a replication sample of 714 German cases (91-108 years) and 890 controls this result was not replicated (p = 0.14) although a similar trend of decreased C4L*Q0 carrier frequency in cases was visible (cases: 7.84%; controls: 10.00%).
| S-EPMC3899116 | biostudies-literature
Project description:Treatment of complement component C4 with C1(-)s and methylamine induces a series of conformation changes such as to generate functional binding sites. A monoclonal antibody (mAb), Al 121/6, which does not inhibit the haemolytic activity of C4 was found to bind to native C4 and C4d, but not to C4b and methylamine-treated C4, unless these C4 derivatives were denatured. These results suggested that a linear epitope for mAb Al 121/6 in the C4d domain is originally located at the surface of C4 and becomes hidden as a result of conformational changes induced by C1(-)s or methylamine treatment. The hidden linear epitope was exposed again upon further cleavage of C4b into C4c and C4d. Trypsin digestion of C4d and its chemical modification with phthalic anhydride suggested that the epitope is located at the C-terminal 13 kDa region of C4d and that lysine residues are involved in the epitope. There is a single lysine residue at 1259 in the 13 kDa C-terminal side of C4d and the synthetic undecapeptide Leu1254-Asp1264 was found to inhibit the binding of C4 to mAb Al 121/6, suggesting that the epitope for mAb Al 121/6 is involved in the sequence. The N-terminal portion of the peptide is partly overlapping, with a highly hydrophobic amino acid sequence spanning residues Ala1249-Leu-Leu-His-Leu-Leu-Leu1255. The surface hydrophobicity of C4 has been reported to decrease upon treatment with C1(-)s and methylamine. So it appears that the hydrophobic sequence spanning Ala1249-Leu1255 may be hidden, together with the linear epitope, into the inner region of C4 upon treatment with C1s and methylamine.
| S-EPMC1132196 | biostudies-other
Project description:An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4·MASP-2 substrate·enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C-CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme-substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen-antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation.
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Project description:Thiol compounds have been investigated as inhibitors of the covalent binding reaction of human complement protein C4 using Sepharose-C1s as a combined activating and binding surface. o- and p-substituted aminothiophenols are equally effective inhibitors, whereas the m-substituted compound is a less potent inhibitor. The anti-hypertensive drug captopril is also shown to inhibit the covalent binding reaction. A comparison of the effects of these compounds on the covalent binding reaction of isolated C4A and C4B has been made. Results suggest that a Pro-to-Leu substitution in C4B is likely to account for the differences in inhibitory potency of C4B compared with C4A observed with the aromatic inhibitors.
| S-EPMC1132247 | biostudies-other
Project description:Schizophrenia is a severe mental illness and a major risk factor for suicide, with approximately 50% of schizophrenia patients attempting and 10% dying from suicide. Although genetic components play a significant role in schizophrenia risk, the underlying genetic risk factors for suicide are poorly understood. The complement component C4 gene, an immune gene involved in the innate immune system and located in the major histocompatibility complex (MHC) region, has been identified to be strongly associated with schizophrenia risk. In addition, recent findings have also suggested that the MHC region has been associated with suicide risk across disorders, making C4 a potential candidate of interest for studying suicidality in schizophrenia patients. Despite growing interest in investigating the association between the C4 gene and schizophrenia, to our knowledge, no work has been done to examine the potential of C4 variants as suicide risk factors in patients with schizophrenia. In this study, we investigated the association between different C4 copy number variants and predicted C4 brain expression with suicidal outcomes (suicide attempts/suicidal ideation). We directly genotyped 434 schizophrenia patients to determine their C4A and C4B copy number variants. We found the C4AS copy number to be marginally and negatively associated with suicide risk, potentially being protective against suicide attempts (OR = 0.49; p = 0.05) and suicidal ideation (OR = 0.65; p = 0.07). Furthermore, sex-stratified analyses revealed that there are no significant differences between males and females. Our preliminary findings encourage additional studies of C4 and potential immune dysregulation in suicide.
| S-EPMC10858919 | biostudies-literature