The purification and characterization of subcomponent C1s of the first component of bovine complement.
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ABSTRACT:
SUBMITTER: Campbell RD
PROVIDER: S-EPMC1161639 | biostudies-other | 1979 Dec
REPOSITORIES: biostudies-other
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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:1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 X 10(13)-4 X 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.
| S-EPMC1162641 | biostudies-other
Project description:Complement component 1q subcomponent binding protein (C1qbp) is a multifunctional protein involved in immune response, energy homeostasis of cells as a plasma membrane receptor, and a nuclear, cytoplasmic or mitochondrial protein. Recent reports suggested its neuronal function, too, possibly in axon maintenance, synaptic function, and neuroplasticity. Therefore, we addressed to identify C1qbp in the rat brain using in situ hybridization histochemistry and immunolabelling at light and electron microscopic level. C1qbp has a topographical distribution in the brain established by the same pattern of C1qbp mRNA-expressing and protein-containing neurons with the highest abundance in the cerebral cortex, anterodorsal thalamic nucleus, hypothalamic paraventricular (PVN) and arcuate nuclei, spinal trigeminal nucleus. Double labelling of C1qbp with the neuronal marker NeuN, with the astrocyte marker S100, and the microglia marker Iba1 demonstrated the presence of C1qbp in neurons but not in glial cells in the normal brain, while C1qbp appeared in microglia following their activation induced by focal ischemic lesion. Only restricted neurons expressed C1qbp, for example, in the PVN, magnocellular neurons selectively contained C1qbp. Further double labelling by using the mitochondria marker Idh3a antibody suggested the mitochondrial localization of C1qbp in the brain, confirmed by correlated light and electron microscopy at 3 different brain regions. Post-embedding immunoelectron microscopy also suggested uneven C1qbp content of mitochondria in different brain areas but also heterogeneity within single neurons. These data suggest a specific function of C1qbp in the brain related to mitochondria, such as the regulation of local energy supply in neuronal cells.
| S-EPMC6418184 | biostudies-literature
Project description:The fourth component of complement, C4, was isolated from bovine plasma in high yield, by using simple purification techniques. The protein, like human component C4, is a beta-globulin with a mol.wt. of about 200 000 and consists of three polypeptide chains, alpha, beta and gamma, with apparent mol. wts. of 98 000, 82 000 and 32 000 respectively. The chains of C4 have been separated by methods previously used for human C4. Their amino acid compositions are very similar to those of the human component, but differences in carbohydrate distribution have been observed. The haemolytic activity of bovine C4 is totally destroyed by incubation with bovine C1s, the activated subcomponent of the first component of complement. Component C4, treated in this way, was shown to be cleaved in the alpha chain, which was decreased in mol.wt. by about 9000, corresponding to the removal of subcomponent C4a.
| S-EPMC1186463 | biostudies-other
Project description:1. C1q, a subcomponent of the first component of complement, has been isolated, in a haemolytically active and soluble form, by ion-exchange chromatography and gel filtration, from human and rabbit sera. Yields ranged from 10 to 25mg/litre of serum and the activity of final preparations was consistently in the range 5x10(3)-15x10(3) C1qH(50) units/mg. 2. The molecular weights of human and rabbit subcomponent C1q were 409600 and 417600, as determined by sedimentation equilibrium studies. 3. Subcomponent C1q from both species was shown to be composed of non-covalently linked subunits of approximately 57000 molecular weight as determined by gel-filtration or sedimentation equilibrium studies in 5.3m-guanidinium chloride. Reduction or oxidation of human and rabbit subcomponent C1q yielded three chains each having a molecular weight of approximately 23000 and which differed slightly in amino acid composition but markedly in carbohydrate content. The oxidized chains were separated, on a preparative scale, by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 4. Both human and rabbit subcomponent C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 8% carbohydrate. Glutamic acid and aspartic acid were the free N-terminal amino acids of human subcomponent C1q whereas only serine was found in rabbit subcomponent C1q. 5. Collagenase digestion of human or rabbit subcomponent C1q caused a rapid loss of haemolytic activity which correlated with the breakdown of collagenous regions in the molecule.
| S-EPMC1174513 | biostudies-other
Project description:1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.
| S-EPMC1172797 | biostudies-other
Project description:1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15-18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52 degrees C (but not at 49 degrees C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.
| S-EPMC1168381 | biostudies-other
Project description:The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 ("a" chain) and 27000 ("b" chain). The amino acid analyses of the "a" chains from each activated subcomponent are similar, as are those of the "b" chains. The N-terminal amino acid sequence of 29 residues of the C-1s "a" chain was determined, but the C-1r "a" chain has blocked N-terminal amino acid. The 20 N-terminal residues of both "b" chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the 'b' chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.
| S-EPMC1164687 | biostudies-other
Project description:The complement component 1, q subcomponent binding protein (C1QBP/p32/HABP1) is a ubiquitously expressed and multicompartmental cellular protein involved in various biological processes. In order to further understand its biological functions, we conducted proteomics analysis of its interactome in this study. An improved sample preparation and mass spectrometric identification strategy was developed combining high-speed centrifugation, formaldehyde labeling, and two-dimensional reverse-phase liquid chromatography. Using this approach, we identified 187 interacting proteins and constructed a highly connected interacting network for C1QBP. Moreover, we explored the interaction between C1QBP and protein kinase C ζ, a key regulator of cell polarity and migration. The results indicated that C1QBP regulated the activity of protein kinase C ζ and modulated EGF-induced cancer cell chemotaxis. In addition, C1QBP was required for breast cancer metastasis in a severe combined immunodeficiency mouse model. Furthermore, C1QBP was observed to be overexpressed in breast cancer tissues, and its expression level was closely linked with distant metastasis and TNM stages. In summary, C1QBP was identified as a novel regulator of cancer metastasis that may serve as a therapeutic target for breast cancer treatment.
| S-EPMC3820933 | biostudies-literature
Project description:The complement system consists of a series of soluble and cell-surface proteins that serve numerous roles in innate immunity, development, and homeostasis. Despite its many functions, the central event in the complement system is the proteolytic activation of the 185 kDa complement component 3 (C3) into its opsonin and anaphylatoxin fragments known as C3b (175 kDa) and C3a (10 kDa), respectively. The C3 protein is comprised of thirteen separate structural domains, several of which undergo extensive structural rearrangement upon activation to C3b. In addition to this, the C-terminal C345c domain found in C3, C3b, and the terminal degradation product, C3c (135 kDa), appears to adopt multiple conformations relative to the remainder of the molecule. To facilitate various structure/function studies, we designed two C3 analogs that could be activated to a C345c-less, C3c-like state following treatment with Tobacco Etch Virus (TEV) protease. We generated stably transfected Chinese Hamster Ovary (CHO) cell lines that secrete approximately 1.5 mg of the highest-expressing C3 analog per liter of conditioned culture medium. We purified this C3 analog by sequential immobilized metal ion affinity and size exclusion chromatographies, activated the protein by digestion with TEV protease, and purified the resulting C3c analog by a final size exclusion chromatography. The conformations and activities of our C3 and C3c analogs were assessed by measuring their binding profiles to known C3/b/c ligands by surface plasmon resonance. Together, this work demonstrates the feasibility of producing a C3 analog that can be site-specifically activated by an exogenous proteolytic enzyme.
| S-EPMC6707835 | biostudies-literature