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Expression and characterization of a recombinant C4b-binding protein lacking the beta-chain.

ABSTRACT: C4b-binding protein (C4BP) is a high-molecular-mass glycoprotein which contains binding sites for complement component C4b, anti-coagulant vitamin K-dependent protein S and serum amyloid P component (SAP). The major form of C4BP in plasma is composed of seven identical alpha-chains and a single beta-chain. We have expressed full-length cDNA for the alpha-chain in a eukaryotic expression system and characterized functional properties of non-beta-chain-containing C4BP. During synthesis, recombinant alpha-chains polymerized into two different high-molecular-mass C4BP forms which were composed of seven or eight alpha-chains. Recombinant C4BP bound C4(H2O) (used instead of C4b) equally as well as native C4BP, functioned equally as well as factor I cofactor in the degradation of C4(H2O) and bound to SAP. In contrast, the recombinant C4BP did not bind protein S and therefore did not inhibit the ability of protein S to function as a cofactor to activated protein C. Tunicamycin treatment of the transfected cells prevented N-linked glycosylation, but did not affect polymerization of the alpha-chains into a high-molecular-mass C4BP. The non-glycosylated C4BP had comparable properties to glycosylated C4BP in several functional assays. These results demonstrate polymerization of C4BP alpha-chains to be independent both of the beta-chain and of the N-linked carbohydrates. Moreover, N-linked carbohydrates and the beta-chain were neither required for the ability of C4BP to bind C4b and to function as factor I cofactor nor for the interaction with SAP.

SUBMITTER: Hardig Y

PROVIDER: S-EPMC1136795 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Project description:The aim of this study was to discover novel biomarkers for pulmonary tuberculosis (TB). Differentially expressed proteins in the serum of patients with TB were screened and identified by iTRAQ-two dimensional liquid chromatography tandem mass spectrometry analysis. A total of 79 abnormal proteins were discovered in patients with TB compared with healthy controls. Of these, significant differences were observed in 47 abnormally expressed proteins between patients with TB or pneumonia and chronic obstructive pulmonary disease (COPD). Patients with TB (n = 136) exhibited significantly higher levels of serum amyloid A (SAA), vitamin K-dependent protein Z (PROZ), and C4b-binding protein β chain (C4BPB) than those in healthy controls (n = 66) (P<0.0001 for each) albeit significantly lower levels compared with those in patients with pneumonia (n = 72) (P<0.0001 for each) or COPD (n = 72) (P<0.0001, P<0.0001, P = 0.0016, respectively). After 6 months of treatment, the levels of SAA and PROZ were significantly increased (P = 0.022, P<0.0001, respectively), whereas the level of C4BPB was significantly decreased (P = 0.0038) in treated TB cases (n = 72). Clinical analysis showed that there were significant differences in blood clotting and lipid indices in patients with TB compared with healthy controls, patients with pneumonia or COPD, and treated TB cases (P<0.05). Correlation analysis revealed significant correlations between PROZ and INR (rs = 0.414, P = 0.044), and between C4BPB and FIB (rs = 0.617, P = 0.0002) in patients with TB. Receiver operating characteristic curve analysis revealed that the area under the curve value of the diagnostic model combining SAA, PROZ, and C4BPB to discriminate the TB group from the healthy control, pneumonia, COPD, and cured TB groups was 0.972, 0.928, 0.957, and 0.969, respectively. Together, these results suggested that SAA, PROZ, and C4BPB may serve as new potential biomarkers for TB. Our study may thus provide experimental data for the differential diagnosis of TB.

Project description:Complement inhibitor C4b-binding protein (C4BP) is synthesized in liver and pancreas and composed of 7 identical alpha chains and one unique beta chain. We showed previously that C4BP binds islet amyloid polypeptide (IAPP) and affects fibril formation in vitro. Now we found that polymeric C4BP inhibited lysis of human erythrocytes incubated with monomeric IAPP while no erythrocyte lysis was observed after incubation with preformed IAPP fibrils. In contrast, monomeric alpha chain of C4BP had significantly reduced activity. Further, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Accordingly, addition of C4BP rescued the ability of INS-1 cells and isolated rat islets to respond to glucose stimulation with insulin secretion, which was impaired in the presence of IAPP alone. C4BP was internalized together with IAPP into INS-1 cells and therefore we aimed to study its effect on gene expression. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison to IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-Î²-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-ÎºB had a similar effect. Taken together, one of the mechanisms by which C4BP protects beta-cells from IAPP cytotoxicity is by enhancing cholesterol synthesis. The INS-1 cells were grown as 5 separate clones for 10 passages before plating in a 12-well plate (Nunc) at 100.000 cells per well and grown in complete RPMI 1640 medium to 70% confluency for approximately 48 h. The cells were then challenged by adding 77 Î¼M monomeric IAPP alone or together with C4BP (0.6 Î¼M). DMSO (1%) used as solvent for IAPP as well as C4BP (0.6 Î¼M) alone were used as controls. RNA was extracted after 10h incubation and analysis carried out using Rat Gene 2.0 array chip (Affymetrix).

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Expression and characterization of a recombinant C4b-binding protein lacking the beta-chain.

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