A simplified assay for the detection of C3a in human plasma employing a monoclonal antibody raised against denatured C3

Nilsson, Bo; Svensson, K.-E.; Inganäs, M.; Nilsson, Ulf

doi:10.1016/0022-1759(88)90229-3

Cited by 68 publications

(82 citation statements)

References 24 publications

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“…To assess whether these residues confer isomerase activity, we determined the SaDsbA catalyzed recovery of active RNase A from oxidized, scrambled RNase A (scRNase) (45). In this assay, the isomerase DsbC yields 80% of active molecules (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Refolding of Scrambled RNase A-The in vitro assay of refolding of scrambled RNase was used to monitor the isomerase activity of SaDsbA (45). scRNase was prepared from reduced denatured RNase by incubating the enzyme (0.5 mg/ml) in 50 mM Tris-HCl, pH 8.5 and 6 M GdmCl for at least 3 days at room temperature and in the dark.…”

Section: Journal Of Biological Chemistry 4263mentioning

confidence: 99%

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Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Heras

Kurz

Jarrott

et al. 2008

Journal of Biological Chemistry

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In Gram-negative bacteria, the introduction of disulfide bonds into folding proteins occurs in the periplasm and is catalyzed by donation of an energetically unstable disulfide from DsbA, which is subsequently re-oxidized through interaction with DsbB. Gram-positive bacteria lack a classic periplasm but nonetheless encode Dsb-like proteins. Staphylococcus aureus encodes just one Dsb protein, a DsbA, and no DsbB. Here we report the crystal structure of S. aureus DsbA (SaDsbA), which incorporates a thioredoxin fold with an inserted helical domain, like its Escherichia coli counterpart EcDsbA, but it lacks the characteristic hydrophobic patch and has a truncated binding groove near the active site. These findings suggest that SaDsbA has a different substrate specificity than EcDsbA. Thermodynamic studies indicate that the oxidized and reduced forms of SaDsbA are energetically equivalent, in contrast to the energetically unstable disulfide form of EcDsbA. Further, the partial complementation of EcDsbA by SaDsbA is independent of EcDsbB and biochemical assays show that SaDsbA does not interact with EcDsbB. The identical stabilities of oxidized and reduced SaDsbA may facilitate direct re-oxidation of the protein by extracellular oxidants, without the need for DsbB.The formation of native disulfide bonds through air oxidation of cysteine pairs is a slow reaction and organisms ranging from bacteria to humans encode enzymatic systems to catalyze the process. In eukaryotes, oxidative folding in the endoplasmic reticulum is primarily catalyzed by protein-disulfide isomerases that are reoxidized by Ero1p/Erv2p proteins (reviewed in Ref. 1). In Escherichia coli, dithiol oxidation takes place in the periplasm through the action of the Dsb 3 (Disulfide bond) family of proteins. Dsb proteins form two distinct pathways, the DsbA-DsbB (oxidative) pathway that introduces disulfides indiscriminately, and the DsbC/DsbG-DsbD (isomerization) pathway that shuffles incorrect disulfides (2, 3).Probably the best-studied Dsb protein is EcDsbA, the primary disulfide catalyst in E. coli (reviewed in Refs. 2, 4). This promiscuously oxidizing protein is a 21-kDa monomer containing a CPHC active site in a thioredoxin (TRX) fold with an inserted helical domain of ϳ80 residues. Upon catalyzing disulfide bond formation in substrate proteins, reduced EcDsbA relies on EcDsbB, a quinone reductase, to recover its catalytically active, and higher energy, oxidized form (5

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Section: Resultsmentioning

confidence: 99%

Section: Journal Of Biological Chemistry 4263mentioning

confidence: 99%

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Heras

Kurz

Jarrott

et al. 2008

Journal of Biological Chemistry

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“…For subsequent analysis the cell suspensions were thawed, diluted in 3 vols of TKM buffer (50 mM Tris-HCI, pH 7.5, 25 mM KCI, 5 mM MgCls) and sonicated in an ice-bath using an MSE 150 probe sonicator at peak-to-peak amplitude of 5-7 pm, for 3 bursts of 15 s with 15 s cooling intervals. The sonicates were assayed for protein [15], for PDI protein by ELISA (see above) and for PDI activity by the standard assay involving reactivation of incorrectly oxidised (scrambled) bovine pancreatic ribonuclease [16,17].…”

Section: Methodsmentioning

confidence: 99%

Induction of expression of protein disulphide‐isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide

et al. 1989

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“…The supernatant was collected after centrifugation at 3300 ϫ g for 30 min. Samples were diluted 1/10 in working buffer and analyzed as described previously (38); mAb 4SD17.3 was used as the capture Ab (20,39). Bound C3a was detected with biotinylated rabbit anti-C3a, diluted 1/150, followed by HRP-conjugated streptavidin (Amersham, Slough, U.K.), diluted 1/500.…”

Section: Enzyme Immunoassay For the Detection Of C3amentioning

confidence: 99%

C3 Adsorbed to a Polymer Surface Can Form an Initiating Alternative Pathway Convertase

Andersson

Ekdahl

Larsson

et al. 2002

The Journal of Immunology

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136

109

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Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.

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A simplified assay for the detection of C3a in human plasma employing a monoclonal antibody raised against denatured C3

Cited by 68 publications

References 24 publications

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Induction of expression of protein disulphide‐isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide

C3 Adsorbed to a Polymer Surface Can Form an Initiating Alternative Pathway Convertase

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