Abstract
DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.
Highlights
Subunit, two in the active site -Cys98-Gly-Tyr-Cys101- and the other two at positions Cys141 and Cys163 [4, 5]
Effects of DsbC on the Reactivation and Aggregation of GdnHCl-denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) upon Dilution—Fig. 2A shows that the reactivation of GAPDH at 2.8 M in the presence of DsbC increases with increasing molar ratios of DsbC/GAPDH from a low spontaneous yield of from 6 to 32% at the ratio of 20, indicating that DsbC is more effective in assisting GAPDH refolding than protein disulfide isomerase (PDI), which at the ratios of 5–10 increases GAPDH reactivation to a maximal level of about 20% [11]
DsbA and DsbC are the two soluble members of Dsb family directly involved in the formation of disulfide bonds
Summary
Vol 274, No 28, Issue of July 9, pp. 19601–19605, 1999 Printed in U.S.A. (Received for publication, September 1, 1998, and in revised form, April 12, 1999). The formation of disulfide bonds is known to be catalyzed by protein disulfide isomerase (PDI) in endoplasmic reticulum for eukaryotes [1] and by proteins of the Dsb family in periplasm for Gram-negative bacteria [2]. DsbC catalyzes the rearrangement of disulfide bonds in concert with DsbA for the formation of native disulfides and has been recognized as a counterpart of eukaryotic PDI [3, 6, 7]. The chaperone activity of PDI greatly increases its efficiency as a foldase in promoting protein folding and in catalyzing the formation of native disulfide bonds [19]. We are interested in examining whether DsbC, like its eukaryotic counterpart PDI, has chaperone activity to promote the formation of correct disulfide bonds during protein folding. We propose to show an even stronger chaperone activity of DsbC than that of PDI for protein folding in vitro with possibly different mechanisms for target protein binding
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