Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2

Abstract

The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S‐methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.

Catalysis of Creatine Kinase Refolding by Protein Disulfide Isomerase Involves Disulfide Cross-link and Dimer to Tetramer Switch

“…These results suggested that the formation of disulfide bonds between PDI and CK contributed to the suppression of CK aggregation during refolding but was not the major driving force. DISCUSSION PDI has been demonstrated to act as a molecular chaperone independent of its isomerase activity (21)(22)(23)(24)(25), and it can exploit the synergy between these functions, especially when disulfide reshuffling is needed in the refolding of the substrates (3,26). Our results showed that WT PDI at high concentrations could act as a protector against aggregation but acted as an inhibitor of reactivation during CK refolding.…”

DsbA-L is a versatile player in adiponectin secretion

“…PDI contains 2 CXXC (C, cysteine; X, any amino acid) active sites necessary for disulfide bond formation and isomerization. However, these sites are not essential for the molecular chaperone activity when PDI assists the refolding of client proteins that do not have a need for disulfide bond formation and isomerization (13). Based on its structural similarities, DsbA-L is categorized as a member of the PDI family.…”

The protein disulphide-isomerase family: unravelling a string of folds

“…For PDI, the extent to which the chaperone and redox\isomerase activities are apparent in the folding of proteins is substrate-dependent, as is the degree of involvement of individual a or ah domains in redox\isomerase activity [69,79,[82][83][84]. Whereas chaperone activity has been shown for disulphide-containing proteins, such as lysozyme [85,86], and independence of the isomerase and chaperone activities has been shown in itro in the refolding of acidic phospholipase A # , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rhodanese [87][88][89], chaperone activity is lacking and does not seem to be required for folding of substrates such as antibody Fab fragments with intact disulphide bonds [90].…”