Mechanism of the antichaperone activity of protein disulfide isomerase: Facilitated assembly of large, insoluble aggregates of denatured lysozyme and PDI

Protein disulfide isomerase (PDI), a folding catalyst and chaperone can, under certain conditions, facilitate the misfolding and aggregation of its substrates. This behavior, termed antichaperone activity [Puig, A., and Gilbert, H. F., (1994) J, Biol. Chem. 269, 25889] may provide a common mechanism for aggregate formation in the cell, both as a normal consequence of cell function or as a consequence of disease. When diluted from the denaturant, reduced, denatured lysozyme (10-50 mu M) remains soluble, although it does aggregate to form an ensemble of species with an average sedimentation coefficient of 23 +/- 5 S (similar to 600 +/- 100 kDa). When low concentrations of PDI (1-5 mu M) are present, the majority (80 +/- 8%) of lysozyme molecules precipitate in large, insoluble aggregates, together with 87 +/- 12% of the PDI. PDI-facilitated aggregation occurs even when disulfide formation is precluded by the presence of dithiothreitol (10 mM). Maximal lysozyme-PDI precipitation occurs at a constant lysozyme/PDI ratio of 10:1 over a range of lysozyme concentrations (10-50 mu M). Concomitant resolubilization of PDI and lysozyme from these aggregates by increasing concentrations of urea suggests that PDI is an integral component of the mixed aggregate, PDI induces lysozyme aggregation by noncovalently cross-linking 23 S lysozyme species to form aggregates that become so large (similar to 38 000 S) that they are cleared from the analytical ultracentrifuge even at low speed (1500 rpm). The rate of insoluble aggregate formation increases with increasing PDI concentration (although a threshold PDI concentration is observed). However, increasing lysozyme concentration slows the rate of aggregation, presumably by depleting PDI from solution. A simple mechanism is proposed that accounts for these unusual aggregation kinetics as well as the switch between antichaperone and chaperone behavior observed at higher concentrations of PDI.