Linear polymerization caused by the defective folding of a non-inhibitory serpin ovalbumin.

Polymerization caused by defective folding of heat-denatured ovalbumin was examined. A compactly misfolded ovalbumin that was produced by cooling heat-denatured protein rapidly tended to aggregate in the presence of salt. Two different forms of aggregates were observed as the concentration of salt was varied: a linear polymer at a physiological concentration and a massive agglomerate at a higher concentration. Salt-induced polymerization depended on the species of anion and the order of effectiveness followed the lyotropic series of Hofmeister. Defective folding of heat-denatured ovalbumin induced the exposure of cysteine residues in sequences located in the interior of the native protein. The misfolded ovalbumin, but not the native protein, bound to bovine BiP and stimulated its ATPase activity with the K(m) of 64 microM and the V(max) of 0.5 nmol/min per milligram. Measurement of surface plasmon resonance revealed that only the misfolded ovalbumin was recognized with the K(d) of 4.12 X 10(-8) M by the Fab fragment of a monoclonal antibody raised against hen ovalbumin, and its epitope was determined to be a hydrophobic segment in the beta-strand of central sheet A. Transmission electron microscopy showed that the linear polymerization was inhibited by the addition of bovine BiP and the Fab fragment. These results demonstrated that the compactly misfolded ovalbumin polymerized through hydrophobic interaction occurring among the areas exposed as a result of defective folding of the heat-denatured protein. Exposure of the region of, or adjacent to, the central beta-sheet A was required for axial contact among the misfolded molecules, suggesting that this process may be explained by reference to the mechanism proposed for loop-sheet polymerization in the Z type variant of a serpin alpha1-antitrypsin.