pH-dependent changes in the in vitro ligand-binding properties and structure of human clusterin

Clusterin is a glycoprotein which is locally overexpressed at sites of tiss ue damage or stress, leading to the proposal that it may be a cytoprotectiv e protein. It has been shown that clusterin has chaperone-like activity, be ing able to protect proteins against precipitation under stress conditions. It has also been shown that local acidosis is common at sites of tissue da mage or stress. We asked whether acidic pH induces structural changes in cl usterin and enhances its ability to bind to other proteins. We found by aff inity chromatography and ELISA that the binding of clusterin to glutathione -S-transferase, IgG, apolipoprotein A-I, and complement protein C9 was enha nced at mildly acidic compared to physiological pH. Analytical ultracentrif ugation and gel filtration studies revealed that clusterin exists in differ ent polymerization states with monomer occurring preferentially at pH 5.5 a nd multimeric species at pH 7.5. Although circular dichroism showed little difference in the alpha-helical and beta-sheet contents of clusterin at pH 5 compared to pH 7.5, evidence for pi-I-dependent structural changes in clu sterin was obtained from fluorescence experiments, pH titrations showed rev ersible changes in the fluorescence of tryptophan residues in clusterin. Th ere was a reversible 2-fold increase in the fluorescence of the extrinsic p robe 4,4'-bis(1-anilinonaphthalene-8-sulfonate) bound to clusterin at pH 5. 5 compared to pH 7.5. There was also a 3.5-fold increase in fluorescence re sonance energy transfer from tryptophan residues in clusterin to 4,4'-bis(1 -anilinonaphthalene-8-sulfonate) at pH 5.5 compared to pH 7.5. These data s uggest that pH-induced changes in the structure of clusterin are responsibl e for its enhanced ability to bind protein ligands at mildly acidic pH.