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

Tim Hochgrebe, Greg J. Pankhurst, Jackie Wilce, Simon B. Easterbrook-Smith

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Clusterin is a glycoprotein which is locally overexpressed at sites of tissue damage or stress, leading to the proposal that it may be a cytoprotective protein. It has been shown that clusterin has chaperone-like activity, being able to protect proteins against precipitation under stress conditions. It has also been shown that local acidosis is common at sites of tissue damage or stress. We asked whether acidic pH induces structural changes in clusterin and enhances its ability to bind to other proteins. We found by affinity chromatography and ELISA that the binding of clusterin to glutathione-S-transferase, IgG, apolipoprotein A-I, and complement protein C9 was enhanced at mildly acidic compared to physiological pH. Analytical ultracentrifugation and gel filtration studies revealed that clusterin exists in different polymerization states with monomer occurring preferentially at pH 5.5 and multimeric species at pH 7.5. Although circular dichroism showed little difference in the α-helical and β-sheet contents of clusterin at pH 5 compared to pH 7.5, evidence for pH-dependent structural changes in clusterin was obtained from fluorescence experiments. pH titrations showed reversible changes in the fluorescence of tryptophan residues in clusterin. There was a reversible 2-fold increase in the fluorescence of the extrinsic probe 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 resonance 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 suggest that pH-induced changes in the structure of clusterin are responsible for its enhanced ability to bind protein ligands at mildly acidic pH.

Original languageEnglish
Pages (from-to)1411-1419
Number of pages9
Issue number6
Publication statusPublished - 15 Feb 2000
Externally publishedYes

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