Year

2002

Degree Name

Doctor of Philosophy

Department

Department of Biological Sciences

Abstract

Clusterin is a widely distributed and highly conserved secreted mammalian glycoprotein whose elevated expression has been detected in a number of disease states (e.g. Scrapie, Alzheimer's and Creutzfeldt-Jakob diseases) that are associated with abnormally high levels of misfolded and/or precipitated proteins. Clusterin, present at concentrations ranging from 35-105 ug/ml in human serum, is known to interact with a variety of proteins and lipids in vitro. These numerous binding interactions have led to a number of biological functions being proposed for clusterin; these include roles in reproduction, lipid transport, endocrine secretion, apoptosis, and complement regulation. In a recent study, it was reported that clusterin has molecular chaperone activity in vitro. Molecular chaperones, as defined by John Ellis, are proteins that function in vivo to specifically interact with and stabilise unfolded or partially unfolded proteins, thereby preventing them from potentially aggregating and precipitating, e.g. during protein folding.

In this study, clusterin was shown to prevent the precipitation of heat-stressed ovotransferrin and γ-crystallin, as well as DTT-reduced ovotransferrin and lysozyme. Analysis by size exclusion chromatography of samples in which clusterin was co-incubated with ovotransferrin or lysozyme undergoing stress-induced denaturation revealed the presence of HMW species in the void volume that was eluted from the column. Subsequent analysis by SDS-PAGE of these HMW species, confirmed the presence of both clusterin and the stressed target protein. Results presented in this report also demonstrate that clusterin protects proteins in (i) diluted human serum from heat-induced precipitation and (ii) undiluted human serum from DTT-mediated precipitation. Other results presented in this thesis indicate that clusterin does not have the ability to hydrolyse ATP and hence, performs its chaperone function in an ATP-independent manner. In addition, clusterin was unable to independently facilitate the reactivation of heat-inactivated ADH and catalase after removal of stress. However, in the presence of a chaperone with refolding capability (i.e. Hsc70) and ATP, clusterin-stabilised ADH and catalase were partially refolded. Taken together, these results raise the possibility that clusterin may inhibit precipitation of human serum proteins in vivo and create a reservoir of inactive protein structures from which folding-competent proteins can be subsequently reactivated by other refolding chaperones.

Clusterin was shown to inhibit the slow precipitation of γ-crystallin and lysozyme, but was unable to prevent these same target proteins from rapid precipitation. Real-time 1H NMR spectroscopic analysis of the interaction between clusterin and a-lactalbumin reveal that clusterin did not alter the rate of a-lactalbumin reduction but did stabilise the less ordered intermediately folded form of the protein. These results suggest that (i) kinetic factors are important in the chaperone action of clusterin and (ii) clusterin binds specifically to slowly aggregating proteins on the irreversible off-folding pathway.

This thesis also presents results to show that elevated temperature (up to 50 °C) does not induce significant changes in the oligomerisation state of clusterin nor does it substantially alter the ability of clusterin to interact with heat-stressed or chemically reduced target proteins. In contrast, incubation at mildly acidic conditions resulted in the dissociation of clusterin oligomers, which led to an increased exposure of hydrophobic regions on clusterin to solution and a concomitant enhancement of its chaperone action. A phenomenon known as acidosis occurs at sites of tissue damage or inflammation where the local pH can drop below 6. Acidosis has been reported to occur at sites of inflammation as well as in many of the diseases to which clusterin has been associated. Taken together, these results suggest that under these conditions, the dissociation and enhanced chaperone actions of clusterin could help to inhibit the aggregation and deposition of inflammatory and/or toxic insoluble protein deposits which would otherwise exacerbate pathology.

At present, the structural regions responsible for the ligand binding and chaperone action of clusterin have not been identified. Sequence analysis of clusterin has revealed several regions that could be functionally important, including three regions of amphipathic a-helices and two coiled-coil domains. In addition, studies have shown that, despite having variable truncations at the C-terminus of the α-chain and the N-terminus of the β-chain, clusterin expressed by yeast Pichia pastoris has similar chaperone activity to human clusterin in vitro, indicating that the sites responsible for the chaperone action of clusterin more likely to be located more towards the N-terminal region of the a-chain and the C-terminal region of the β-chain. In order to identify the functional sites of clusterin and to the above statements, five proline-substitution and five truncation clusterin mutants, as well as wild-type clusterin, were developed. These mutants, including wild type clusterin, were expressed in transiently transfected Spodoptera frugiperda (Sf9) insect cells. Sf9 cells were chosen as the host for the expression of wild type and mutant clusterin due to their reported ability to express high levels of recombinant proteins and perform post-translational modifications in a manner that is similar to mammalian cells. However, Western blot analysis showed that the expressed proteins were not expressed at high levels and had molecular masses that were approximately 15 kDa smaller than their expected sizes. Since the Sf9 transfectants were not cloned, the Sf9 cell cultures may have contained a large number of non-transformants/non-secretors that may have outnumbered secreting transformants and hence, explain why the yield was so low. The size irregularity can be explained by incomplete glycosylation of the expressed proteins or by truncations caused by protease actions. Whatever the case may be, these results clearly indicate that Sf9 insect cells are unsuitable for producing properly processed wild-type or mutant clusterin. Therefore, to produce properly processed wild-type and mutated clusterin for the study of clusterin's structure-function relationship, a different expression system will be required.

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