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Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.

The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg·mL-1 ) is reduced by coincubation with clusterin (10 µg·mL-1 ). In contrast, addition of clusterin (0.1 mg·mL-1 ) to preformed apoC-II amyloid fibrils (0.3 mg·mL-1 ) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid. [ABSTRACT FROM AUTHOR]

Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of α-lactalbumin, γ-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of α-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced α-lactalbumin. Reduction-induced changes in the conformation of α-lactalbumin, in the absence and presence of clusterin, were monitored by real-time 1H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of α-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of α-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and γ-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein. [Copyright &y& Elsevier]

Examines the role of clusterin in the stabilization of stressed proteins in a state competent for refolding using heat shock protein 70. Correlation between clusterin expression and types of diseases; Inhibition of stress-induced precipitation of human serum protein; Effect of ATP on clusterin ability to inhibit stress-induced protein precipitation.

Provides information on clusterin, a secreted glycoprotein. Details on the structure and distribution of clusterin; Ideas about the action of clusterin; Information on the sites of action of clusterin.

Haptoglobin (Hp) is an acidic glycoprotein present in most body fluids of humans and other mammals. Although the functions of Hp are not yet fully understood, the available evidence indicates that it is likely to play an important role in suppressing inflammatory responses. Some earlier work suggested that Hp might be a newly identified member of a small group of extracellular chaperones found at significant levels in human body fluids. Previously, the only well-characterized member of this group was clusterin, which shares functional similarities with the small heat-shock proteins. We report here that Hp specifically inhibited the precipitation of a variety of proteins induced by either heat or oxidation, including proteins in unfractionated human serum. We also show that, like clusterin, Hp (i) inhibits the precipitation of stressed proteins by forming solubilized high molecular weight complexes with them, (ii) cannot protect enzymes from heat-induced loss of function, and (iii) lacks ATPase activity and the ability to independently refold proteins following stresses. Furthermore, we show that Hp has maximum chaperone activity at mildly alkaline pH and, unlike clusterin, does not undergo significant changes in oligomerization state coincident with pH-induced changes in chaperone activity. Our results raise the possibility that Hp may exert an anti-inflammatory action in vivo by inhibiting the inappropriate self-association of "damaged" (misfolded) extracellular proteins. [ABSTRACT FROM AUTHOR]

Reports on the expression of recombinant human clusterin in the yeast Pichia pastoris. Proteolytic truncations of the resultant protein; Comparison of the chaperone and ligand binding activities of the recombinant product with those of clusterin purified from human serum; Testing of the binding of clusterin to ligands.

Provides information on an experimental study reporting on the prediction that clusterin is a heparin-binding protein. Methodology used to conduct the experiment; Information on the binding of reduced and alkylated clusterin to Heparin-Sepharose; Results of the study; Discussion on the results.