Abstract
Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of Coomassie Blue-stained SDS-PAGE gels indicated that all three HMW CLU-client protein complexes had an approximate mass ratio of 1:2 (CLU:client protein). SEC indicated that all three clients formed complexes with CLU>or=4x10(7) Da; however, DLS estimated HMW CLU-FGN to have a diameter of 108.57+/-18.09 nm, while HMW CLU-CS and HMW CLU-GST were smaller with estimated diameters of 51.06+/-6.87 nm and 52.61+/-7.71 nm, respectively. Measurements of bisANS fluorescence suggest that the chaperone action of CLU involves preventing the exposure to aqueous solvent of hydrophobic regions that are normally exposed by the client protein during heat-induced unfolding. CD analysis indicated that, depending on the individual client protein, CLU may interact with a variety of intermediates on protein unfolding pathways with different amounts of native secondary structure. In vivo, soluble complexes like those studied here are likely to serve as vehicles to dispose of otherwise dangerous aggregation-prone misfolded extracellular proteins.
Highlights
A large number of alternative functions have been proposed for clusterin (CLU),4 the potent chaperone activity of this protein (9 –13) and its constitutive presence in many biological fluids suggests that it is likely to be important in extracellular protein folding quality control
Under the same conditions, when CLU was present with citrate synthase (CS) at a near equimolar concentration, no increases in turbidity were detected, indicating that CS was stabilized in solution
Incubation of FGN alone at 45 °C resulted in progressive precipitation of the protein after an initial lag phase of ϳ200 min, no further increases in turbidity were measured after 800 min (Fig. 1B)
Summary
Materials—4,4Ј-Bis(1-anilino-8-naphthalene sulfonate; bisANS), bovine serum albumin (BSA), CS, and FGN were all obtained from Sigma-Aldrich. The proteins were redissolved in an appropriate volume of filtered (0.45 m) PBS and the absorbance at 280 nm of several aliquots measured using a SpectraMax Plus 384 microplate reader (Molecular Devices). The average absorbance at 280 nm and the known protein concentration were used to calculate the extinction coefficient using Beer’s law These proteins, as well as SECpurified HMW CLU-CS, HMW CLU-FGN, and HMW CLUGST complexes were reduced by boiling in SDS-PAGE loading buffer containing 100 mM dithiothreitol and 1% (v/v) -mercaptoethanol and separated on a 12% SDS gel. The average optical density/mm of the major bands was used to construct a standard curve for each protein Using these standard curves, the relative amounts of CLU and FGN, CS or GST present in the SEC-purified HMW complexes were calculated.
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