Structural Characterization of Clusterin-Chaperone Client Protein Complexes^*

Materials

4,4′-Bis(1-anilino-8-naphthalene sulfonate; bisANS), bovine serum albumin (BSA), CS, and FGN were all obtained from Sigma-Aldrich. All buffer salts and H₂O₂ were obtained from Ajax Chemical Co. Human blood was obtained as a kind gift from Wollongong Hospital (Wollongong, NSW, Australia) and processed to yield plasma, which was stored frozen at −20 °C until used. CLU was purified from human plasma by immunoaffinity chromatography as previously described (23). GST was expressed in Escherichia coli using the vector pGEX-2T (without an insert; Invitrogen) as previously described (24) and purified using a glutathione-Sepharose^TM High Performance column according to the manufacturer's directions (GE Healthcare).

Precipitation Assays

CS (6.0 μm), FGN (6.0 μm), or GST (20 μm) were incubated at 41, 45, or 60 °C, respectively, in phosphate-buffered saline (PBS, 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH₂PO₄, and 8 mm Na₂HPO₄, pH 7.4) containing 0.1% (w/v) NaN₃ (PBS/Az) in the presence or absence of CLU (6.6, 6.5, and 3.2 μm, respectively) or the control protein, BSA (at the same respective molar concentrations). The solutions were added in triplicate 100-μl aliquots to a 384-well plate (Greiner Bio-one). Protein precipitation was monitored by measuring the absorbance at 360 nm (A_{360 nm}; an indication of turbidity) with a FLUOstar Optima incubator microplate reader (BMG Labtechnologies).

Preparation of Preheated Protein Controls

Preheated control proteins were prepared by incubating client proteins or CLU (alone) at the same temperature and for the same duration used to form the HMW complexes. Preheated controls were filtered (0.45 μm) and the clarified solutions assayed for residual protein content using the bicinchoninic microprotein assay (25), or the respective extinction coefficients at 280 nm.

Size Exclusion Chromatography (SEC)

SEC was carried out using a Superose^TM 6 10/300 column (GE Healthcare) at the recommended flow rate of 0.5 ml/min and the absorbance at 280 nm continuously monitored using an ÄKTA FPLC system (GE Healthcare). Mass standards were from a commercial high molecular weight calibration kit (GE Healthcare). All buffers and samples were filtered (0.45 μm) before use. SEC-purified HMW complexes were collected from fractions between 7–8 ml in the peak corresponding to the size exclusion limit of ≥ 4 × 10⁷ Da. The integrity of the complexes was checked periodically by reanalysis using the same column.

Sandwich ELISA

The wells of an ELISA plate (Greiner Bio-one) were coated with purified G7 anti-CLU antibody (23), then blocked with 1% (w/v) BSA in PBS (BSA/PBS). Mixtures of CLU and client protein were heated as described under “Precipitation Assays,” and fractionated by SEC (as described above). After washing with PBS, proteins eluting at the exclusion limit (≥4 × 10⁷ Da), preheated CLU or client proteins, or a mixture of CLU and individual client proteins were incubated in the wells of the ELISA plate (diluted to 50 μg/ml in BSA/PBS). Then a primary antibody (or antiserum) reactive with the client protein, diluted in BSA/PBS, was added following the manufacturer's instructions. Finally an appropriate horseradish peroxidase-conjugated secondary antibody diluted in BSA/PBS was added following the manufacturer's instructions. All incubations were carried out for 1 h at 37 °C with shaking and washing was performed with PBS. After a final wash, ortho-phenylenediamine at 2.5 mg/ml in 50 mm citric acid, 100 mm Na₂HPO₄, pH 5, was added. The absorbance at 490 nm (A_{490 nm}) was measured using a SpectraMax Plus³⁸⁴ microplate reader (Molecular Devices). Nonspecific binding was assessed using a species-matched polyclonal antibody or serum of irrelevant specificity and the appropriate secondary antibody. Primary antibodies used were sheep polyclonal anti-CS (IgG fraction, Abcam), goat anti-FGN antiserum, and rabbit polyclonal anti-GST (IgG fraction, Chemicon). The respective controls were polyclonal anti-apolipoprotein A1 (IgG fraction, Abcam), normal goat serum (Sigma-Aldrich), and purified normal rabbit IgG fraction (Sigma-Aldrich).

Dynamic Light Scattering (DLS)

Solutions of SEC-purified HMW CLU-CS, HMW CLU-FGN, and HMW CLU-GST complexes, CLU, FGN, CS, or GST (controls were preheated or untreated), or mixtures of these proteins, were made between 0.1 and 1.0 mg/ml in PBS and filtered (0.45 μm). Triplicate samples were examined in low volume plastic cuvettes using a Zetasizer Nano ZS (Malvern). Particle diameters were recorded as a frequency distribution curve, and the average diameter and range (average peak and width of 9 normally distributed curves) reported.

Densitometry

Several milligrams of CS, FGN, GST, or CLU were extensively dialyzed against distilled water and then freeze-dried. The recovered protein was weighed using a XS205 Dual Range analytical balance (Mettler Toledo). The proteins were redissolved in an appropriate volume of filtered (0.45 μm) 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 SEC-purified HMW CLU-CS, HMW CLU-FGN, and HMW CLU-GST complexes were reduced by boiling in SDS-PAGE loading buffer containing 100 mm dithiothreitol and 1% (v/v) β-mercaptoethanol and separated on a 12% SDS gel. Six sample wells containing unknown amounts of reduced HMW CLU-CS, HMW CLU-FGN, or HMW CLU-GST complexes, and triplicate wells containing 0.5–6 μg of reduced CS, FGN, GST, or CLU were also loaded onto the gel. Following Coomassie Blue staining and destaining, the major bands corresponding to reduced CS, FGN, GST, and CLU were analyzed using a GS 800 calibrated densitometer (Bio-Rad) and Quantity One software (Bio-Rad). The average optical density/mm² 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.

4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic Acid (bisANS)

For bisANS analyses, CLU client proteins (0.5 mg/ml CS, 2 mg/ml FGN, or 0.5 mg/ml GST), or mixtures of client proteins (at the same concentrations) and CLU (at 0.4 mg/ml for experiments using CS or FGN, or at 0.2 mg/ml for experiments using GST) were incubated under the same conditions used to form HMW CLU-client protein complexes (see above). At specified time points, samples were taken from the solutions and snap-frozen in liquid nitrogen. Following completion of the time courses, all samples were thawed and immediately diluted in PBS containing bisANS to give final concentrations of 50 μg/ml client protein and 10 μm bisANS. Samples containing CLU alone were diluted to give equivalent concentrations to that present in the (CLU + client protein) samples (i.e. 15 μg/ml or 30 μg/ml). Fluorescence was measured on a FLUOstar Optima fluorescence plate reader using excitation and emission windows of 360 ± 10 and 490 ± 10 nm, respectively.

Circular Dichroism (CD)

For CD analyses, samples were analyzed as previously described (26); all samples were in 10 mm Na₂HPO₄, pH 7.4. Individual proteins were analyzed before and after heating (the latter were residual soluble protein) at the following concentrations: CLU at 164 μg/ml, CS at 119 μg/ml, FGN at 164 μg/ml, and GST at 110 μg/ml. In separate experiments, spectra were acquired for HMW complexes of CLU-CS (119 μg/ml), CLU-FGN (164 μg/ml), CLU-GST (110 μg/ml), and solutions of CLU or the individual client proteins at concentrations corresponding to those present in the complexes analyzed (calculated on the basis of the mass ratios of CLU:client protein in the complexes). Estimates of secondary structure were obtained using the program CDSSTR (27).

Thioflavin T Analyses

For thioflavin T fluorescence assays, all samples were prepared at 50 μg/ml in PBS and contained 62.5 μm thioflavin T; fluorescence was measured using a FLUOstar Optima microplate reader (BMG Labtechnologies), with an excitation wavelength of 440 nm and an emission wavelength of 490 nm (slit-width 10 nm). Lysozyme amyloid was formed as described in Ref. 28.

In Vitro Formation of CLU-Client Protein Complexes

When incubated alone at 41 °C, 6 μm CS showed a progressive increase in turbidity (increasing A_{360 nm}; Fig. 1A) from 0 to about 500 min. No further increase in turbidity was observed after ∼500 min. Under the same conditions, when CLU was present with CS at a near equimolar concentration, no increases in turbidity were detected, indicating that CS was stabilized in solution. At the same concentration, BSA had little effect on the precipitation of CS. Incubation of FGN alone at 45 °C resulted in progressive precipitation of the protein after an initial lag phase of ∼200 min, no further increases in turbidity were measured after 800 min (Fig. 1B). Under the same conditions, co-incubation of 6.5 μm CLU with 6.0 μm FGN completely abolished any increase in turbidity; in contrast, 6.5 μm BSA had little effect on the precipitation of FGN. Incubation of 20 μm GST at 60 °C resulted in rapid precipitation of the protein after a lag phase of ∼25 min (Fig. 1C). After 40 min, the protein solution reached maximum turbidity and prolonged heating had no further effect. Co-incubation of 20 μm GST with 3.2 μm CLU almost completely inhibited the precipitation of GST under the same conditions. This was in contrast to co-incubation with 3.2 μm BSA, where the protein solution exhibited a similar precipitation profile to that observed when GST was incubated alone. However, the maximum turbidity was marginally less in the presence of BSA. Both CLU and BSA were stable when incubated alone at 41–60 °C (data only shown for 60 °C; Fig. 1C). As first reported by Humphreys et al. (9) the effect of CLU on protein precipitation was dose-dependent for all client proteins (data not shown).

Production of Preheated Protein Controls

To produce preheated protein controls, solutions of individual proteins were heated as described above and insoluble protein removed by filtration through a 0.45-μm filter. There was no difference in the SEC profiles of unheated CS, FGN, GST, or CLU and the residual corresponding proteins remaining in solution after the respective heat treatments (Fig. 2). Also, when the residual, soluble heat-treated proteins were mixed, very little interaction was detected by SEC between CLU and CS (Fig. 2A), CLU and FGN (Fig. 2B), or CLU and GST (Fig. 2C).

Detection of HMW Complexes by SEC

SEC fractionation of the heat-stressed mixtures of CLU and CS (41 °C), CLU and FGN (45 °C), or CLU and GST (60 °C) showed that they contained HMW species eluting at the exclusion limit of the column (≥4 × 10⁷ Da) that were absent from the same mixtures left unheated, and from solutions of the individual proteins (Fig. 2). Compared with FGN and GST, co-incubation of CS with CLU at 41 °C produced proportionally less HMW species; however, it was evident that complexes comparable in mass to those formed at higher temperatures by CLU and FGN or GST were also formed by CLU and CS. The exclusion limit peak was collected in each case and represented putative HMW CLU-client protein complexes. The identity of these complexes was confirmed by sandwich ELISA (see below). These complexes were stored in PBS/Az at 4 °C, and their integrity checked at intervals by SEC; under these conditions they remained stable for months.

Sandwich ELISA to Confirm the Identity of CLU-Client Protein Complexes

Sandwich ELISA designed to capture CLU and subsequently detect CS, FGN, or GST was used to confirm the identity of the putative complexes purified by SEC. Relative to the samples containing the HMW complexes, little absorbance was obtained for control samples (Fig. 3).

Estimates of Stoichiometry within Complexes

To estimate the stoichiometry of individual proteins within the HMW complexes, SEC-purified complexes were separated by SDS-PAGE under reducing conditions, the gels stained with Coomassie Blue, and the intensity of individual protein bands quantified by densitometry. After standard curves were generated for each protein, the approximate mass ratios and molar ratios for each complex were calculated. The mass ratio of CLU to client protein was similar for CS, FGN, and GST complexes (about 1:2 in each case). However, the molar ratios were very different; HMW CLU-CS complexes contained approximately the same number of molecules of CS and CLU, HMW CLU-FGN complexes contained about 3 molecules of CLU for every FGN molecule, while CLU-GST complexes contained 5 molecules of GST for each molecule of CLU (Table 1). These estimated CLU:client protein ratios were employed when selecting controls for structural studies of the HMW complexes.

Size Estimation by Dynamic Light Scattering

The results of DLS were consistent between 0.1 and 1 mg/ml; representative results obtained at 0.5 mg/ml are shown (Fig. 4). In contrast to SEC, DLS was unable to resolve the differently sized oligomers of CLU in solution; DLS analysis of CLU samples indicated a normally distributed particle size. There was no apparent difference in size between native and preheated control proteins. The limitations of DLS in determining the respective diameters of particles of similar size, resulted in a peak corresponding to an intermediate size (compared with the individual proteins) when mixtures of residual preheated CLU and CS FGN or GST were analyzed. DLS indicated that SEC purified HMW CLU-CS and HMW CLU-GST were approximately six times larger than either of their respective components, while HMW CLU-FGN was more than 8 times larger than CLU or FGN (Fig. 4).

BisANS and Thioflavin T Fluorescence Assays

The bisANS fluorescence of individual soluble preheated client proteins showed time-dependent changes. For CS, there was a gradual increase in fluorescence peaking at 6 h, before declining at 8 h (Fig. 5A). FGN showed transient peak fluorescence at 1–2 h before returning to the level of the unheated control (Fig. 5B). For GST, the fluorescence was increased over the period 1–3 h before declining at 4 h (Fig. 5C). In contrast, the bisANS fluorescence of solutions of CLU heated alone only displayed a small increase at 1 h and 6 h after heating at 45 °C (Fig. 5); CLU did not precipitate under any of these conditions (data not shown). In all cases, at times when preheated soluble client protein showed increased bisANS fluorescence, the corresponding fluorescence of the respective preheated mixtures of CLU and client protein showed a lesser change. This suggests that the interaction between CLU and partially unfolded client proteins in theses mixed solutions reduces the extent to which hydrophobic regions on the client proteins are exposed to solvent.

Thioflavin T analyses were carried out to determine if SEC-purified complexes possessed any amyloid-like characteristics (i.e. contained β-sheet rich structures). SEC-purified HMW CLU-FGN and CLU-GST complexes and the relevant native and preheated protein controls all produced less than 8% of the fluorescence arising from a sample of lysozyme amyloid present at the same mass concentration (data not shown).

Circular Dichroism Spectrophotometry

The CD spectrum for unheated CLU indicated high α-helical content with minima at ∼208 nm and ∼222 nm. The molar ellipticity at these wavelengths slightly increased when CLU was preheated at 43 or 60 °C. Under these conditions, CDSSTR analysis predicted a large decrease in α-helical content, a similarly large increase in predicted β-sheet content and smaller increases in the predicted contents of β-turn and unordered structure (Fig. 6A and Table 2). However, heating at 41 °C only produced small increases in the predicted content of β-sheet and β-turn structure (Fig. 6A and Table 2). Preheated CS had a CD spectrum superimposable on that of unheated CS (Fig. 6B). In contrast, following heating at 45 °C, FGN showed a change in minima from ∼222 nm to ∼200 nm, consistent with the following predicted changes: a small decrease in α-helical content, a moderate decrease in β-sheet, and small increases in β-turn and unordered structure content (Fig. 6C and Table 2). After heating at 60 °C, GST also showed a change in minima from that typical of high α-helical to a more disordered structure. CDSSTR predicted a substantial decrease in α-helical content and much smaller increases in β-sheet, β-turn, and unordered structure content (Fig. 6D and Table 2). Comparing the CD spectra of SEC-purified HMW CLU-CS complexes with those of mixtures of the native or soluble preheated client proteins suggested that secondary structure was significantly altered in HMW CLU-CS complexes (Fig. 6E). This was largely attributed to a predicted loss of more than half the native α-helical content, compared with the unheated proteins (Table 2). Analysis of the spectrum of a mixture of soluble preheated CS and preheated CLU predicted that, relative to the CLU-CS complex, the proteins had a smaller decrease in α-helical content and an increase in unordered structure. In contrast the CD spectrum for HMW CLU-FGN complexes was virtually superimposable on that of a mixture of native CLU and FGN (Fig. 6F and Table 2). However, a corresponding mixture of preheated CLU and preheated FGN had a very different CD spectrum, consistent with the loss of predicted structure for the individual proteins following heating. It was evident that the formation of HMW CLU-GST complexes was accompanied by changes in secondary structure (Fig. 6G). For these complexes, relative to a mixture of the native proteins, there were predicted significant losses of α-helical content, a large increase in β-sheet content, and small increases in β-turn and unordered structure (Table 2). The CD spectrum and predicted losses of secondary structure were similar for HMW CLU-GST complexes and a mixture of soluble preheated GST and CLU.