Structural Characterization of Clusterin-Chaperone Client Protein Complexes

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 ≥ 4 × 107 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. 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 ≥ 4 × 107 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. Controlled unfolding is important in many biological processes including protein translocation, degradation by proteases, and regulation of enzyme activity. Uncontrolled unfolding and the consequent accumulation of insoluble protein aggregates is implicated in the pathology of many diseases including Alzheimer disease and type II diabetes and is promoted by various stresses such as oxidative stress (1Davies K.J. Delsignore M.E. J. Biol. Chem. 1987; 262: 9908-9913Abstract Full Text PDF PubMed Google Scholar), shear stress (2Ker Y.C. Chen R.H. Lebenson Wiss. Technol. 1998; 31: 107-113Crossref Scopus (14) Google Scholar), and thermal stress (3Day R. Bennion B.J. Ham S. Daggett V. J. Mol. Biol. 2002; 322: 189-203Crossref PubMed Scopus (304) Google Scholar). Cells have extensive quality control mechanisms to ensure that intracellular proteins are maintained predominantly in their native conformations. Molecular chaperones are known to play a central role in these systems by targeting unfolded proteins for refolding or degradation (4Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2792) Google Scholar, 5Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3121) Google Scholar, 6Höhfeld J. Cyr D.M. Patterson C. EMBO Rep. 2001; 2: 885-890Crossref PubMed Scopus (288) Google Scholar, 7Muchowski P.J. Neuron. 2002; 35: 9-12Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). However, little is known about the existence of corresponding systems for protein folding quality control in the extracellular environment (8Yerbury J.J. Stewart E.M. Wyatt A.R. Wilson M.R. EMBO Rep. 2005; 6: 1131-1136Crossref PubMed Scopus (97) Google Scholar). A large number of alternative functions have been proposed for clusterin (CLU), 4The abbreviations used are: CLUclusterinHMWhigh molecular weightCScitrate synthaseFGNfibrinogenGSTglutathione S-transferasesHSPsmall heat shock proteinHspheat shock proteinLDLlow density lipoproteinIgGγimmunoglobulin γSECsize exclusion chromatographyG7anti-human clusterin monoclonal antibodyFPLCfast protein liquid chromatographyBSAbovine serum albuminELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salinebisANS4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acidAFUarbitrary fluorescence unitsDLSdynamic light scattering. nevertheless, the potent chaperone activity of this protein (9Humphreys D.T. Carver J.A. Easterbrook-Smith S.B. Wilson M.R. J. Biol. Chem. 1999; 274: 6875-6881Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 10Poon S. Rybchyn M.S. Easterbrook-Smith S.B. Carver J.A. Pankhurst G.J. Wilson M.R. J. Biol. Chem. 2002; 277: 39532-39540Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Poon S. Easterbrook-Smith S.B. Rybchyn M.S. Carver J.A. Wilson M.R. Biochemistry. 2000; 39: 15953-15960Crossref PubMed Scopus (206) Google Scholar, 12Poon S. Treweek T.M. Wilson M.R. Easterbrook-Smith S.B. Carver J.A. FEBS Lett. 2002; 513: 259-266Crossref PubMed Scopus (110) Google Scholar, 13Lakins J.N. Poon S. Easterbrook-Smith S.B. Carver J.A. Tenniswood M.P. Wilson M.R. Biochemistry. 2002; 41: 282-291Crossref PubMed Scopus (52) Google Scholar) and its constitutive presence in many biological fluids suggests that it is likely to be important in extracellular protein folding quality control. Recently haptoglobin (14Yerbury J.J. Rybchyn M.S. Easterbrook-Smith S.B. Henriques C. Wilson M.R. Biochemistry. 2005; 44: 10914-10925Crossref PubMed Scopus (85) Google Scholar) and α2-macroglobulin (15Yerbury J.J. Kumita J.R. Meehan S. Dobson C.M. Wilson M.R. J. Biol. Chem. 2009; 284: 4246-4254Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16French K. Yerbury J.J. Wilson M.R. Biochemistry. 2008; 47: 1176-1185Crossref PubMed Scopus (79) Google Scholar) have also been identified as extracellular chaperones. All three proteins exhibit small heat shock protein (sHsp)-like activity, preferentially binding to stressed client proteins to prevent their precipitation in an ATP-independent manner (9Humphreys D.T. Carver J.A. Easterbrook-Smith S.B. Wilson M.R. J. Biol. Chem. 1999; 274: 6875-6881Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 11Poon S. Easterbrook-Smith S.B. Rybchyn M.S. Carver J.A. Wilson M.R. Biochemistry. 2000; 39: 15953-15960Crossref PubMed Scopus (206) Google Scholar, 14Yerbury J.J. Rybchyn M.S. Easterbrook-Smith S.B. Henriques C. Wilson M.R. Biochemistry. 2005; 44: 10914-10925Crossref PubMed Scopus (85) Google Scholar, 16French K. Yerbury J.J. Wilson M.R. Biochemistry. 2008; 47: 1176-1185Crossref PubMed Scopus (79) Google Scholar). When acting alone, extracellular chaperones lack refolding activity; however it has been shown that CLU can hold partially unfolded proteins in a state competent for refolding by Hsc70 (11Poon S. Easterbrook-Smith S.B. Rybchyn M.S. Carver J.A. Wilson M.R. Biochemistry. 2000; 39: 15953-15960Crossref PubMed Scopus (206) Google Scholar). clusterin high molecular weight citrate synthase fibrinogen glutathione S-transferase small heat shock protein heat shock protein low density lipoprotein immunoglobulin γ size exclusion chromatography anti-human clusterin monoclonal antibody fast protein liquid chromatography bovine serum albumin enzyme-linked immunosorbent assay phosphate-buffered saline 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid arbitrary fluorescence units dynamic light scattering. CLU is found associated with extracellular protein deposits in numerous diseases including drusen in age-related macular degeneration (17Crabb J.W. Miyagi M. Gu X. Shadrach K. West K.A. Sakaguchi H. Kamei M. Hasan A. Yan L. Rayborn M.E. Salomon R.G. Hollyfield J.G. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 14682-14687Crossref PubMed Scopus (994) Google Scholar), renal immunoglobulin deposits in kidney disease (18French L.E. Tschopp J. Schifferli J.A. Clin. Exp. Immunol. 1992; 88: 389-393Crossref PubMed Scopus (43) Google Scholar), Lewy bodies in Parkinson disease (19Sasaki K. Doh-ura K. Wakisaka Y. Iwaki T. Acta Neuropathol. 2002; 104: 225-230Crossref PubMed Scopus (88) Google Scholar), prion deposits in Creutzfeldt-Jakob disease (20Freixes M. Puig B. Rodríguez A. Torrejón-Escribano B. Blanco R. Ferrer I. Acta Neuropathol. 2004; 108: 295-301Crossref PubMed Scopus (44) Google Scholar), and amyloid plaques in Alzheimer disease (21Calero M. Rostagno A. Matsubara E. Zlokovic B. Frangione B. Ghiso J. Microsc. Res. Tech. 2000; 50: 305-315Crossref PubMed Scopus (210) Google Scholar). Knock-out studies have shown that CLU-deficient mice accumulate insoluble protein deposits in the kidneys and develop progressive glomerulopathy (22Rosenberg M.E. Girton R. Finkel D. Chmielewski D. Barrie 3rd, A. Witte D.P. Zhu G. Bissler J.J. Harmony J.A. Aronow B.J. Mol. Cell Biol. 2002; 22: 1893-1902Crossref PubMed Scopus (89) Google Scholar). These findings suggest a role for CLU in the clearance of extracellular misfolded proteins; however, the mechanism by which this may occur has yet to be determined. Currently, little is known about the physical characteristics of the soluble complexes formed during the interaction of CLU with chaperone client proteins (9Humphreys D.T. Carver J.A. Easterbrook-Smith S.B. Wilson M.R. J. Biol. Chem. 1999; 274: 6875-6881Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 10Poon S. Rybchyn M.S. Easterbrook-Smith S.B. Carver J.A. Pankhurst G.J. Wilson M.R. J. Biol. Chem. 2002; 277: 39532-39540Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Poon S. Easterbrook-Smith S.B. Rybchyn M.S. Carver J.A. Wilson M.R. Biochemistry. 2000; 39: 15953-15960Crossref PubMed Scopus (206) Google Scholar, 12Poon S. Treweek T.M. Wilson M.R. Easterbrook-Smith S.B. Carver J.A. FEBS Lett. 2002; 513: 259-266Crossref PubMed Scopus (110) Google Scholar). This is the first study to investigate the physical properties of CLU-client protein complexes. The present study provides new insights into the properties of complexes formed in vitro between CLU and citrate synthase (CS), fibrinogen (FGN), and glutathione S-transferase (GST). 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 (23Wilson M.R. Easterbrook-Smith S.B. Biochim. Biophys. Acta. 1992; 1159: 319-326Crossref PubMed Scopus (76) Google Scholar). GST was expressed in Escherichia coli using the vector pGEX-2T (without an insert; Invitrogen) as previously described (24Heuer K.H. Mackay J.P. Podzebenko P. Bains N.P. Weiss A.S. King Easterbrook-Smith S.B. Biochemistry. 1996; 35: PubMed Scopus Google Scholar) and purified using a to the FGN or GST were at or in phosphate-buffered saline and in the presence or of CLU and or the control protein, the The were in to a precipitation was by the at nm an of with a control proteins were by client proteins or CLU at the and for the used to the HMW complexes. were and the for protein using the assay B.J. PubMed Scopus Google Scholar), or the at SEC was using a at the of and the at nm using an were from a high molecular weight All and were HMW complexes were from between in the corresponding to the size exclusion of ≥ 4 × 107 The of the complexes was by using the The of an were with purified antibody (23Wilson M.R. Easterbrook-Smith S.B. Biochim. Biophys. Acta. 1992; 1159: 319-326Crossref PubMed Scopus (76) Google Scholar), with in of CLU and client protein were as described and by SEC described with proteins at the exclusion × 107 CLU or client or a of CLU and individual client proteins were in the of the to in a antibody with the client protein, in was the an secondary antibody in was the All were for at °C with and was with a at in was The at nm was using a binding was using a antibody or serum of and the secondary used were and The were serum and purified of HMW HMW and HMW CLU-GST CS, or GST were or or of these were between and in and were in low using a diameters were as a and the diameter and and of normally of CS, or CLU were and The protein was using a The proteins were in an of and the at nm of using a The at nm and the known protein were used to the using These as as HMW HMW and HMW CLU-GST complexes were by in SDS-PAGE buffer and and on a amounts of HMW HMW or HMW CLU-GST and of CS, or CLU were also the Coomassie and the corresponding to CS, and CLU were using a and The of the was used to a for protein. these the amounts of CLU and or GST present in the HMW complexes were bisANS CLU client proteins CS, or or of client proteins the and CLU for using or or at for using were the used to HMW CLU-client protein complexes were from the and in liquid of the all were and in bisANS to of client protein and CLU were to to that present in the client or was on a fluorescence using and of ± and ± nm, respectively. CD were as previously described E.M. J.A. Easterbrook-Smith S.B. D. C. S. Wilson M.R. Biochemistry. PubMed Scopus Google all were in proteins were and were soluble at the CLU at at FGN at and GST at In were for HMW complexes of CLU-CS CLU-FGN CLU-GST and of CLU or the individual client proteins at corresponding to those present in the complexes on the of the mass of protein in the of secondary were obtained using the 1999; 35: PubMed Scopus Google Scholar). fluorescence all were at in and fluorescence was using a with an of nm and an of nm amyloid was formed as described in J.J. Poon S. Meehan S. B. Kumita J.R. Dobson C.M. Wilson M.R. J. PubMed Scopus Google When at a progressive in from to about in was the CLU was present with at a in were that was in the had little on the precipitation of of FGN at °C in progressive precipitation of the protein an of in were the of CLU with FGN in in had little on the precipitation of of GST at °C in precipitation of the protein a of the protein and had of GST with CLU the precipitation of GST the This was in to with the protein a precipitation to that GST was However, the was in the presence of CLU and were at °C shown for first by the of CLU on protein precipitation was for all client proteins protein of individual proteins were as described and insoluble protein by a was in the SEC of CS, or CLU and the corresponding proteins in the heat the soluble proteins were little interaction was by SEC between CLU and CLU and FGN or CLU and GST SEC of the of CLU and CLU and FGN or CLU and GST that HMW at the exclusion of the × 107 that were from the and from of the individual proteins with FGN and of with CLU at °C HMW however, it was that complexes in mass to those formed at by CLU and FGN or GST were also formed by CLU and The exclusion was in and HMW CLU-client protein complexes. The of these complexes was by These complexes were stored in at 4 and their at by these for to CLU and CS, or GST was used to the of the complexes purified by to the the HMW little was obtained for control the of individual proteins the HMW complexes were by SDS-PAGE the gels with Coomassie and the of individual protein by were generated for protein, the approximate mass and for were The mass ratio of CLU to client protein was for CS, and GST complexes 1:2 in However, the were HMW CLU-CS complexes the number of of and HMW CLU-FGN complexes about of CLU for FGN while CLU-GST complexes of GST for of CLU These estimated protein were for studies of the HMW mass and of client protein to CLU in HMW HMW and HMW CLU-GST ratio ratio in a new The of DLS were between and obtained at are shown In to DLS was to the of CLU in DLS analysis of CLU indicated a normally was in size between native and control proteins. The of DLS in the diameters of of size, in a corresponding to an size with the individual of CLU and FGN or GST were DLS indicated that SEC purified HMW CLU-CS and HMW CLU-GST were than of their while HMW CLU-FGN was than than CLU or FGN The bisANS fluorescence of individual soluble client proteins CS, was a in fluorescence at at FGN fluorescence at to the of the control the fluorescence was the at 4 In the bisANS fluorescence of of CLU a small at and at °C CLU of these In all at soluble client protein bisANS fluorescence, the corresponding fluorescence of the of CLU and client protein a This suggests that the interaction between CLU and partially unfolded client proteins in the to which hydrophobic regions on the client proteins are exposed to were to complexes characteristics HMW CLU-FGN and CLU-GST complexes and the native and protein all than of the fluorescence from a of amyloid present at the mass The CD for CLU indicated high with at nm and The at these CLU was at or these analysis a large in a large in and smaller in the of and and However, at °C small in the of and and had a CD on that of In at FGN a in from nm to nm, with the a small in a in and small in and and at GST also a in from that of high to a structure. a in and smaller in and and the CD of HMW CLU-CS complexes with those of of the native or soluble client proteins that secondary was in HMW CLU-CS complexes This was to a of than the native with the proteins of the of a of soluble and CLU that, to the CLU-CS the proteins had a smaller in and an in structure. In the CD for HMW CLU-FGN complexes was on that of a of native CLU and FGN and However, a corresponding of CLU and FGN had a different CD with the of for the individual proteins was that the of HMW CLU-GST complexes was by in secondary these to a of the native were of a large in and small in and The CD and of secondary were for HMW CLU-GST complexes and a of soluble GST and of secondary ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a new FGN extracellular and and GST are different proteins with a large in mass and and the complexes with CLU were formed using different or in all the estimated mass of protein in the complexes was about 1:2 In in CLU formed soluble complexes in which it about its mass in the of client protein. The ratio of protein was different for type of that the of CLU and client protein the of the complexes and their However, of a number of client proteins is to this SEC indicated that all of client protein generated complexes of × 107 in vitro DLS that CLU-FGN complexes had a diameter that of CLU-CS or CLU-GST complexes this the soluble CLU-client protein complexes are large a size to Measurements of bisANS fluorescence indicated that heat CS, and GST to to However, at for FGN and 4 of the of had to that of the This may be at these a of the client protein had from and was to The bisANS fluorescence of of CLU at and °C the A and were was large or in the bisANS fluorescence of CLU at °C A in all three is that the bisANS fluorescence of of CLU and client protein during than was for corresponding of client protein the shown suggest that with CLU the to which client proteins exposed to it likely that the molecular in the of CLU-client protein complexes hydrophobic regions on the client protein from exposure to prevent that otherwise occur to in their has implicated the binding of CLU to regions of exposed on client proteins as an of its chaperone action S. Rybchyn M.S. Easterbrook-Smith S.B. Carver J.A. Pankhurst G.J. Wilson M.R. J. Biol. Chem. 2002; 277: 39532-39540Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). the CD indicated that HMW CLU-FGN complexes had a of the various secondary structures from that of a of native CLU and FGN at the The likely of this is that, the the interaction between CLU and FGN in a of secondary the was different complexes formed between CLU and or In these the of in the complexes was the or than that in the corresponding of CLU and soluble client protein. However, the complexes had and and than the corresponding of previously proteins The may be to the of the client proteins and the of secondary of unfolded of also it is likely that in extracellular fluids CLU-client protein complexes as a mechanism to the of insoluble protein which can to a variety of disease (8Yerbury J.J. Stewart E.M. Wyatt A.R. Wilson M.R. EMBO Rep. 2005; 6: 1131-1136Crossref PubMed Scopus (97) Google Scholar). here suggest that CLU may interact with unfolding proteins at different their unfolding on the at which this interaction CLU may the native secondary structures of the client protein or the client in The between CLU and the client protein are likely to CLU regions of exposed such as and shear stress are likely to protein aggregation in with low of purified proteins in (2Ker Y.C. Chen R.H. Lebenson Wiss. Technol. 1998; 31: 107-113Crossref Scopus (14) Google Scholar, Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. 2000; PubMed Scopus Google Scholar). like many studies of chaperone used to client proteins to and interact with CLU in CLU is heat and to at °C its chaperone action (9Humphreys D.T. Carver J.A. Easterbrook-Smith S.B. Wilson M.R. J. Biol. Chem. 1999; 274: 6875-6881Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 12Poon S. Treweek T.M. Wilson M.R. 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Google Scholar). the heat stress used to the precipitation of is the for their in vitro, all three of complexes the a protein mass ratio of large size × 107 by and diameters of nm by and exposed on the client protein with client protein to that these characteristics important insights into the properties of CLU-client protein complexes in and to of the chaperone action of In have shown that human plasma is by for at the plasma CLU-FGN complexes by and by SEC CLU and FGN are present in corresponding to of × 107 R. in it likely that CLU-client protein complexes generated in in plasma are likely to of the here for complexes formed in vitro from purified proteins. is to CLU-client protein complexes from plasma by immunoaffinity chromatography the at proteins can be and also to large deposits which can with Cell Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, B. Biol. 1998; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. Sci. Full Text PDF PubMed Scopus Google Scholar, C. FEBS Lett. 2001; PubMed Scopus Google Scholar). In vivo, the of misfolded proteins into soluble complexes with extracellular chaperones like CLU is likely to be the first in preventing from forming or otherwise the of have proposed that these soluble complexes are from the by and degradation (8Yerbury J.J. Stewart E.M. Wyatt A.R. Wilson M.R. EMBO Rep. 2005; 6: 1131-1136Crossref PubMed Scopus (97) Google Scholar, M.R. Yerbury J.J. Poon S. Mol. 2008; PubMed Scopus Google Scholar). be important to that this in a this is the Wollongong Hospital for human blood used in this

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