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Although clusterin was identified morethan 16 years ago, its function has re-mained elusive. It was first described in1983 as a secreted glycoprotein in ramrete testis fluid that enhanced aggre-gation (‘clustering’) of a variety of cells invitro1. Many homologs in other specieswere subsequently discovered. Typically,each ‘discovery’ of clusterin in a differentspecies or by a different research groupled to it being assigned another name. Bythe early 1990s clusterin was knownunder many aliases2, some of which per-sist in the literature. However, the in-augural international workshop on clus-terin (Cambridge, 1992) agreed to thename clusterin, in deference to the origi-nal reports of the protein’s properties.

Clusterin structure and distributionClusterin is a 75–80 kDa disulfide-

linked heterodimeric protein with 30%of its mass being N-linked carbohydrate.It is encoded by a single gene and thetranslated product is internally cleavedto produce its a and b subunits prior tosecretion from the cell. Sequence analy-ses predict that clusterin contains adisulfide-linked core region flanked bysections of the a and b subunits contain-ing three amphipathic a helices and twocoiled-coil a helices (Fig. 1).

The breadth of its biological distribu-tion is striking. In animal tissues clusterinmRNA is nearly ubiquitous, being foundin locales as diverse as the rat prostategland and quail neuroretinal cells. Acrossspecies, clusterin maintains a high levelof sequence homology (70–80% betweenmammals)2. There is extensive evidenceof a correlation between clusterin ex-pression and diseases (e.g. Alzheimer’sdisease, gliomas) or pathological stress(e.g. pressure or ischemic kidney insult)2.

Previous ideas about clusterin’s functionUnravelling the function of clusterin

has been an elusive goal, as it has beenascribed many functions.

Controlling cell–cell and cell–substratum in-teractions. Although clusterin was namedafter its ability to mediate clustering ofcells1 and exogenous clusterin pro-motes formation of ‘nodules’ in culturedporcine cells5, the relevance of these

observations to in vivo physiology re-mains to be established.

Regulating apoptosis. This idea was pop-ular following the discovery that clus-terin mRNA was upregulated in apop-totic rat prostate6. However, subsequentstudies showed that clusterin expres-sion was not increased in other modelsof apoptosis.

Acting as a ‘membrane policeman’. It wassuggested that clusterin protects cellsat fluid–tissue interfaces from stress7.Given that heat shock proteins (HSPs)protect cells from a variety of stresses,this idea is consistent with recent datasuggesting that clusterin is a novel typeof heat shock protein (see below).

Transporting lipids. A fraction of clus-terin in human plasma is associatedwith high-density lipoprotein (HDL) par-ticles. Clusterin is secreted from HepG2cells associated with lipids8, and exoge-nous clusterin can promote the in vitroefflux of cholesterol from lipid-loaded

mouse macrophages9. It remains to beestablished whether clusterin has alipid transport role in vivo.

Regulating complement. Several reportsshow that in vitro clusterin can influ-ence complement-mediated cytolysisbrought about by purified complementcomponents2. Hence, the notion thatclusterin is a complement regulator waspopular. However, our recent work sug-gests that under physiological condi-tions clusterin is unlikely to be an im-portant complement regulator10.

Thus, until recently, the biologicalfunction(s) of clusterin was clouded inuncertainty and confounded by an in-creasing list of putative functions. Partof the reason for this and, we suggest,part of the answer lies in the diversity ofclusterin-binding ligands (Table 1).

Clusterin binds to many different biologicalligands

Clusterin binds to a wide array of bio-logical ligands (Table 1). In many cases,the discoverers of a new ligand also proposed a new function for clusterin,associated with the ligand. For example,when we reported that clusterin binds toIgG, we suggested that clusterin’s func-tion might involve association with IgG(Ref. 18). Arguably, the sheer number ofclusterin-binding ligands makes it unlikely that each relates to a separatefunction. A reasonable hypothesis is thatmany of these interactions result from asingle underlying property of clusterin,related to a primary function. The firsttangible clue for this function came froma study of the clusterin promoter24.

Is clusterin a functional heat shock protein?Mammalian heat shock proteins are a

diverse group of intracellular proteinsthat protect cells against heat and otherstresses. Individual HSPs differ in theirpatterns of expression and mode of action

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Clusterin is a secretedmammalian chaperone

Ti BS

NC CN

α chain

β chain

Figure 1Predicted structure of clusterin. Thecartoon shows mature human clusterinafter cleavage of its 22-mer signal peptideand internal cleavage at itsArg227–Ser228 bond, generating the achain (residues 23–227) and b chain(residues 228–449). The three predictedamphipathic a helices (yellow ovals,residues 173–184, 234–250 and424–441) were identified by helical wheelanalysis3. The two predicted coiled-coila helices (blue rectangles, residues40–99 and 318–350) were identifiedusing the COILS algorithm4. The centraldisulfide-rich region (green ovals) spansresidues 102–129 in the a chain andresidues 285–313 in the b chain.

Table 1. Reported clusterin-binding ligands

Ligand Refs

apoA-I 11b-amyloid peptide 12Complement components 13Glutathione-S-transferase 14gp330/megalin 15,16Heparin 17Immunoglobulins 18Lipids 8Paraoxonase 19Prion peptide 20SIC (streptococcal inhibitor of 21complement)

Staphylococcus aureus cell surface 22TGF-b receptor 23

0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(99)01534-0 95

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but they are all chaperones in that theybind to exposed hydrophobic regions ofproteins that are either incompletelyfolded or are partly unfolded as the re-sult of stress. In this way, chaperones canstabilize conformations of proteins thatare otherwise unstable. This is believedto be the primary function of the smallHSPs (sHSPs; see Ref. 25). In vivo, differ-ent HSPs are thought to cooperate in sta-bilizing partially unfolded proteins (e.g.via the action of the sHSPs) and then re-folding them (e.g. via the action ofHSP70). These combined actions mightprotect cells from stress by preservingthe integrity of critical proteins26.

In 1997 it was reported that a 14-bp element in the clusterin promoter, the sequence of which is strictly conserved invertebrates, is specifically recognized bytranscription factor HSF1 (heat shock factor 1) and can mediate heat-shock-induced transcription in transient expres-sion assays. This suggests that clusterin isa heat shock protein24. In a recent review,the same suggestion was made and amodel was proposed in which clusterinacts as a ‘biological detergent’, binding tohydrophobic complexes and denaturedproteins to aid their uptake or clearance27.However, this simple model did not incor-porate either the concept that, like allknown mammalian HSPs, clusterin couldbe a chaperone, or recently publisheddata showing that clusterin can potentlyinhibit stress-induced protein precipita-tion14. It is clear that, like HSPs, clusterinexpression is induced by heat shock27.However, if clusterin indeed functions as aHSP, it should stabilize stressed proteinsand, acting alone or in concert with otherHSPs, protect cells from stress.

The first of these criteria was satisfiedby a recent report that clusterin formshigh-molecular-weight ‘solubilized’ com-plexes with heat- or reduction-stressed

proteins, inhibiting their precipitation14.On a molar basis, clusterin is substantiallymore potent than sHSPs at inhibitingstress-induced protein precipitation14,28.

Figure 2Sequence alignment of clusterin and aB-crystallin. The alignment shown concernsthe C-terminal halves of matched specieshomologs of clusterin and aB-crystallin.The sequences were from the SwissProtdatabase and the alignment was carriedout using the Multalin algorithm34. Theresidues in red are identical or highlyconser ved between clusterin and aB-crystallin, and the residues in blue areidentical between the species homologsof aB-crystallin. The boxed regions of aB-cr ystallin show regions identified asbeing part of its chaperone active sitefrom chemical crosslinking to alcoholdehydrogenase (ADH, orange box) andfrom photochemical crosslinking to 4,49-bis-(1-anilinoaphthalene-8-sulfonate)(bisANS, green box)35,36. The overallsequence similarity between aB-crystallinand residues 230–422 of clusterin is17% (33/193 residues identical or highlyconserved).

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Clusterin did not protect the two enzymestested, catalase and glutathione-S-trans-ferase, from heat-induced loss of func-tion14, nor did it promote recovery of en-zyme activity following heat stress (D.Humphreys, unpublished). Thus, clus-terin can stabilize stressed proteins butcannot catalyse protein refolding – prop-erties shared with sHSPs.

Regarding the second criterion for afunctional HSP, it appears likely thatclusterin can protect cells from manyenvironmental stresses. LNCaP cellstransfected with anti-sense clusterincDNA are sensitized to tumor necrosisfactor a (TNFa)-mediated death29.Furthermore, overexpression of clusterinprotects LNCaP (Ref. 29) and L929(Ref. 30) cells from TNFa. Althoughstudies involving overexpression of proteins must be interpreted with cau-tion, taken together, these results sug-gest that clusterin protects cells fromthe cytotoxicity of TNFa. A correlationbetween clusterin expression and cellsurvival was reported for a variety ofmodels of apoptosis, ranging from UV irradiation of U937 cells to dexametha-sone treatment of CEM-C7 cells31.Recently, it was shown that A431 cellstransfected with anti-sense clusterincDNA are more susceptible to heatshock or oxidative stress than con-trols32. Finally, it has been shown thatexogenous clusterin can protect LLC-PK1 cells from oxidative stress33 and protects a variety of human cell linesfrom heat stress (S. Poon, unpublished).These results strongly suggest that clusterin is a cytoprotective moleculeand support the hypothesis that clusterin is a functional HSP.

In this context, it is interesting to notethat there are limited sequence similari-ties between residues 230–422 of clus-terin and the aB-crystallins (a type ofHSP; Fig. 2). The overall sequence simi-larity is low at 17% (33/193 residuesidentical or highly conserved), however,it is noteworthy that there is 25% simi-larity between residues 286–343 of clusterin and residues 57–107 of aB-crystallin. This region of aB-crystallinforms part of its chaperone activesite35,36. The equivalent region of clusterincontains a discrete motif, the coiled-coila helix in the b chain (Fig. 1).

To summarize: (i) at least one com-mon element, HSF1, can control expres-sion of both clusterin and HSPs; (ii) likeHSPs, clusterin can inhibit stress-induced protein precipitation and appears likely to protect cells fromstresses; and (iii) clusterin shares some

sequence similarity with HSPs. Thus, atthe levels of gene control and proteinfunction, clusterin shares many similari-ties with HSPs, an established group ofchaperones. Amino acid sequence analyses indicate that there could be adistant evolutionary relationship betweenclusterin and HSPs (represented by aB-crystallin) but also suggest that clusterinis a unique protein without any closely related family members yet identified.

Site of actionClusterin is constitutively secreted by

mammalian cells, although stress mightinduce intracellular forms of the pro-tein. A truncated form, possibly result-ing from differential translation, hasbeen reported in transforming growthfactor b (TGF-b)-treated cells37; corrobo-ration of this or comparable observationsby independent laboratories would beof interest. Furthermore, a recent studyhas clearly demonstrated that in celllysates clusterin associates with a DNA-binding protein known as Ku70 and in-hibits binding of Ku70 to a short, 32P-labelled double-stranded oligonucleotide38.Purified clusterin and Ku70 were alsoclearly shown to bind to each other38.These findings are interesting and raisethe possibility that clusterin might inter-act with Ku70 within cells. However, allthese results could also be explainedwith the hypothesis that, when liber-ated by detergent-mediated cell lysisfrom its normal intracellular compart-ments (e.g. the lumen of the endoplas-mic reticulum), clusterin binds to re-gions of exposed hydrophobicity onKu70, thereby inhibiting its interactionswith DNA. Finally, it was recently reported that clusterin is normally intracellular in chickens39.

For mammalian cells, although an in-tracellular action of clusterin duringstress remains possible, more evidenceis required to establish this. Thus, asclusterin is in most physiological set-tings a secreted protein, and in light ofdirect demonstrations of an extracellu-lar cytoprotective action, we suggestthat clusterin acts in vivo as an extracel-lular chaperone. Known mammalianHSPs exert their chaperone effects intra-cellularly to protect cells. Secreted HSPshave been identified in yeast and plants:following heat shock, soybean seeds se-crete a 168-kDa glycoprotein (basic 7Sglobulin)40 and the yeast Hansenula poly-morpha secretes a 198-kDa glycoprotein41.However, in neither case are the functionsof these HSPs known. There are no previ-ous reports of mammalian proteins being

secreted in response to heat or acting asextracellular chaperones. Thus, clusterincould be the first identified secreted mammalian chaperone.

In the extracellular milieu, in additionto its apparent direct effects on cell vi-ability, clusterin might bind to damagedor toxic molecules, or both, and targetthem for removal via megalin. Megalin,an endocytic receptor expressed by avariety of cells, can internalize clusterin,which is then degraded15,16.

Challenges aheadThe realization that clusterin is a

novel HSP with chaperone activity is anexciting breakthrough. However, manyquestions remain. We know little abouthow clusterin exerts its potent chaper-one action to stabilize stressed proteins.Another area of interest is clusterin’ssite of action; can clusterin act both in-tracellularly and extracellularly to pro-tect cells from stresses? Further, there isthe question of the targets that clusterinacts on during stress: does clusterinprotect membrane proteins or exert adirect effect on lipids to stabilize cellmembranes? Finally, does extracellularclusterin act as a hydrophobic ‘sink’, se-questering toxic or damaged molecules,directing them away from sensitive cel-lular sites to a safe disposal route?Obtaining the answers to these ques-tions will keep many of us busy for yearsto come.

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MARK R.WILSON

Dept of BiologicalSciences,University ofWollongong,NorthfieldsAvenue,Wollongong, NSW Australia2522.Email:mrw@uow.edu.au

SIMON B.EASTERBROOK-SMITH

Dept ofBiochemistry,University ofSydney, Sydney,NSW Australia2006.