Clusterin (1. Introduction)

Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death ^Ref . Clusterin is induced de novo during the regression of the prostate, the mammary gland and other hormone-dependent tissues after hormone ablation, and is over-expressed in several human neurodegenerative diseases including Alzheimer‘s di­sease, epilepsy and retinitis pigmentosa ^Ref .
Clusterin is a heterodimeric glycoprotein produced by a wide array of tissues and found in most biologic fluids. A number of physiologic functions have been proposed for clusterin based on its distribution and in vitro properties. These include the above mentioned complement regulation and lipid transport, as well as sperm maturation, initiation of apoptosis, endocrine secretion, membrane protection, and promotion of cell interactions. A prominent and defining feature of clusterin is its induction in such disease states as glomerulonephritis, polycystic kidney disease, renal tubular injury, neurodegenerative conditions including Alzheimer‘s di­sease, atherosclerosis, and myocardial infarction. The expression of clusterin in these states is puzzling, from the specific molecular species and cellular pathways eliciting such expression, to the roles subserved by clusterin once induced ^Ref .
Overall, the evidence suggests that function of clusterin is to protect surviving cells after damage. This protection may result from a detergent- and chaperon-like action of the protein ^{Ref Ref} .

Clusterin has originally been purified from rat rete testis fluid by conventional techniques and by immunoaffinity chromatography.the molecule is characterized as a glycoprotein having a molecular mass of approximately 80,000 Da and an isoelectric point of 3.6. The purified protein retains the capacity to elicit clustering of cells in an in vitro assay. Under reducing conditions in the presence of sodium dodecyl sulfate, clusterin dissociates into subunits of about 40,000, designated alpha (34–36 kDa), and beta (36–39 kDa). It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. All the cysteine residues are involved in interchain (alpha-beta) disulphide bonds. There are no free cysteine residues ^Ref . Disulfide bonds were determined between Cys58(alpha)Cys107(b­eta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there is no free sulfhydryl groups in the clusterin molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in clusterin are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of clusterin ^Ref .When deglycosylated, the molecular mass of the alpha-subunit is 24 kDa and that of the beta subunit is 28 kDa, suggesting that approximately 30% of the mass of each subunit is carbohydrate. The amino acid compositions of clusterin alpha and beta beta are very similar; however, the sequences of the first 30-amino acid residues are distinct. Some antibodies react with both subunits whereas others with only one of them ^Ref .

Heterogeneities in apparent molecular mass were eliminated after treatment of clusterin with neuraminidase. Gel filtration chromatography revealed that clusterin exists in dimeric and tetrameric forms under conditions of neutral pH and low salt concentrations. In the presence of 6 M urea, only the monomeric form is evident, with an apparent molecular mass of approximately 85,000 Da. Clusterin, which was found to contain 4.5% glucosamine, binds to concanavalin A-sepharose and also to wheat germ agglutinin sepharose ^Ref .
Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17–27% carbohydrate by weight, the alpha subunit contains 0–30% carbohydrate and the beta subunit contains 27–30% carbohydrate. The most abundant glycoforms are bisialobiantennary without fucose and the least abundant were monosialobian­tennary, trisialotrian­tennary with two fucose and/or tetrasialotri­antennary. No evidence was found for the presence of O-linked or sulfated oligosaccharides ^Ref .
Clusterin is initially produced as a single chain, intracellular precursor of 58 kDa which contains N-linked oligosaccharide. The precursor is converted to an intracellular 70 kDa glycoprotein, which becomes the major intracellular form of clusterin prior to secretion. Maturation of the 58 kDa precursor involves conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurres at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of clusterin occurres rapidly, with a half-time of 30–35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of clusterin. The unglycosylated clusterin species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation ^Ref .
Clusterin is expressed at high levels in an array of specialized cell types of adult and fetal mouse tissues and in similar cell types of human tissues. Most of these cell types are highly secretory and form the cellular interfaces of many fluid compartments. This group includes epithelial boundary cells of the esophagus, biliary ducts, gallbladder, urinary bladder, ureter, kidney distal convoluted tubules, gastric glands, Brunner‘s glands, choroid plexus, ependyma, ocular ciliary body, endometrium, cervix, vagina, testis, epididymus, and visceral yolk sac. Several nonepithelial secretory cell types that express high levels of clusterin also line fluid compartments, such as synovial lining cells and ovarian granulosa cells. In the context of its known biochemical properties, this expression pattern suggests that localized synthesis of clusterin serves to protect a variety of secretory, mucosal, and other barrier cells from surface-active components of the extracellular environment ^Ref .

Clusterin has chaperone-like activity

At physiological concentrations, clusterin potently protected glutathione S-transferase and catalase from heat-induced precipitation and alpha-lactalbumin and bovine serum albumin from precipitation induced by reduction with dithiothreitol. Enzyme-linked immunosorbent assay data showed that clusterin bound preferentially to heat-stressed glutathione S-transferase and to dithiothreitol-treated bovine serum albumin and alpha-lactalbumin. Size exclusion chromatography and SDS-polyacrylamide gel electrophoresis analyses showed that clusterin formed high molecular weight complexes (HMW) with all four proteins tested. Small heat shock proteins (sHSP) also act in this way to prevent protein precipitation and protect cells from heat and other stresses. The stoichiometric subunit molar ratios of clusterin:stressed protein during formation of HMW complexes (which for the four proteins tested ranged from 1.0:1.3 to 1.0:11) is less than the reported ratios for sHSP-mediated formation of HMW complexes (1.0:1.0 or greater), indicating that clusterin is a very efficient chaperone. These results suggest that clusterin may play a sHSP-like role in cytoprotection ^Ref .

Clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally divergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, it was shown that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, other reports suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins ^Ref .
The interactions of clusterin with different structural forms of alpha-lactalbumin, gamma-crystallin and lysozyme were studied. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of alpha-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced alpha-lactalbumin. Reduction-induced changes in the conformation of alpha-lactalbumin, in the absence and presence of clusterin, were monitored by real-time (1)H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of alpha-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of alpha-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 gamma-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, these 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 ^Ref .

Using sequence analyses, it was shown that clusterin likely contains three long regions of natively disordered or molten globule-like structures containing putative amphipathic alpha-helices. Natively disordered regions with amphipathic helices form a dynamic, molten globule-like binding site provide clusterin the ability to bind to a variety of molecules ^Ref .

Local acidosis is common at sites of tissue damage or stress. It was found by affinity chromatography and ELISA that the binding of clusterin to glutathione-S-transferase, IgG, apolipoprotein A-I, and complement protein C9 was enhanced at mildly acidic compared to physiological pH. Analytical ultracentrifugation and gel filtration studies revealed that clusterin exists in different polymerization states with monomer occurring preferentially at pH 5.5 and multimeric species at pH 7.5. Although circular dichroism showed little difference in the alpha-helical and beta-sheet contents of clusterin at pH 5 compared to pH 7.5, evidence for pH-dependent structural changes in clusterin was obtained from fluorescence experiments. pH titrations showed reversible changes in the fluorescence of tryptophan residues in clusterin. There was a reversible 2-fold increase in the fluorescence of the extrinsic probe 4, 4‘-bis(1-anilinonaphthalene-8-sulfonate) bound to clusterin at pH 5.5 compared to pH 7.5. There was also a 3.5-fold increase in fluorescence resonance energy transfer from tryptophan residues in clusterin to 4,4‘-bis(1-anilinonaphthalene-8-sulfonate) at pH 5.5 compared to pH 7.5. These data suggest that pH-induced changes in the structure of clusterin are responsible for its enhanced ability to bind protein ligands at mildly acidic pH ^Ref .

Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. A model was proposed in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution ^Ref .

Truncated, nonglycosylated, nuclear clusterin variant

In addition to the well characterized secreted form of the protein, there exists an intracellular, nuclear form of clusterin. This intracellular form of the protein was found to be induced to accumulate in the nucleus of two epithelial cell lines (HepG2 and CCL64) in response to treatment with transforming growth factor beta (TGF beta). It was demonstrated in vitro that clusterin protein can be translated from two in-frame ATG sites. Initiation from the first ATG encodes for the secretory form of clusterin and initiation from the second ATG, located 33 amino acids downstream of the first and lacking the hydrophobic signal sequence, encodes for a truncated clusterin protein. This shorter form of clusterin is not recognized by microsomes and therefore not glycosylated, and it was postulated that it is retained intracellularly and targeted to the nucleus due to the presence of an SV40-like nuclear localization sequence (NLS). This mechanism of nuclear targeting of apoJ occurs in cells since the protein isolated from nuclei of TGF beta-treated cells and the in vitro-translated truncated form are identical by V8 protease analysis.these results suggest that the diverse physiological responses attributed to clusterin may be elicited through a common molecular mechanism involving a previously uncharacterized intracellular form of the protein ^Ref .
Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. Endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following ir, and induces apoptosis ^Ref .
During the course of a study to examine the effect of cycloheximide on apoptosis-related genes, the variant rat clusterin mRNA deficient of the exon 5 was found. The putative protein encoded by the variant clusterin mRNA is only constituted from the N-terminal one-third portion of the ordinary clusterin protein. The expression of the variant form was increased dramatically by cycloheximide treatment, while that of the ordinary form was not affected very much. The similar phenomenon was also observed by the use of other types of protein synthesis inhibitors, anisomycin and emetine. The enhancement of expression of the variant was observed in the rat treated with heat shock as well. The variant form was presumably generated by the exon skip mechanism. Systematic analyses of cycloheximide effect on the alternative splicing at various splicing junctions were performed. However, cycloheximide did not exhibit any remarkable effects on other types of alternative splicing, including exon skip in beta A4-amyloid protein precursor (APP) gene, alternative donor selection in Fas antigen gene and alternative acceptor selection in catechol O-methyltransferase (COMT) gene. These results indicated that the induction of exon skip by both protein synthesis inhibition and heat shock treatment occurs in a limited number of genes, if not only in clusterin ^Ref .
A cDNA clone designated p116 was isolated from rat seminal vesicles. A sequence study suggested the expression of a transcript predicting an alternative form of clusterin. RT-PCR demonstrated the androgen-dependent expression of p116 transcript in the seminal vesicles, ventral prostate, the liver and the thymus. Since clusterin has been suggested as a classical molecular marker of apoptosis, p116 transcript newly identified in the this study might provide a useful probe to further understand the roles of clusterin and its related proteins during the course of apoptotic process not only in male accessory sex organs, but also in many types of cells undergoing apoptosis ^Ref .
A similar variant mRNA lacking exon 5 was also induced by heat shock treatment of the human culture cell line HepG2. On the other hand, in mouse cell line L929, heat shock treatment induced a variant clusterin mRNA lacking only a small region located in exon 5. However, irrespective of the difference of mechanism of variant production, all the variant clusterin mRNA species derived from each animal species encoded a putative protein constituted from the N-terminal one-third of the clusterin protein attached to a C-terminal clusterin unrelated tail. In humans, the variant clusterin species was not detected in normal tissues but was present in certain kinds of tumour cells. These results indicate that the splicing variants were induced as a direct result of heat shock treatment on cells per se and that the phenomenon of heat shock induction was observed in culture cells derived from different animal species ^Ref .

Clusterin can evade the secretory pathway

In live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin ^Ref .

Clusterin and HDL

Clusterin was also termed apolipoprotein J (apoJ), and was purified from human plasma by immunoaffinity chromatography and found to be associated with high density lipoproteins (HDL) and specifically with subclasses of HDL which also contain apoAI and cholesteryl ester transfer protein activity ^Ref .
Apolipoprotein J (apoJ)-containing high-density lipoproteins (HDL), isolated from human plasma by immunoaffinity chromatography, are associated with apoAI and a protein of approximately 44 kDa. The 44-kDa protein, a monomeric glycoyslated polypeptide, was identified by N-terminal sequencing as serum paraoxonase. Not all of the plasma paraoxonase is associated with apoJ. Both oxidation states of paraoxonase bind to apoJ with equal affinity. These data combined with other evidence suggest that the plasma link of apoJ with paraoxonase might be implicated as a predictor of vascular damage ^Ref .
Purified apoJ added directly to apoJ-depleted plasma can interact with apoAI or with apoAI-containing lipoproteins, as evidenced by the association of apoAI with apoJ that is reisolated by immunoaffinity chromatography. The amount of apoAI associated with apoJ increases linearly with increasing amount of apoJ added, over the range of apoJ concentrations tested. No other known apolipoprotein is associated with apoJ. By two-dimensional electrophoretic analysis, the lipoproteins containing both apoJ and apoAI have approximate molecular masses of 350–400 kDa. Taken together, the results suggest that the interaction between apoJ and apoAI is physiologically important and that lipoproteins which contain both apoJ and apoAI can be produced in the plasma. ApoJ-HDL and apoJ/apoAI-HDL may have different functions and metabolic fates or may represent different stages of apoJ catabolism ^Ref .
It was demonstrateh that HepG2 human hepatocellular carcinoma cells secrete apoJ in association with a significant amount of lipid, providing unequivocal evidence that apoJ can transport lipids. The HepG2 cell line has provided important clues about the structural organization of nascent lipoprotein particles. HepG2 cell apoJ-containing lipoproteins are dense and heterogenous in size, ranging from 100 to 910 kDa. Plasma and HepG2 cell apoJ-lipoproteins differ in size distribution. Both have alpha 2 electrophoretic mobility, although their average mobilities differ within the alpha 2 region. In contrast to plasma apoJ-HDL which contain little triglyceride and which can associate with apoA-I, HepG2 cell apoJ-lipoproteins are rich in triglyceride and lack apoA-I. By implication, nascent apoJ-lipoproteins undergo plasma remodeling that results in triglyceride depletion and apoA-I association. We propose that the metabolic consequences of this remodeling play an important role in lipid homeostasis in localized tissue environments, particularly where organs are isolated from the blood by cellular barriers such as in testis and brain. In such tissues, apoJ is expressed constitutively in high level compared to other lipid transport proteins ^Ref .

Clusterin is a positive acute phase protein

Endotoxin (LPS), tumor necrosis factor (TNF), and interleukin (IL)-1 increased hepatic mRNA and serum protein levels of clusterin in syrian hamsters. Hepatic clusterin mRNA levels increased 10– to 15-fold with doses of LPS from 0.1 to 100 micrograms/100 g body weight within 4 h and were elevated for > or = 24 h. Serum clusterin concentrations were significantly increased by 16 h and further elevated to 3.3 times that of control, 24 h after LPS administration. Serum clusterin was associated with high density lipoprotein and increased fivefold in this fraction, after LPS administration. Hepatic clusterin mRNA levels increased 3.5– and 4.6-fold, with TNF and IL-1, respectively, and 8.2-fold with a combination of TNF and IL-1. Serum clusterin concentrations were increased 2.3-fold by TNF, 79% by IL-1, and 2.9-fold with a combination of TNF and IL-1. These results demonstrate that apoJ is a positive acute phase protein ^Ref .

Clusterin receptors

LRP-2

Clusterin has three independent classes of binding sites for 1) LRP-2, 2) stressed proteins, and 3) ustressed ligands, respectively, and the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the beta-chain. It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation. This hypothesis is supported by the demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules ^Ref .
Glycoprotein 330 (gp330), low density lipoprotein receptor-related protein-2/megalin (LRP-2) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to clusterin with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to clusterin transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to clusterin in either type of assay. The binding of gp330 to clusterin could be competitively inhibited with excess clusterin as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein- a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-clusterin. Only cells that expressed gp330 endocytosed and degraded radiolabeled clusterin. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade clusterin. Cellular internalization and degradation of radiolabeled clusterin could be inhibited with unlabeled clusterin, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate clusterin endocytosis leading to lysosomal degradation ^Ref .
After endocytosis, the LRP-2 is recycled back to the cell surface while clusterin is delivered to the lysosomes for degradation. To provide additional evidence implicating LRP-2 in clusterin endocytosis, a receptor-associated protein (RAP), an antagonist of apo J binding to LRP-2, was injected into the efferent duct lumen. Subsequent immunocytological analysis of the efferent duct showed that the RAP treatment abolished the endocytosis of apo J by the nonciliated cells. Taken together, these data indicate that LRP-2 is a likely mediator of apoJ endocytosis by the nonciliated efferent duct cells ^Ref .

TGF-betaRI and II

Clusterin interacts with both the type I (RI) and type II (RII) TGF beta receptors but does not interact with the epidermal growth factor (EGF) receptor. The interaction between RII and clusterin occurs through the C-terminal 127 amino acids of RII. Deletion of this region, which contains the kinase insert 2 domain, abrogates binding to clusterin. The binding of clusterin to either the RI and the RII receptors is direct, not requiring other proteins, and is not specific for the alpha or beta subunit of apoJ since both subunits are effective in competing for binding. RI and RII fusion proteins are capable of precipitating the 60 kDa intracellular form of apoJ from [35S]methionine-labeled cellular lysates, suggesting that this form of the protein may play some role in TGF beta signaling or TGF beta receptor processing ^Ref .
Clusterin is also a modulator of TGF-beta signaling by regulating Smad2/3 proteins. Overexpression of clusterin enhanced TGF-beta-induced transcriptional activity and increased the amount of Smad2/3 proteins, while clusterin sIRNA repressed TGF-beta-induced transcriptional activity and decreased the amount of Smad2/3 proteins in Hep3B cells. Clusterin is also involved in Smad2/3 stability at the protein level. These findings suggest that CLU regulates TGFbeta signaling pathway by modulating the stability of Smad2/3 proteins ^Ref  .