Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands.

Although serum albumin has an established function as a transport protein, evidence is emerging that serum albumin may also have a role as a molecular chaperone. Using established techniques to characterize chaperone interactions, this study demonstrates that bovine serum albumin: 1) preferentially binds stressed over unstressed client proteins; 2) forms stable, soluble, high molecular weight complexes with stressed client proteins; 3) reduces the aggregation of client proteins when it is present at physiological levels; and 4) inhibits amyloid formation by both WT and L55P transthyretin. Although the antiaggregatory effect of serum albumin is maintained in the presence of physiological levels of Ca(2+) and Cu(2+), the presence of free fatty acids significantly alters this activity: stabilizing serum albumin at normal levels but diminishing chaperone-like activity at high concentrations.

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values.

Characterization of the binding of Cu(II) and Zn(II) to transthyretin: effects on amyloid formation.

Although metal ions can promote amyloid formation from many proteins, their effects on the formation of amyloid from transthyretin have not been previously studied. We therefore screened the effects of Cu(II), Zn(II), Al(III), and Fe(III) on amyloid formation from wild-type (WT) transthyretin as well as its V30M, L55P, and T119M mutants. Cu(II) and Zn(II) promoted amyloid formation from the L55P mutant of transthyretin at pH 6.

Effects of glycosylation on the structure and function of the extracellular chaperone clusterin.

Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation.

The acute phase protein haptoglobin is a mammalian extracellular chaperone with an action similar to clusterin.

Haptoglobin (Hp) is an acidic glycoprotein present in most body fluids of humans and other mammals. Although the functions of Hp are not yet fully understood, the available evidence indicates that it is likely to play an important role in suppressing inflammatory responses. Some earlier work suggested that Hp might be a newly identified member of a small group of extracellular chaperones found at significant levels in human body fluids.

Mildly acidic pH activates the extracellular molecular chaperone clusterin.

Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH.

Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin.

The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II.

Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off-folding pathway.

Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of alpha-lactalbumin, gamma-crystallin and lysozyme.

Evidence that clusterin has discrete chaperone and ligand binding sites.

Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins.Clusterin also binds to many different unstressed ligands including the cell surface receptor low-density lipoprotein receptor-related protein-2 (LRP-2). It is unknown whether clusterin binds to all of these many ligands via one or more binding sites.

Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state.

We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol.

pH-dependent changes in the in vitro ligand-binding properties and structure of human clusterin.

Clusterin is a glycoprotein which is locally overexpressed at sites of tissue damage or stress, leading to the proposal that it may be a cytoprotective protein. It has been shown that clusterin has chaperone-like activity, being able to protect proteins against precipitation under stress conditions. It has also been shown that local acidosis is common at sites of tissue damage or stress.

A reexamination of the role of clusterin as a complement regulator.

Clusterin is a highly conserved glycoprotein which has been proposed to protect host cells against complement-mediated cytolysis. We tested the hypothesis that clusterin is a complement regulator using erythrocytes and cells which had been stably transfected with a membrane-anchored form of clusterin as targets for complement-mediated cytolysis. Clusterin gave dose-dependent protection of antibody-coated sheep erythrocytes against complement-mediated lysis by diluted normal human serum.

Clusterin has chaperone-like activity similar to that of small heat shock proteins.

Clusterin is a highly conserved protein which is expressed at increased levels by many cell types in response to a broad variety of stress conditions. A genuine physiological function for clusterin has not yet been established. The results presented here demonstrate for the first time that clusterin has chaperone-like activity.

Characterization of the heparin-binding properties of human clusterin.

Clusterin is a highly conserved mammalian glycoprotein which has been predicted to contain heparin-binding sites. We tested this prediction by studying the interactions between heparin and clusterin using ELISA and heparin affinity chromatography methodologies. Two forms of biotinylated heparin were used in ELISA: heparin which had been directly biotinylated with a biotin-N-hydroxysuccinimide ester and heparin which had been activated using epichlorohydrin and 1,6-diaminohexane prior to biotinylation.

Apolipoprotein J (clusterin) induces cholesterol export from macrophage-foam cells: a potential anti-atherogenic function?

Apolipoprotein J (apo J) is a secreted glycoprotein of which the exact function remains a matter for speculation. Apo J has been implicated in such diverse processes as sperm maturation, regulation of complement activation, programmed cell death, tissue remodelling and lipid transport. In this study a possible role for apo J in lipid transport was explored.

Effects of clusterin overexpression on TNFalpha- and TGFbeta-mediated death of L929 cells.

Clusterin is a widely distributed and highly conserved protein for which many functions have been proposed. We used transfected L929 cells to study the effect of clusterin expression on the regulation of cell death signals. We showed that high levels of clusterin expression, about 0.

Histidine-rich glycoprotein binds to human IgG and C1q and inhibits the formation of insoluble immune complexes.

Purification of the complement component C1q from human serum using an established method resulted in the copurification of two 30 kDa proteins with an N-terminal sequence identical to human histidine-rich glycoprotein (HRG). Therefore, to explore the possibility that HRG can interact with C1q, we examined the ability of 81 kDa (native) and the 30 kDa proteins (presumably proteolytic N-terminal fragments of HRG) to bind to C1q, using both ELISA and optical biosensor techniques. Both forms of HRG were found to bind to the human complement component C1q and also to purified human and rabbit IgG by ELISA.

C1q is a nucleotide binding protein and is responsible for the ability of clusterin preparations to promote immune complex formation.

Clusterin prepared from human serum by monoclonal antibody affinity chromatography was devoid of the ability to increase the rates of formation of insoluble immune complexes associated with clusterin preparations obtained by polyclonal IgG affinity chromatography. Clusterin did not bind to AMP-Sepharose but the protein responsible for increasing the rates of formation of insoluble immune complexes did bind to this affinity matrix. This protein was identified as complement protein C1q on the basis of its behaviour on SDS/PAGE and reactivity in sandwich ELISA with monoclonal antibodies specific for C1q.

The formation of insoluble immune complexes between ovalbumin and anti-ovalbumin IgG occurs in at least two distinct phases dependent on reactant concentration and ionic strength.

The mechanism regulating the formation of insoluble immune complexes (IIC) in serum in certain disease states is not well understood. Ovalbumin and rabbit anti-ovalbumin IgG was used to study the formation of IIC in vitro in a stirred reaction vessel; and the radii of IIC that formed was determined by light scattering techniques. Using an initial IgG concentration of 1 mg/ml at equivalence antigen:antibody ratio IIC formation was detected within 5 s, and the complexes increased in radii to approx.

Development of a sensitive peptide-based immunoassay: application to detection of the Jun and Fos oncoproteins.

c-Jun and c-Fos belong to the bZIP class of transcriptional activator proteins, many of which have been implicated in the neoplastic transformation of cells. We are interested in engineering dominant-negative leucine zipper (LZ) peptides as a means of sequestering these proteins in vivo in order to suppress their transcriptional regulatory activity. Toward this end, we have developed a novel immunoassay for measuring the dimerization affinities of dimeric Jun and Fos complexes.

The effects of histidine residue modification on the immune precipitating ability of rabbit IgG.

Treatment of anti-ovalbumin rabbit IgG with diethylpyrocarbonate (DEPC) at concentrations up to 100 microM led to a progressive decrease in the rates of formation of insoluble immune complexes, without affecting the final extent of immune complex formation. DEPC concentrations approximately 10-fold higher were needed to give comparable decreases in the rates of immune complex formation by F(ab')2. Treatment of DEPC-treated IgG with hydroxylamine led to substantial restoration of the rates of formation of insoluble immune complexes.

A light-scattering method for measuring the sizes of insoluble immune complexes.

A turbidimetric method for measuring the diameters of insoluble immune complexes, based on the wavelength dependence of their ability to scatter light, was developed. The method was validated by demonstrating that it gave experimental values for the diameters of polystyrene microspheres which were in good agreement with independently known values of these. The method was used to measure the diameters of ovalbumin:anti-ovalbumin IgG immune complexes, giving values consistent with literature measurements of the sizes of other IgG-containing immune complexes.

Enzyme complex amplification--a signal amplification method for use in enzyme immunoassays.

An amplification system for enzyme immunoassays, in which complexes of streptavidin, biotinylated horseradish peroxidase, antibody are used to detect biotinylated target molecules was developed. In enzyme-linked immunosorbent assay (ELISA) experiments this enzyme complex enhancement (ECA) system gave up to a better than two orders of magnitude increase in sensitivity, compared to a horseradish peroxidase-conjugated streptavidin detection system. The ECA system was shown to be applicable to direct detection of biotinylated antigens, as well as in ELISA employing biotinylated antibodies and in sandwich ELISA.

Clusterin binds by a multivalent mechanism to the Fc and Fab regions of IgG.

Clusterin was purified from human serum by IgG and monoclonal antibody affinity chromatography. SDS-PAGE and immunoblotting revealed no major differences between clusterin prepared in these two ways. An ELISA method for measuring the binding of clusterin to immunoglobulins was developed.