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. Mouse peritoneal macrophages were incubated with acetylated low-density lipoprotein (AcLDL) to produce foam cells containing cholesterol and cholesteryl esters. Incubation of the foam cells with physiological concentrations of purified apo J led to a dose-dependent export of cholesterol. The appearance of cholesterol in the medium was associated predominantly with a decline in intracellular cholesteryl esters rather than intracellular free cholesterol. The kinetics of cholesterol release to apo J were similar to apo A-I, an established promoter of cholesterol efflux. Apo J was also shown to induce phospholipid efflux from cells, whereas the cholesterol exported to the medium was associated with the apo J. Studies using foam cells from apo E-null mice showed that the cholesterol exported to the medium was independent of apo E production by the cells. These results present the first evidence that apo J can promote cholesterol efflux from foam cells and indicates that it might have a function in cellular cholesterol homoeostasis in both normal and pathological situations.

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 0.2 pg clusterin secreted per cell per 48 h period, specifically protected L929 cells from TNFalpha-mediated cytotoxicity, while low expression (about 4 fg/cell/48 h) had no effect. However, clusterin expression did not provide transfected L929 cells with protection against death mediated by colchicine, staurosporine or azide. High level expression of clusterin in transfected L929 cells also potentiated the cytotoxicity of TGFbeta. It had previously been shown that exposure of L929 cells to TGFbeta provides protection against TNFalpha. We showed that this protective effect is not additive to that of clusterin expression. One interpretation of this data is that it suggests that clusterin and TGFbeta may act via a common mechanism to provide protection against the cytotoxicity of TNFalpha. Our results indicate that an intracellular action of clusterin protein is responsible for protection against TNFalpha cytotoxicity. Exposure to TNFalpha induces an increase in the level of cell-associated clusterin and specifically in the level of a novel clusterin molecule, which when analyzed under reducing conditions by SDS/PAGE and immunoblotting appears as two closely spaced bands at about 36 and 38.5 kDa. When analyzed under the same conditions, the normal form of intracellular clusterin, which is present with or without exposure to TNFalpha, appears as two poorly resolved bands at about 43-45 kDa. Since the novel form of clusterin is also expressed in cells exposed to TGFbeta, colchicine, staurosporine, and azide, it may result from toxin-induced disruption of processes of normal cellular protein production.

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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. We asked whether acidic pH induces structural changes in clusterin and enhances its ability to bind to other proteins. We 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.

The study of protein interactions using mass spectrometry (MS) for identification of the components of purified protein complexes is leading to the description of increasingly valuable data on protein function. Commonly proteins in a given complex are identified via MS analysis of in-gel digests of gel electrophoretically separated proteins. In this study, we have evaluated the use of an approach employing the digest of the whole protein complex to identify directly the proteins present in a purification of the CD4 receptor complex. We used a cysteinyl affinity capture method to reduce the complexity of the peptide mixture that was obtained from the tryptic digest of the whole protein complex to the rather limited mixture of only cysteine-containing peptides. Here we report the use of this approach with MS for identification of the CD4 receptor complex components CD4 and p56lck, along with several other proteins present in the detergent-solubilized fractions from the purification. We have been able to identify these proteins using peptide sequence data obtained from cysteine-containing peptides. With appropriate control experiments, we have demonstrated the specific nature of the CD4-p56lck interaction. In contrast, the other proteins identified are shown to arise from nonspecific interactions during the affinity chromatography purification suggesting a possible loss of specific interactions during the chromatography procedure. We found that the complexity of the mixture was reduced such that only 10% of the peptides derived from tryptic digest of the identified proteins were detected. This represents only one-third of the cysteine-containing peptides, however, suggesting that this approach does not enable detection of all individual proteins.

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