Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants.

The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin. Recent independent studies are now exploring the potential use of clusterin as a therapeutic in a variety of disease contexts. In the past, the extensive post-translational processing of clusterin, coupled with its potent binding to essentially any misfolded protein, have meant that its expression as a fully functional recombinant protein has been very difficult. We report here the first rapid and high-yield system for the expression and purification of fully post-translationally modified and chaperone-active clusterin. Only 5-6 days is required from initial transfection to harvest of the protein-free culture medium containing the recombinant product. Purification to near-homogeneity can then be accomplished in a single affinity purification step and the yield for wild type human clusterin is of the order of 30-40 mg per litre of culture. We have also shown that this system can be used to quickly express and purify custom-designed clusterin mutants. These advances dramatically increase the feasibility of detailed structure-function analysis of the clusterin molecule and will facilitate identification of those specific regions responsible for the interactions of clusterin with receptors and other molecules.

Clusterin Binds to Aß1-42 Oligomers with High Affinity and Interferes with Peptide Aggregation by Inhibiting Primary and Secondary Nucleation.

The aggregation of amyloid ß protein (Aß) is a fundamental pathogenic mechanism leading to the neuronal damage present in Alzheimer disease, and soluble Aß oligomers are thought to be a major toxic culprit. Thus, better knowledge and specific targeting of the pathways that lead to these noxious species may result in valuable therapeutic strategies. We characterized some effects of the molecular chaperone clusterin, providing new and more detailed evidence of its potential neuroprotective effects. Using a classical thioflavin T assay, we observed a dose-dependent inhibition of the aggregation process. The global analysis of time courses under different conditions demonstrated that clusterin has no effect on the elongation rate but mainly interferes with the nucleation processes (both primary and secondary), reducing the number of nuclei available for further fibril growth. Then, using a recently developed immunoassay based on surface plasmon resonance, we obtained direct evidence of a high-affinity (KD= 1 nm) interaction of clusterin with biologically relevant Aß1-42oligomers, selectively captured on the sensor chip. Moreover, with the same technology, we observed that substoichiometric concentrations of clusterin prevent oligomer interaction with the antibody 4G8, suggesting that the chaperone shields hydrophobic residues exposed on the oligomeric assemblies. Finally, we found that preincubation with clusterin antagonizes the toxic effects of Aß1-42oligomers, as evaluated in a recently developedin vivomodel inCaenorhabditis elegans.These data substantiate the interaction of clusterin with biologically active regions exposed on nuclei/oligomers of Aß1-42, providing a molecular basis for the neuroprotective effects of the chaperone.

Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach.

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

Expression and purification of chaperone-active recombinant clusterin.

Clusterin was the first described secreted mammalian chaperone and is implicated as being a key player in both intra- and extracellular proteostasis. Its unique combination of structural features and biological chaperone activity has, however, previously made it very challenging to express and purify the protein in a correctly processed and chaperone-active form. While there are multiple reports in the literature describing the use of recombinant clusterin, all of these reports suffer from one or more of the following shortcomings: details of the methods used to produce the protein are poorly described, the product is incompletely (if at all) characterised, and purity (if shown) is in many cases inadequate. The current report provides the first well validated method to economically produce pure chaperone-active recombinant clusterin. The method was developed after trialling expression in cultured bacterial, yeast, insect and mammalian cells, and involves the expression of recombinant clusterin from stably transfected HEK293 cells in protein-free medium. The product is expressed at between 7.5 and 10 µg/ml of culture, and is readily purified by a combination of immunoaffinity, cation exchange and size exclusion chromatography. The purified product was shown to be glycosylated, correctly proteolytically cleaved into α- and ß-subunits, and have chaperone activity similar to that of human plasma clusterin. This new method creates the opportunity to use mutagenesis and metabolic labelling approaches in future studies to delineate functionally important sites within clusterin, and also provides a theoretically unlimited supply of recombinant clusterin which may in the future find applications in the development of therapeutics.

[The expression and purification of TRPM2 protein in E.coli and preparation of its polyclonal antibody].

OBJECTIVE: To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific polyclonal antibody against human transient receptor potential melastatin 2(TRPM2) protein. METHODS: The DNA fragment encoding evolutionarily conserved N-terminus (1-334 amino acids) of TRPM2 was amplified by PCR. The amplicon was then subcloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL21 (DE3) cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with 1 mmol/L IPTG and purified by GST affinity chromatography. The purified protein was mixed with complete Freund's adjuvant and used to immunize the New Zealand white rabbits with classical 4-injection protocol to generate specific anti-TRPM2 polyclonal antibody. The specificity and titer of the anti-TRPM2 antibody was analyzed by Western blotting. RESULTS: The cDNA fragment encoding N-terminus of human TRPM2 was amplified by PCR and directionally subcloned into pGEX-4T3 plasmid. Under the induction of IPTG, we observed the expression of GST-TRPM2N fusion protein. Polyclonal antibody against human TRPM2 protein was successfully prepared in the rabbits immunized with the purified GST-TRPM2N fusion protein. And the preliminary analysis showed that the anti-TRPM2 antibody could specifically identify transiently expressed TRPM2-EE in HEK293 cells. CONCLUSION: The specific polyclonal antibody against human TRPM2 protein has been successfully prepared, which facilitates our future research on the function of TRPM2 channel.

Beneficial effects of omega-3 fatty acids in the proteome of high-density lipoprotein proteome.

BACKGROUND: Omega-3 poly-unsaturated fatty acids (ω-3 PUFAs) have demonstrated to be beneficial in the prevention of cardiovascular disease, however, the mechanisms by which they perform their cardiovascular protection have not been clarified. Intriguingly, some of these protective effects have also been linked to HDL. The hypothesis of this study was that ω-3 PUFAs could modify the protein cargo of HDL particle in a triglyceride non-dependent mode. The objective of the study was to compare the proteome of HDL before and after ω-3 PUFAs supplemented diet. METHODS: A comparative proteomic analysis in 6 smoker subjects HDL before and after a 5 weeks ω-3 PUFAs enriched diet has been performed. RESULTS: Among the altered proteins, clusterin, paraoxonase, and apoAI were found to increase, while fibronectin, α-1-antitrypsin, complement C1r subcomponent and complement factor H decreased after diet supplementation with ω-3 PUFAs. Immunodetection assays confirmed these results. The up-regulated proteins are related to anti-oxidant, anti-inflammatory and anti-atherosclerotic properties of HDL, while the down-regulated proteins are related to regulation of complement activation and acute phase response. CONCLUSIONS: Despite the low number of subjects included in the study, our findings demonstrate that ω-3 PUFAs supplementation modifies lipoprotein containing apoAI (LpAI) proteome and suggest that these protein changes improve the functionality of the particle.

Presence, localization, and origin of clusterin in normal human spermatozoa.

PURPOSE: Clusterin in mammalian semen is a secretory form of clusterin (sCLU) with the heterodimeric structure. It is secreted by the epididymis and seminal vesicle. It is generally agreed that clusterin mainly exists on the surface of abnormal spermatozoa and is implicated in decreased sperm motility, sperm aggregation and infertility. However, few studies observe clusterin in normal spermatozoa, which is presumed to be a novel form. Up to now, the systematical information about the presence, localization, origin and function of clusterin in normal human spermatozoa has yet not been established. The aim of our current study is to systematically research clusterin in normal human spermatozoa. METHODS: We detected the presence of clusterin via western blot, explored the localization of clusterin using immunofluorescence, and investigated the origin and distribution of clusterin in human testis by western blot and immunohistochemistry. RESULTS: We found native clusterin in the inner plasma membrane of normal human spermatozoa. It was derived from the testis and showed similar molecular weight and heterodimeric structure compared with sCLU in semen and on the surface of abnormal spermatozoa. CONCLUSION: Clusterin in normal spermatozoa should be self-synthesized during the later stage of spermatogenesis. The different localization and origin suggested that the clusterin observed by us may be a novel form compared with conventional sCLU on the surface of abnormal spermatozoa.

Protective effect of clusterin on blood-retinal barrier breakdown in diabetic retinopathy.

Purpose. To investigate whether clusterin attenuates blood-retinal barrier (BRB) breakdown in diabetic retinopathy. Methods. Mice with streptozotocin-induced diabetes and advanced glycation end product-treated human retinal microvascular endothelial cells (HRMECs) were used to determine the effect of clusterin on vascular permeability and tight junction protein expression, through perfusion of retinal vessels with FITC-bovine serum albumin, a [(3)H]sucrose permeability assay, a cell viability assay, Western blot analysis, immunocytochemistry, immunohistochemistry, and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling. Results. Up to 20 mug/mL of clusterin, which is 20 times the effective therapeutic concentration, did not affect the viability of the HRMECs. Moreover, it caused no toxicity in the retina. It effectively inhibited vascular endothelial growth factor-induced hyperpermeability in the HRMECs and the retinas. The antipermeability activity of clusterin was related to the restoration of tight junction proteins. Finally, it was shown to reduce leakage from the vessels in the diabetic retinas and to restore the expression of the tight junction proteins. Conclusions. The data suggest that clusterin, a well-known antipermeability factor naturally secreted by cells, may have therapeutic potential in the treatment of diabetic BRB breakdown.

Cancer cell-derived clusterin modulates the phosphatidylinositol 3'-kinase-Akt pathway through attenuation of insulin-like growth factor 1 during serum deprivation.

Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.

Serum antibodies against prostasomal clusterin in prostate cancer patients.

OBJECTIVE: Clusterin is a ubiquitous secretory sulphated glycoprotein present in prostasomes. It is an anti-apoptotic mediator in prostate cancer and is among the most frequently occurring prostasomal proteins immunogenic in prostate cancer patients. The aim of the present study was to investigate the occurrence of anti-clusterin antibodies in the serum of patients with prostate cancer and whether there is a relationship between anti-clusterin antibody titres and other clinico-pathological variables. MATERIAL AND METHODS: Serum samples were collected from 391 consecutive patients with suspected prostate cancer (150 benign prostate and 241 prostate cancer). The patients' serum samples were used in an ELISA where microtitre wells were coated with purified clusterin from serum of a healthy volunteer. Flow cytometric studies of clusterin and prostasomes were performed. RESULTS: Flow cytometric analyses revealed the presence of clusterin on the surface of seminal prostasomes. Anti-clusterin ELISA titres in sera of patients did not differ significantly from those of a control group. A significant "inverse" correlation existed between anti-clusterin ELISA titres and lymph node metastases (p = 0.047), but only 11 out of 161 patients had metastases. These titres correlated significantly with total prostate (p = 0.021) and transitional zone (p = 0.015) volumes of the patients. CONCLUSIONS: The correlation between serum anti-clusterin antibody titres and other clinico-pathological variables was generally weak in prostate cancer patients, although clusterin has been assigned an important role in tumourigenesis and progression of prostate cancer. However, the anti-clusterin antibody titre appeared to be related to prostate volume, correlating to both transitional zone volume and total volume of the prostate.

Characterization of an eppin protein complex from human semen and spermatozoa.

Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.

The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures.

Clusterin is an extracellular chaperone present in all disease-associated extracellular amyloid deposits, but its roles in amyloid formation and protein deposition in vivo are poorly understood. The current study initially aimed to characterize the effects of clusterin on amyloid formation in vitro by a panel of eight protein substrates. Two of the substrates (Alzheimer's beta peptide and a PI3-SH3 domain) were then used in further experiments to examine the effects of clusterin on amyloid cytotoxicity and to probe the mechanism of clusterin action. We show that clusterin exerts potent effects on amyloid formation, the nature and extent of which vary greatly with the clusterin:substrate ratio, and provide evidence that these effects are exerted via interactions with prefibrillar species that share common structural features. Proamyloidogenic effects of clusterin appear to be restricted to conditions in which the substrate protein is present at a very large molar excess; under these same conditions, clusterin coincorporates with substrate protein into insoluble aggregates. However, when clusterin is present at much higher but still substoichiometric levels (e.g., a molar ratio of clusterin:substrate=1:10), it potently inhibits amyloid formation and provides substantial cytoprotection. These findings suggest that clusterin is an important element in the control of extracellular protein misfolding.