Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry

Fairburn, Barbara; Muthana, Munitta; Hopkinson, Kay; Slack, Laura K.; Mirza, Shabana; Georgiou, A. S.; Espigares, Elena; Wong, Chi Huey; Pockley, A. Graham

doi:10.1016/j.biochi.2006.04.004

Cited by 13 publications

(10 citation statements)

References 56 publications

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“…The purification of HSPC4 typically involves a sequence of ammonium sulphate precipitation, affinity chromatography on Concanavalin A-Sepharose, passage through a P10 size exclusion column and a final purification using anion affinity chromatography (Wearsch and Nicchitta 1996;Srivastava 1997;Fairburn et al 2006). It has been reported that gp96 purified using a Concanavalin A affinity purification step contains sub-stoichiometric amounts of Concanavalin A which can promote the formation of HSPC4-ConA oligomers, enhance the immunological activity of HSPC4 as a consequence (Monks et al 2005) and itself activate T cells due to its lectin-mediated mitogenic activity (ArnoldSchild et al 2000).…”

Section: Tuberculosis Hsp70 and Host Immunity (Tl)mentioning

confidence: 99%

Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants

Henderson

Calderwood

Coates

et al. 2010

Cell Stress and Chaperones

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In recent years, it has been hypothesised that a new signalling system may exist in vertebrates in which secreted molecular chaperones form a dynamic continuum between the cellular stress response and corresponding homeostatic physiological mechanisms. This hypothesis seems to be supported by the finding that many molecular chaperones are released from cells and act as extracellular signals for a range of cells. However, this nascent field of biological research seems to suffer from an excessive criticism that the biological activities of molecular chaperones are due to undefined components of the microbial expression hosts used to generate recombinant versions of these proteins. In this article, a number of the proponents of the cell signalling actions of molecular chaperones take this criticism head-on. They show that sufficient evidence exists to support fully the hypothesis that molecular chaperones have cell-cell signalling actions that are likely to be part of the homeostatic mechanism of the vertebrate.

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Section: Tuberculosis Hsp70 and Host Immunity (Tl)mentioning

confidence: 99%

Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants

Henderson

Calderwood

Coates

et al. 2010

Cell Stress and Chaperones

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“…Gp96 was purified from Wistar rat livers essentially as described (Srivastava 1997;Fairburn et al 2006). Briefly, livers were homogenized in phosphate-buffered saline (PBS) containing the protease inhibitor phenylmethylsulfonylfluouride (0.2 mM, 60 mL of buffer per 30 g of tissue) with a Waring Laboratory Science laboratory blender and stainless steel minicontainer (Christison Particle Technologies Ltd, Gateshead, UK).…”

Section: Purification Of Gp96mentioning

confidence: 99%

The stress protein gp96 is not an activator of resting rat bone marrow–derived dendritic cells, but is a costimulator and activator of CD3+ T cells

Mirza¹,

Muthana²,

Fairburn³

et al. 2006

Cell Stress Chaper

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Although low doses of tumor-derived stress protein gp96 elicit protective immunity to the tumor from which it is isolated, protection is lost at high doses because of the induction of immunoregulatory CD4 ϩ T cells. This study evaluated the influence of gp96 on resting rat bone marrow-derived dendritic cells (BMDCs) and purified CD3 ϩ T cells. In contrast to previous reports, gp96 had no effect on adhesion and costimulatory molecule expression by BMDCs, nor did it influence interleukin (IL)-10 and IL-12 secretion or their allostimulatory capacity. Gp96 did not bind to BMDCs but dose-dependently bound to CD4 ϩ and CD8 ϩ T cells. At low concentrations (1 and 25 g/mL), gp96 acted as a costimulator of CD3 ϩ T cells, inducing proliferation and the secretion of interferon (IFN)-␥ and IL-10. Gp96 also increased the proliferation of CD28-costimulated CD3 ϩ T cells and their secretion of IFN-␥, IL-4, and IL-10. Gp96 had no effect at higher concentrations (50 and 100 g/mL), despite the occurrence of cell surface binding at these concentrations. These findings indicate that gp96 can act as a costimulatory molecule for CD3 ϩ T cells, and an observed increase in the IL-10:IFN-␥ secretion ratio induced by gp96 suggests that it might, at appropriate concentrations, promote a regulatory T-helper 2 (Th2)-like phenotype.

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“…This purification approach reproducibly yields approximately 30-60 μg gp96 per gram wet weight of liver tissue with an endotoxin content which is routinely less than 0.001 EU (0.1 pg) per μg gp96 [64]. Analysis of gp96 preparations using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry reveals that the procedure for purifying gp96 is reproducible, as similar protein profiles are observed in replicate gels of gp96 preparations [64].…”

mentioning

confidence: 96%

“…Gp96 can be purified from Wistar rat and C57BL/6 mouse livers as described below [64]. All laboratory equipment used in the purification procedure should be baked overnight at 220°C and all solutions should be prepared using autoclaved ultrapure H 2 O (e.g.…”

Section: Preparation and Analysis Of Purified Gp96mentioning

confidence: 99%

“…Analysis of gp96 preparations using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry reveals that the procedure for purifying gp96 is reproducible, as similar protein profiles are observed in replicate gels of gp96 preparations [64]. The purity of the preparations is typically around 70%, with minor copurified proteins of varying molecular weights and mobilities being present [64].…”

mentioning

confidence: 99%

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A non-receptor-mediated mechanism for internalization of molecular chaperones

Pockley

Fairburn

Mirza

et al. 2007

Methods

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The evolving realization that stress proteins, which have for many years been considered to be exclusively intracellular molecules under normal conditions, can be released from viable cells via a number of potential routes/pathways has prompted interest into their extracellular biology and intercellular signaling properties. That the stress proteins Hsp60, Hsp70 and gp96 can elicit both proand anti-inflammatory effects suggests that these molecules play a key role in the maintenance of immunological homeostasis, and a better understanding of the immunobiology of extracellular stress proteins might reveal new and more effective approaches for controlling and managing infectious disease, inflammatory disease and cancer. A number of cell surface receptors for stress proteins have been identified, and the intracellular consequences of these cell surface receptor-ligand interactions have been characterized. To date, studies into the intercellular signaling properties of stress proteins and their interactions with antigen presenting cells have focused on specific receptor-mediated uptake, and have not considered the fact that such cells can also take up proteins via non-specific endocytosis/pinocytosis. Herein we present a methodological approach for assessing receptormediated and non-receptor mediated uptake of gp96 by rat bone marrow-derived dendritic cells.

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Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry

Cited by 13 publications

References 56 publications

Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants

Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants

The stress protein gp96 is not an activator of resting rat bone marrow–derived dendritic cells, but is a costimulator and activator of CD3+ T cells

A non-receptor-mediated mechanism for internalization of molecular chaperones

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