Doctor of Philosophy
School of Biological Sciences
Dabbs, Rebecca A., The potential of clusterin to act as an immune modulator, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2013. http://ro.uow.edu.au/theses/4019
Clusterin (CLU) was the first described extracellular mammalian chaperone and can interact with misfolded proteins to stabilise them in a soluble form, until the bound protein can be re-folded or degraded. In the last twenty years there has been an interest in using chaperones as adjuvants of the immune system. Numerous, mainly intracellular, chaperones have been investigated in this context and some are able to elicit a specific, cytotoxic T lymphocyte (CTL) response and in some cases a humoral response against bound antigens.
The first aim of this project was to develop a chaperone-active form of recombinant CLU (rCLU), as forms of rCLU described in published reports or currently commercially available have not been thoroughly characterised or tested for chaperone activity, and in many cases are impure. A bacterial expression system was first investigated which yielded high molecular weight aggregates of rCLU; the protein was not glycosylated or cleaved into α- and β-chains, and lacked chaperone activity. A mammalian expression system was next trialled; previous attempts in our laboratory to produce pure rCLU by mammalian expression failed due to the chaperone activity of CLU resulting in it binding to misfolded proteins in the culture medium, which then co-purified with the rCLU. Therefore an innovative approach was required to overcome this problem, whereby a polyclonal culture of HEK293 cells expressing rCLU were allowed to adhere to the surface of tissue culture flasks in medium containing foetal calf serum (FCS), before replacement with FCS-free media. Under these conditions cells remained attached to the flask surface and secreted rCLU into the medium until dead. Using this approach, rCLU was successfully expressed and purified, and shown to have chaperone activity comparable to that of wild type (wt)CLU; there were only minor client protein-specific differences in activity between the rCLU and wtCLU that may be attributable to differences in glycosylation between the two forms of CLU.
The mechanism by which chemically linked complexes formed between CLU and ovalbumin (i.e. CLU-OVA) were bound and internalised by a range of cells was next investigated. CLU-OVA and wtCLU showed significantly higher binding to murine bone marrow derived macrophages, human granulocytes, monocytes and to a lower extent lymphocytes, than OVA or control ligands. Confocal microscopy studies indicated that CLU-OVA was rapidly internalised by murine bone marrow derived macrophages, in less than 15 min, and subsequently co-localised with both early endosomes and lysosomes. The binding of CLU-OVA was inhibitable by fucoidan and polyinosinic acid, implicating scavenger receptors in the uptake CLU-OVA into these cell types.
The final aim of the project was to determine whether CLU could act as an adjuvant of the immune system, to elicit both a CTL response and a humoral response when bound to the model antigen OVA. The results obtained from an in vivo cytolysis assay suggest that injection with CLU-OVA or CLU-OVA and a strong adjuvant (Complete Freund’s Adjuvant; CFA) is able to stimulate an OVA-specific CTL response. Relative to mice immunised with OVA or PBS alone, the levels of interleukin 2 and interferon gamma contained within CD8+ T cells isolated from the draining lymph nodes and secreted from cells obtained from the draining lymph nodes or spleen were significantly greater in mice immunised with CLU-OVA + CFA, suggesting that the CD8+ T cells from this treatment group were undergoing the highest levels of proliferation and had the highest gain of (CTL) effector function. These experiments, however, need to be repeated with more replicates to confirm these interpretations. Following immunisation with CLU-OVA or CLU/OVA (complexes formed using the chaperone action of CLU), mice mounted a mature immune response to OVA of a predominantly IgG1 isotype. Immunisation with CLU-OVA and CLU/OVA also significantly enhanced the production of OVA-specific IgM antibodies compared to mice immunised with OVA, or other controls.
The results from this study provide a practical method to successfully express and purify chaperone active, correctly post-translationally modified rCLU, thereby opening the path for future studies to identify functional sites within CLU. It also provides a theoretically unlimited supply of rCLU which may find future application in the development of therapeutics. Similar to the results obtained for intracellular chaperones that act as adjuvants of the immune system, CLU appears to direct bound proteins for uptake by a wide range of cells via scavenger receptors. Further work is required to definitively establish whether CLU can induce an antigen specific CTL response, as is suggested by the results of this study. If CLU is able to activate both a CTL and an antibody response to bound antigen, then CLU may provide a useful vector for the development of vaccines to overcome the current limitations of vaccines based on the use of intracellular chaperones.