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Chaperonin 60: Molecular, cellular and structural studies to define its role as an information molecule

Khan, Sahar; (2000) Chaperonin 60: Molecular, cellular and structural studies to define its role as an information molecule. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Experimental evidence obtained in this thesis establishes that the purified, recombinant, E. coli chaperonin 60 protein, GroEL, is a potent stimulator of cytokine production by human peripheral blood mononuclear cells (PBMCs) and that this cytokine inducing activity is not due to LPS, or to the proteins that contaminate "purified' GroEL. Surprisingly, trypsinization of GroEL failed to inhibit the cytokine-inducing activity of this protein, and by use of reverse phase HPLC, MALDI-MS and N-terminal sequencing, the bioactive GroEL tryptic peptides were identified. The question as to whether physical interaction was required between GroEL and PBMCs in order to activate the cells was answered by flow cytometry, which demonstrated that leukocytes responding to GroEL, bind the protein whereas cells unresponsive to GroEL do not. Single- and double-labelling experiments revealed that only a proportion of CD-14 positive monocytes in PBMCs bind GroEL. GroEL was also shown to stimulate macrophage cells and cell lines, which suggests that the proportion of monocytes observed to bind GroEL may have a more macrophage-like morphology. Affinity chromatography of surface biotinylated PBMCs on GroEL affinity matrices indicates a 100-150kDa and a 35-40kDa band as possible receptors for GroEL, or as part of the GroEL receptor complex. It has also been demonstrated that GroEL does not stimulate cells via CD14 (a known LPS receptor) as this protein remains active in the presence of anti-CD14 antibodies and is unable to stimulate the CD14 positive cell line Mono Mac-6. These studies support the hypothesis that chaperonin 60 is a bacterial virulence factor able to interact with the host's immune system and induce inflammation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Chaperonin 60: Molecular, cellular and structural studies to define its role as an information molecule
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10104620
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