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Title: A study of the structure and biological activity of the Eranthis hyemalis type II ribosome inactivating protein
Author: McConnell, Marie-Therese
ISNI:       0000 0004 6058 9734
Awarding Body: Canterbury Christ Church University
Current Institution: Canterbury Christ Church University
Date of Award: 2016
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Plant lectins and Ribosome inactivating proteins (RIPs) have been studied for well over a century. The first published work on the canonical RIP, Ricin, from Ricinis communis being characterised by Stillmark in his doctoral thesis in 1888. These proteins are characterised by the ability to selectively and reversibly bind to the carbohydrate groups of glycoconjugates, agglutinate erythrocytes and, in the case of RIPs, act as RNA glycosidases, depurinating a specific adenine residue within the highly conserved ricin/sarcin loop of the 28S ribosomal sub unit. Lectins and RIPs are used extensively in biotechnology and biomedical research. Winter Aconite Lectin (EHL) extracted from the corms of the Eranthis hyemalis plant was previously identified as having the characteristics of a Type II RIP, and therefore provided a novel target for functional characterisation studies. Homologous proteins have shown potential as immunotoxins and diagnostic tools in cancer research, mainly due to the changes in glycosylation patterns of tumour cells such as, expression of the Tn antigen rarely found in healthy tissue. As EHL has a glycomic binding profile that has highest affinity for N-acetyl-Dgalactosamine, the defining carbohydrate unit of the Tn antigen, it therefore offers an interesting target for exploring the biological structure, function and effects on model organisms and cell lines. The first aim of the research reported within this thesis was to establish an extraction and purification protocol, which would provide a homogeneous, pure and consistent yield of EHL in sufficient quantity to allow further study of the characteristics and structure. Once established, toxicity studies were carried out using Caenorhabditis elegans to examine the effect of EHL in a biological system. The data collected indicated that EHL binds to the amphid neurons in C.elegans and causes a wide variety of concentration dependent effects including abnormal dauer formation. In order to elucidate the structure of the protein, crystallographic screening was undertaken. A number of large single crystals produced diffraction data. A model structure was constructed using molecular replacement for phasing. The model is presented, as a work in progress with interesting observations as to potential glycosylation sites and active site residues. A preliminary investigation into the viability of a conjugation of EHL to gold nanoparticles was undertaken in collaboration with The Bioscope group (University of Lisbon) and the conjugate was also tested for biological activity. Initial results indicated that the nanoparticles caused a cessation of the toxic effect of EHL. In the final stages of the research presented herein, EHL was tested for antineoplastic activity against a number of cancer cell lines using a variety of methods to develop a viable protocol for future studies. Preliminary results indicate that EHL may act as a mitogenic agent on some cell lines, which also presents an opportunity for onwards research.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH0301 Biology (General)