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Title: Characterisation of a recombinant phosphorylcholine-free form of the immunomodulatory filarial nematode secreted product, ES-62
Author: Watson, C.
ISNI:       0000 0001 3564 3223
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2007
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The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. The gene for ES-62 was expressed in the Pichia pastoris recombinant gene expression system, and an abundant supply of recombinant protein was available for use. When the recombinant protein's biochemical features were compared with parasite-derived tetrameric ES-62, the former was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Differences were found in the recombinant protein's sensitivity to a panel of enzymes when compared to the parasite-derived material, and recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. Evidence was also observed suggesting that although the recombinant material appeared to act similarly to the parasite-derived material in terms of modulation of cytokine production by bone marrow-derived dendritic cells (bmDCs), the mechanism by which this result was achieved appeared to be different from that elucidated for the parasite-derived material: it seemed that the recombinant protein's mode of action was independent of TLR4 or MyD88, in comparison to the parasite-derived ES-62. It was also observed that the recombinant material appeared to have a higher content of mannose than the original protein. Recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycan composition; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and the finger was pointed at the changes in secondary structure. A major take-away message from the data is that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available