title: Characterisation of a recombinant phosphorylcholine-free form of the immunomodulatory filarial nematode secreted product, ES-62
Creators: name: Watson, Caitlin
Publisher: name: University of Glasgow
Year: 2007

description: 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. In order to further investigate this, I attempted to produce ES-62 lacking PC by using a variety of enzymes and enzyme inhibitors. The resultant material was, unfortunately, never 100 % PC-free, and the results were also highly variable. As an alternative approach it was therefore decided to place the gene for ES 62 into the Pichia pastoris recombinant gene expression system. It was hoped that the gene expression system, being eukaryotic in nature, would side-step solubility issues previously encountered when an Escherichia co//-based system was attempted, as it would be able to perform the post-translational modifications necessary for production of a correctly folded protein, whilst avoiding hyperglycosylation issues frequently encountered when using a Saccharomyces cerevisiae-based system. The procedure was successful, and an abundant supply of recombinant protein was available for use. When the recombinant protein's biochemical features were compared with parasite-derived ES-62, which has been shown to be tetrameric in form, the former was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein including a monoclonal antibody (mAb) that recognised a conformational determinant. However, subsequent to this, 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, an unexpected finding considering the evidence that Pichia Is much less likely to hyperglycosylate than other yeast-based recombinant gene expression systems such as S.cerevisiae. In an attempt to explain the differences observed, 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 tine 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.