title: Structure and function studies of the filarial nematode excretory-secretory glycoprotein ES-62
Creators: name: Ackerman, Claire Jennifer
Publisher: name: University of Glasgow
Year: 2002

description: ES-62 is a filarial nematode excretory secretory glycoprotein of mass 57.8 kDa, from the rodent filarial nematode Acanthocheilonema viteae. ES-62 has been shown to possess immunomodulatory capabilities, some of which can be attributed to the phosphorylcholine moieties that are attached to certain of the carbohydrate chains of ES-62. Studies of the effects of ES-62 on B lymphocytes have shown that ES-62 downregulates B lymphocyte activation by selectively targeting protein kinases, such as ErkMAPKinase, downstream of the B cell receptor resulting in immunosuppression of the host immune system. ES-62 has been shown to have a similar effect in T lymphocytes. This thesis describes the search for structural information about ES-62. Three main techniques were employed to characterise the low resolution structure of ES-62; bioinformatics, analytical ultracentrifugation (AUC) and small angle X- ray scattering (SAXS). Bioinformatics techniques identified six proteins homologous to ES-62 on the basis of primary structure and six proteins homologous on the basis of secondary structure. Homology modelling of ES-62 in its entirety was not possible due to a lack of structural information about the six proteins homologous to ES-62 on the basis of amino acid sequence. However, residues 252-343 of ES-62 were modelled due to the homology of this region with residues 74-168 of a leucy1 aminopeptidase from Aeromonas proteolytica for which the structure is known. AUC and SAXS techniques demonstrated that ES-62 exists mainly in a tetrameric state and is slightly elongated in structure. A low resolution structure of ES-62 was also obtained using DAMMIN (Svergun,1999), a computer program which allows the ab initio determination of a three-dimensional structure from the small angle scattering curve of a protein. In addition, this thesis describes the establishment of two recombinant ES-62 expression systems, one of which was unsuccessful due to the aggregation of the expressed protein and the other, while producing rES-62, was contaminated by the presence of a compound absorbing at 260 nm resulting in the rES-62 produced being unsuitable for biochemical or biophysical studies. The search for the receptor through which ES-62 interacts with cells and the fate of ES-62 following this interaction are also discussed. These studies demonstrate that ES-62 binds to specific proteins on the surface of different immune system cells but unfortunately attempts to identify these proteins have not so far yielded a definitive answer. ES-62 has also been shown to locate to both the nucleus and cytoplasm following interaction with cells and appears to be present in different forms in these two locations. This demonstrates that ES-62 is internalised following receptor binding and may induce some of its immunomodulatory effects through interactions with intracellular proteins. Its presence in the nucleus of cells could explain the modulation of transcription factors shown to occur in B lymphocytes following exposure to ES-62.