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Title: Nuclear translocation and transferrin-transferrin receptor interaction of IPSE/[alpha}-1, a secretory glycoprotein from Schistosoma mansoni
Author: Kaur, Ishwinder
ISNI:       0000 0004 2682 5457
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2009
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Helminthic parasites have evolved with immune modulating machinery to manipulate their host's immune response and thus survive unscathed for years and in some cases even decades. However, the underlying molecular mechanisms governing the host-parasite relationship are still largely unknown. Therefore detailed investigation and evaluation of parasitic molecules is desirable. One such molecule worthy of attention is IPSE/a-1 (lnterleukin-4 inducing principle from schistosome eggs). IPSE/a-1 is a secretory glycoprotein produced exclusively by the egg stage of Schistosoma mansoni, which activates human basophils in non-antigen specific IgE dependent mechanisn;t. Sequence analyses of IPSE/a-1 using bioinformatic subcellular localisation prediction tools revealed a putative nuclear localisation signal (NLS) at the C-terminus. The present work was conducted to confirm whether this sequence ('125-PKRRRTY-131') was both necessary and sufficient for nuclear localisation of IPSE/a-1 and other heterologous GFP proteins. A plasmid encoding EGFP-IPSE/a-1, as well as a truncated mutant lacking amino acids 125-134, was transfected into Huh7 and U2-0S cell lines, and fluorescence of the fusion protein was determined by confocal laser scanning microscopy. EGFP-IPSE/a-1 was found to be exclusively nuclear, whereas the mutant displayed both nuclear and cytoplasmic staining. Furthermore, insertion of the IPSE/a-1 NLS into a tetra-EGFP construct showed nuclear localisation, and alanine scanning mutagenesis revealed a requirement for the KRRR residues. IPSE/a-l also binds to transferrin, which lead to downstream effect on cellular proliferation. Besides, fluorescence microscopy revealed that recombinant IPSE/a-l protein added exogenously to culture medium was internalized by variant Chinese hamster ovary (CRO) cells expressing the human transferrin receptor and was found in the nuclei of these cells Western blotting further confirmed this temporal relocalisation of IPSE/a-l from cytosolic to nuclear fractions. In addition, IPSE/a-l exhibited a DNA-binding activity that appeared to be dependent on the C-terminal NLS sequence. In summary, the main achievement of this work is the identification and characterization of a NLS in IPSE/a-l that is functional in mammalian cells, which will form the basis for further investigations into the biological significance of this nuclear targeting and DNA interaction e.g. IPSE/a-l may function as transcription factor in the nucleus. The properties of IPSE/a-l described here also make it an interesting potential vehicle for intracellular and nuclear drug delivery.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available