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Characterisation of the mouse glycosyl phosphatidylinositol-phospholipase D (GPI-PLD) gene

Flores-Borja, Fabian; (2003) Characterisation of the mouse glycosyl phosphatidylinositol-phospholipase D (GPI-PLD) gene. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a serum protein presumed to cleave GPI-anchored proteins on the cell surface. As the initial characterisation of GPI-PLD suggested that more than one gene coded for this enzyme, the work described in this thesis characterised the mouse GPI-PLD gene. A full-length clone isolated from a mouse liver cDNA library was sequenced. Although it showed high homology with the recently reported sequence from an islet cell-derived GPI-PLD gene, a combination of Southern blot and RNAse protection assays demonstrated that only one GPI-PLD gene, with a sequence identical to the liver cDNA, is present in the mouse genome. The main sources of GPI-PLD are liver and brain, although RT-PCR showed expression in macrophage and pancreatic cell lines, suggesting that most cells express GPI-PLD. High GPI-PLD expression levels in liver were detected in CBA/Ca and diabetic-NOD mice, whilst obese (ob/ob) and insulin-resistant (db/db) mice showed relatively lower levels of expression. These results suggest a possible role for insulin in the regulation of GPI-PLD expression. Co-transfection of GPI-PLD and placental alkaline phosphatase (PLAP) resulted in cleavage of PLAP from the cell surface. Co-expression of anti-sense GPI-PLD demonstrated that PLAP cleavage was catalysed by transfected GPI-PLD. However, no evidence of endogenous GPI-PLD activity on endogenous GPI-anchored proteins was obtained. To study the cell-specific localisation of GPI-PLD expression plasmids encoding GPI- PLD fused to different tags (GFP, Flag and Myc) were constructed. A significant amount of GPI-PLD produced following transfection of these plasmids into mammalian cell lines remains cell associated. A combination of sucrose gradients subcellular fractionation and microscopy analysis suggest that GPI-PLD is associated with a Golgi-related compartment. No association with caveolae was observed even following co-expression with wild type or mutant forms of caveolin-3. These results support the hypothesis of an intracellular site of action for GPI-PLD.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterisation of the mouse glycosyl phosphatidylinositol-phospholipase D (GPI-PLD) gene
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Phospholipase
URI: https://discovery.ucl.ac.uk/id/eprint/10102628
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