Coley, Andrew Mark; (1997) Physical and biochemical characterisation of leucocyte membrane complexes containing GP1-anchored proteins. Doctoral thesis (Ph.D), University of London.

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Abstract

This thesis is a study of the biochemical constituents and physical properties of leukocyte membrane complexes. The complexes studied are rich in a subset of surface glycoproteins which are anchored to the membrane by a glycosylphosphatidylinositol (GPI) moiety. The proteins have no apparent contact with the intracellular environment yet they are able to transduce cellular activation signals. The approach taken in this project was to characterise the physical properties of CD14, Thy-1, and CD59 containing complexes with respect to their solubility in non-ionic detergents. The post-translational acquisition of these physical properties by membrane proteins was also studied and was found to be dependent on the presence of a GPI anchor. The relative amounts of membrane proteins contained within the complexes was established under different solubilising conditions. The association of intracellular signalling enzymes with GPI-anchored proteins has previously been described. This was confirmed for CD14 and the nature of association was investigated both biochemically and microscopically. The results demonstrate that src-family kinase molecules are present in low density complexes present in detergent lysates and that they are associated with GPI-anchored proteins. Kinases are removed from the complexes in a temperature dependent fashion but GPI- anchored proteins remain associated indicating different mechanisms of association with the complexes. Analysis of the lipid heterogeneity within the complexes was performed under different solubilising temperatures, the results of which led to the conclusion that the physical properties of the GPI-anchored proteins within the complexes reflected those of the lipids contained in the complexes. The distribution of GPI-anchored proteins on the cell surface was examined by confocal microscopy in capping experiments, as was their association with specific kinase enzymes and their catalytic products, in an attempt to demonstrate their physical and functional association in vivo. Finally plasmids encoding GPI-anchored and transmembrane forms of the green fluorescent protein were generated. The cellular distribution of the products of the two constructs were investigated in transiently transfected Cos-7 cells. Results demonstrate an altered cellular distribution of the products of the two newly generated plasmids when compared with the product of the native GFP plasmid indicating that these will provide to be useful tools in the study of GPI-anchored protein maturation and behaviour at the cell surface

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