Principal Supervisors: Dr Sarah L. Horswell and Liam R. Cox, School of Chemistry
Co-supervisor: G. Besra
PhD project title: Understanding the interactions between glycosylceramindes, saposins and different regions of the cell membrane
University of Registration: Birmingham
The human immune system is capable of responding to a wide range of molecular stimuli, to produce a specific immune response. Whilst much of the immunology literature has focused on the role of peptide antigens in generating an immune response, a set of T cells known as invariant natural killer T-cells (iNKT) are activated by a very different class of molecules, namely lipids. The mechanism of activation involves a protein known as CD1d, which binds a range of lipids. Depending on the structure of the lipid, the resulting CD1d protein–lipid complex can be recognised by receptors located on the cell surface of iNKT cells. This recognition event serves to activate the iNKT cell to produce and then secrete a wide range of cytokines, thus initiating an immune response. The structure of the lipid that is ‘presented’ by the CD1d protein to the iNKT cell receptor has been shown to affect the secreted cytokine profile, which in turn dictates the nature of the resulting immune response, opening up the possibility of using this mechanism of immune system activation to target a range of diseases. The objective of this project is to elucidate the mechanism by which promising glycosylceramide drug molecules interact with cell membranes. These glycolipids locate into cell membranes, from which they are extracted by saposins, which then load the lipids on to the CD1d molecule for subsequent presentation to iNKT cells. That the saposin molecule extracts lipids from a cell membrane raises some important questions about the location of the lipids within the membrane and their effect upon its local structure: the way in which these molecules partition into the cell membrane is not yet understood.
We propose a study of the thermodynamic phase behaviour of several glycosylceramides and of their mixtures with model membranes of controlled composition that mimic natural membranes. In this project we shall focus on mimics of lipid raft regions of the cell plasma membrane. These measurements will inform structural studies of the incorporation of the glycosyl ceramides with the model membranes, which will be used to determine the nature of the interaction between the glycosylceramide and lipid matrix (e.g. are intermolecular hydrogen-bonding effects significant or is packing determined predominantly by geometric considerations?) and its effect on the membrane structure. In this way we anticipate being able to address questions such as why some molecules partition into lipid rafts and some do not, how and why they can be extracted, and why some molecules are more effective than others in effecting an immune response.
The project will involve initially synthesis of lipids, including isotopically labelled lipids and fluorophore-labelled lipids. This will be followed by physical measurements of the phase behaviour and structure of monolayers at the air/water interface and of solid-supported bilayers. These measurements will involve atomic force microscopy (to image bilayers and to determine mechanical properties), x-ray reflectivity (to determine structure and solvation of the headgroup regions and also alterations in lipid ordering within layers) and neutron reflectivity (to determine structure in the direction normal to the plane of the bilayer – isotopic substitution will be a very powerful means of labelling specific positions within the bilayer). Vibrational spectroscopy will provide information on orientation of headgroups and hydrocarbon chains, organisation of molecules and intermolecular interactions. Infrared spectroscopy and Raman scattering provide complementary information and will be used in conjunction with one another. Again, isotopic substitution will be beneficial for these studies. A parallel project within the group (in collaboration with Mechanical Engineering) is developing new spectroscopic techniques for investigating lipid layers and there is scope for the MIBTP2 student to make use of these techniques as part of his/her project. The information gained from these measurements will be fed back into the design of new molecules. Some testing of promising molecules in cell lines will be possible towards the later stages of the project.
- V. Lawson, Turned on by danger: activation of CD1d-restricted invariant natural killer T cells Immunology 2012, 137, 20-27.
- J. Wojno, J.-P. Jukes, H. Ghadbane, D. Shepherd, G.S. Besra, V. Cerundolo, L.R. Cox, Amide analogues of threitol ceramide and a-galactosyl ceramide as CD1d agonists for modulating iNKT-cell-mediated cytokine production ACS Chemical biology 2012, 7, 847-855.
- J.-P. Jukes, U. Gileadi, H. Ghadbane, T. F. Yu, D. Shepherd, L. R. Cox, G. S. Besra, V. Cerundolo, Eur. J. Immunol. 2016, 46, 1224–1234.
- A.R. Hillman, K.S. Ryder, E. Madrid, A.W. Burley, R.J. Wiltshire, J. Merotra, M. Grau, S.L. Horswell, A. Glidle, R.M. Dalgliesh, A. Hughes, R. Cubitt, A. Wildes, Structure and dynamics of phospholipid bilayer films under electrochemical control Faraday Discussions 2010, 145, 357–379.
- E. Madrid and S.L. Horswell, Effect of Headgroup on the Physicochemical Properties of Phospholipid Bilayers in Electric Fields: Size Matters Langmuir 2013, 29, 1695–1708. 5. E. Madrid and S.L. Horswell, Effect of Electric Field on Structure and Dynamics of Bilayers Formed From Anionic Phospholipids. Electrochimica Acta 2014, 146, 850–860.
BBSRC Strategic Research Priority: Food Security
Techniques that will be undertaken during the project:
- Synthetic techniques for molecules of biological interest (primarily phospholipids, glycolipids and sphingolipids, including specific labelling of molecules with fluorophores and/or isotopes)
- Measurement of thermodynamic phase behaviour of model membrane systems and deposition of model membranes onto a variety of substrates with a variety of means
- Microscopy of model membrane systems: Brewster angle microscopy (at Diamond), atomic force microscopy, fluorescence microscopy
- Infrared Spectroscopy and Raman Scattering of model membrane systems and its application to intermolecular interactions between drugs and target membranes
- X-ray reflectivity and neutron reflectivity (subject to beamtime applications)
- Potentially cell line testing toward the end of the project