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Title: Molecular Characterisation of the Chemokine CXCL16 and its Receptor CXCR6
Author: Petit , Sarah Joanna
ISNI:       0000 0004 2668 6063
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2008
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The chemokine CXCL16 is selectively expressed by antigen presenting cells and unli ke most chemokines, is attached to the cell surface by means of a mucin stalk. This allows CXCL16 to function as an adhesion molecule, binding leukocytes such as T-cells which display the cognate receptor, CXCR6. Cleavage of CXCL16 by metalloproteinases can also release a soluble form of CXCLI6 which can induce the migration of CXCR6 expressing cells. In addition, membrane-bound CXCL16 can also ftinction as a scavenger receptor for a variety of ligands, including oxidised low density lipoprotein. Collectively, these functions support a role for both CXCL16 and CXCR6 in the process of atherosclerosis. The CXCL16:CXCR6 relationship appears to be monogamous and the nature of this interaction was investigated by a programme of mutagenesis used to examine CXCR6 function in vitro, assessed by adhesion, chemotaxis and ligand binding assays. CXCL16 function was also examined, in particular, the effects of a single nucleotide polymorphism associated with disease. In a third arm of the project, recombinant CXCL16 was produced in E. Coli using two different systems and subjected to assays of biological function. In contrast to other chemokine receptors, mutation of the CXCR6 N-terminus and inhibition of post-translational modifications were without effect on function. Likewise, N-terminal extension of CXCL16 by 24 amino-acids resulted in a protein with decent potency and efficacy in chemotaxis and not as anticipated, a CXCR6 antagonist. Cells expressing the CXCR6 mutants Asp-176-Asn and Glu274- Gln were unable to interact with soluble CXCLI6, suggesting that these residues are critical for ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the Glu-274-Gln mutant was able ·to promote robust adhesion to membrane-anchored CXCL16, suggesting that the soluble and membrane-bound forms of CXCL16 possess distinct conformations. Glycosylation of CXCL16 was found to be critical for cell-cell adhesion function, and a Ala-181-Val polymorphic variant of CXCL16 was found to have no capacity for mediating cell-cell adhesion, suggesting a potential relevance of this mutation in disease. Collectively, the data suggests that the CXCL16:CXCR6 interaction is distinct from other chemokine-chemokine interactions described in the literature, and that current models for chemokine receptor activation, in which the chemokine N-terminus ligand protrudes into an intrahelical pocket, do not apply to the CXCR6:CXCLI6 interaction. This may have implications for successful antagonism of the receptor by small molecules.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available