D. Phil. Thesis
Protein structures and interactions at the leukocyte cell surface
University College
Trinity Term, 2002
Abstract
Leukocyte cell-cell communication relies on interactions between proteins expressed at the cell surface. An understanding of this process requires insights into both the complement of proteins involved and the nature of their interactions. Addressing both issues, detailed studies of the structures and binding properties of a sample set of proteins, the CD2 subset of the immunoglobulin superfamily, and a serial analysis of gene expression (SAGE)-based study of the gene expression patterns of a cytotoxic T cell clone, were undertaken. Structural and mutational studies of the rat CD48-CD2 interaction showed that low affinity, high specificity recognition at the cell surface by these molecules is characterised by poor shape complementarity and multiple electrostatic contacts, extending a paradigm established for human CD2-CD58 binding. However, these studies revealed a degree of heterogeneity in the detailed structural mechanisms involved. The differences observed accounted for the triple specificity of rat CD48, which was shown to bind two distinct CD244-like proteins in rat. Analysis of another member of the CD2 subset, CD150, supported the hypothesis that this family evolved from a single homophilic ancestor. A systematic search of the human genome was undertaken to identify the remaining members of this subset, revealing three novel genes and four pseudogenes. The identification of new members of this established family suggested that many leukocyte molecules might remain to be characterised. This was confirmed with the SAGE analysis, which showed that at least 40% of the molecules involved in T-cell immune functions are functionally uncharacterised. Although we now have a good understanding of the nature of protein interactions at the leukocyte cell surface, a complete understanding of the full network of interactions involved remains a rather distant goal.
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Full thesis: “Protein structures and interactions at the leukocyte cell surface.”
Chapter 1: Introduction
Chapter 2: Cloning of rat CD244 (2B4) homologues and analysis of their interaction with CD48
Chapter 3: Production of proteins for structural analysis
Chapter 4: CD48 structure and interactions with CD2
Chapter 5: Homophilic interaction of CD150 (SLAM)
Chapter 6: Extending the CD2 subset of the IgSF
Chapter 7: Serial analysis of gene expression in CTL
Chapter 8: General Discussion
Abbreviations, Appendices & Bibliography