Ioannou, Y.; (2007) Investigating the molecular targets of antiphospholipid antibodies. Doctoral thesis , University of London.

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Abstract

The antiphospholipid syndrome (APS) is characterised by the presence of pathogenic antiphospholipid antibodies (aPL) that bind phospholipid via the N-terminal domain I (DI) of the protein cofactor beta2-glycoprotein I (beta2GPI). This thesis aims to investigate and identify the nature of this interaction at the molecular level. In order to study DI, a system for expressing and purifying sufficient quantities of this protein was developed using Escherichia coli (E.coli) as the expression host. This was achieved primarily by synthesising a gene with all codons optimised for E.coli, tight regulation of expression and recovery of DI from the periplasm to facilitate folding. Recombinant purified DI bound clinically relevant human monoclonal and polyclonal purified IgG aPL in both solid an fluid phase assays. Hypotheses were generated that identified candidate epitopes for aPL binding as potentially involving residues D8, D9, E23, E26, E27, R39, G40, R43 and the DI-II interlinker region. In total, 15 mutants of DI targeting these areas were created and computer modelling employed to predict the likely structural effects of these mutations upon DI. Expressed DI mutants were then tested in both solid and fluid phase assays for binding to polyclonal IgG derived from patients with APS and compared to wild-type DI. Some mutations, such as those targeting R39, caused loss of binding to aPL in the fluid phase whilst others caused enhanced binding over wild-type DI. In conclusion, this thesis demonstrates that DI of 62GPI may be expressed using E.coli and binds clinically relevant IgG aPL. Detailed mutational studies support the concept that aPL bind discontinuous epitopes on DI involving regions D8 and D9, R39 to R43 and the DI-II interlinker which are in close proximity to each other in the tertiary structure. The ability to produce a mutant of DI with enhanced binding over wild-type holds therapeutic potential.

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