Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

An investigation into the role of Bcl-3 in toll-like receptor signalling

Through the recognition of potentially harmful stimuli, Toll-like receptors (TLRs) initiate the innate immune response and induce the expression of hundreds of immune and pro-inflammatory genes. TLRs are critical in mounting a defence against invading pathogens however, strict control of TLR signalling is vital to prevent host damage from excessive or prolonged immune activation. In this thesis the role of the IκB protein Bcl (B-cell lymphoma)-3 in the regulation of TLR signalling is investigated. Bcl3-/- mice and cells are hyper responsive to TLR stimulation and are defective in LPS tolerance. Bcl-3 interacts with and blocks the ubiquitination of homodimers of the NF-κB subunit, p50. Through stabilisation of inhibitory p50 homodimers, Bcl-3 negatively regulates NF-κB dependent inflammatory gene transcription following TLR activation. Firstly, we investigated the nature of the interaction between Bcl-3 and p50 and using peptide array technology. Key amino acids required for the formation of the p50:Bcl-3 immunosuppressor complex were identified. Furthermore, we demonstrate for the first time that interaction between Bcl-3 and p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3. Using the data generated from peptide array analysis we then generated cell permeable peptides designed to mimic Bcl-3 function and stabilise p50 homodimers. These Bcl-3 derived peptides are potent inhibitors of NF-κB dependent transcription activity in vitro and provide a solid basis for the development of novel gene-specific approaches in the treatment of inflammatory diseases. Secondly, we demonstrate that Bcl-3 mediated regulation of TLR signalling is not limited to NF-κB and identify the MAK3K Tumour Progression Locus (Tpl)-2 as a new binding partner of Bcl-3. Our data establishes role for Bcl-3 as a negative regulator of the MAPK-ERK pathway.