No description available.

No description available.

Structure and sequence can be used to trace the evolution of a protein domain over the course of several hundred million years. Here it is shown that the immunoglobulin I-set is present in sub-vertebrates, whereas the C1, C2, V and I-sets are found in vertebrates. This implies that the I-set is the ancestral set and that the Cl, C2 and V-sets arose from I-set ancestors. The I-set arose at least 800 million years ago, and the C1, C2 and V-sets diverged from the I-set some 400 million years ago. Phylogenetic analysis of immunoglobulin kinases shows that they have arisen by at least two independent associations with immunoglobulin-like domains, once with receptor tyrosine kinases and once with intracellular serine/threonine muscle kinases. Prokaryotic examples of the immunoglobulin superfamily are discovered in two bacterial proteins and a bacteriophage T4 protein. These are probably the products of horizontal transmission from animals. Outline structures for the extracellular immunoglobulin-like domains of fibroblast growth factor receptor and the L1 cell adhesion molecule are presented. These outline structures describe the relative positions of residues in the domains, their major secondary structures and gives a guide to the extent residues are accessible to the solvent. These outline structures are used to provide molecular explanations for mutations that lead to human disease. All proteins contain key residue sites and mutations at these sites will disrupt structure, reduce stability and in some cases alter the conformation of functional sites. The majority of mutations in fibroblast growth factor receptors and L1 occur at key residue sites. In myelin protein zero, an immunoglobulin disease protein of known structure, the majority of mutations occur in, or near, functional sites.

No description available.

No description available.

The MHC class I antigen presentation pathway plays a critical role in defense against intracellular pathogens. Viruses use many different strategies to evade the class I machinery. The subject of my thesis is the characterisation of the KK3 protein from the Kaposi’s Sarcoma-associated herpes virus. I show that KK3 subverts the MHC class I antigen presentation pathway by downregulating MHC class I complexes from the plasma membrane via a novel mechanism. The 322 amino acid KK3 is a type III transmembrane protein that contains an N-terminal RING-variant domain and a unique C-terminal tail with a number of predicted SH3 domains, myristoylation and phosphorylation sites. The RING-variant motif is characterised by seven cysteines and one histidine in the order C₄HC₃ and share both sequence and structural homology with RING finger domains found in a class of cellular E3 ubiquitin ligases. Following homodimerisation, KK3 associates with MHC class I molecules and promotes their ubiquitination after export from the edoplasmic reticulum. Ubiquitination requires the KK3 RING-variant domain and provides the signal for class I internalisation at the plasma membrane via clathrin-coated vesicles. Once internalised, ubiquitinated MHC class I molecules are targeted in a TSG101 dependent manner to the late endocytic pathway for degradation. KK3 is the first viral gene product that subverts the trafficking of a host protein via the ubiquitin-dependent endosomal sorting machinery. KK3 and its mouse specific homologue MK3 of MHV-68 are the first members of a new subclass of E3 ubiquitin ligases containing a RING-variant domain. The RING-variant domain can be found in a number of viral cellular homologues. Following structural and functional comparison of classical RING domain E3 ligases with the RING-variant proteins, the emergence of a new class of transmembrane E3 ubiquitin ligases is predicted.

FcγRI associates with the homodimeric γ-chain of the high affinity receptor for IgE (Fc<SUB>γ</SUB>RI). This receptor subunit has a cytoplasmic consensus sequence identified in several immunoreceptor signalling polypeptides known as the Immunoreceptor Tyrosine Activation Motif (ITAM). Here a functional role of this association is demonstrated. Whilst COS-7 cells transfected with Fc<SUB>γ</SUB>RI alone were unable to mediate phagocytosis, cells co-transfected with Fc<SUB>γ</SUB>RI and γ-chain could mediate phagocytosis in a tyrosine kinase dependent manner. Tail truncation mutants of Fc<SUB>γ</SUB>RI demonstrate that this interaction between Fc<SUB>γ</SUB>RI and γ-chain is within the transmembrane domain. Recognition of multiple IgG molecules on the opsonised target by Fc<SUB>γ</SUB>RI results in clustering of this receptor. This serves to aggregate the ITAMs of the associated γ-chain through non-covalent interactions between the two molecules thus initiating the phagocytic signal transduction cascade. Using a panel of chimeric receptors, the importance of the cytoplasmic ITAM was confirmed and the ligand independence of phagocytosis demonstrated. Replacing the γ-chain cysteine with an alanine had no effect on the ability of this subunit to transduce the phagocytic signal or to enhance the binding affinity of Fc<SUB>γ</SUB>RI for IgG. However, the strength of the physical association between γ-chain and Fc<SUB>γ</SUB>RI appears to be diminished, (as assessed by co-immunoprecipitation data). In addition to defining the molecular determinants for Fc receptor mediated phagocytosis, these results suggest a valuable model system for investigating protein-protein interactions within the plasma-membrane.

To specifically investigate the necessity for Tim1 and Tim3 during an ovalbumin- (OVA) induced, type 2-mediated allergic lung model, <i>havcr1^-/- </i>and <i>havcr2^-/- </i>mice were backcrossed to the BALB/c background over six generations. The allergic lung response was assessed using unrestrained whole body plethysmography to test lung function and, by analysing, cellular infiltration into the bronchoalveolar lavage (BAL), mucus production in the lung, eosinophilia, the cytokine and proliferative response of OVA-restimulated lymph node cells, and OVA-specific serum immunoglobulin production. The data demonstrate that whilst Tim3 is expressed in the lung by CD4⁺CD25⁺, and CD11c⁺ cells, it is not essential for any aspect of the allergic lung response tested. Tim1 was also expressed in the lung following an OVA-induced type 2 immune response, but only on CD19⁺B cells. Whilst Tim1 was not essential for many aspects of the allergic lung response which were tested, OVA treated <i>havcr1^-/- </i>mice did show a statistically significant deficit in blood and BAL eosinophils. To further understand the role of Tim3 in the naïve immune response, <i>in vitro, </i>a Tim3-human IgG₁ fusion protein (Tim3Ig) was generated. Similarly, as Tim5 is not expressed in naïve tissue, a Tim5-human IgG₁ fusion protein (Tim5Ig) was also generated as a control. Flow cytometric analysis using Tim3Ig led to the detection of an unknown binding partner for Tim3Ig on CD19/B220⁺ B cells, but not on CD3⁺ T cells. The interaction of Tim3Ig with B cells led to a Toll4- and FcgRII receptor-independent increase in proliferation and upregulation of the lymphocyte activation marker CD69, in a naive, as well as in an anti-IgM stimulated B cell population. These data demonstrate the identification of a novel function for Tim3 in the regulation of B cell proliferation and activation.

No description available.

The aim of this study was to identify dendritic cells in the chicken through the development of a set of markers and probes to potential dendritic cell markers in the chicken. In the absence of markers to avian dendritic cell markers published sequence databases were used to identify three candidate markers that showed significant homologies to CD83, fascin and DEC205 all potential dendritic cell markers in humans and mice. These markers were chosen for the following reasons: CD83 is currently the best marker of mature human and murine dendritic cells; fascin-1 is an actin bundling protein highly conserved between species, with expression restricted in leukocytes to dendritic cells; and DEC205 is a C-type lectin whose expression is also restricted to dendritic cells. CD83 was expressed in immunologically relevant tissues, had low levels of expression in the bursa and was not detectable on endothelial cell types. Based upon these distributions, CD83 was selected as the most suitable marker of dendritic cells in the chicken and an anti-chicken CD83 polyclonal serum and monoclonal antibodies were used to assess the expression of CD83 at the protein level. A map of CD83 expression was created using immunohistochemistry techniques upon a variety of tissues including spleen thymus and bursa derived from out-bred chickens and specific pathogen free chickens. This data revealed that whilst some aspects of CD83 immunobiology were conserved such as the upregulation of CD83 expression under inflammatory conditions, the distribution of CD83⁺ cells was uniquely distributed amongst the B cell areas of the chicken immune system in contrast to the T cell associated distribution of humans and mice. The expression of CD83 by specific cellular lineages was determined using commercially available lineage specific monoclonal antibodies and two-colour fluorescent microscopy. This established a dendritic cell type with features that are unique to the chicken.