CD4 is a co-receptor for binding of T cells to antigen-presenting cells (APC) and the primary receptor for human immunodeficiency virus-I. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cysl30 in one monomer and Cysl59 in the other one (Cys133 and Cysl62 in the mouse CD4). Thus, we hypothesized that the domain swapping of D2 in CD4 regulates its co-receptor function of antigenspecific T cell activation. We found that mild reduction of the extracellular part of human CD4 resulted in formation of disulfide-linked dimers. We then tested the functional significance of dimer formation for co-receptor function using the engineered Jurkat T cell system by expressing wild-type or disulfide-bond mutant mouse CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function as assessed by antigen-specific IL-2 production. Exogenous wild type thioredoxin, but not redox-inactive thioredoxin, could inhibit the CD4-mediated IL-2 production, suggesting that the redox state of D2 disulfide bond is controlled by this oxidoreductase. Furthermore, structural modeling of the complex ofthe T cell receptor and domain-swapped CD4 dimer bound to class II major histocompatibility complex and antigen supports the domain-swapped dimer as the immune co-receptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation isalso accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred functional co-receptor for binding to APC. Strategies to promote dimerisation of CD4 should, therefore, enhance the immune response, while inhibiting dimer formation is predicted to be immunosuppressive.

Members of the membrane-spanning four-domains, subfamily A (MS4A) family are small polypeptides that share the structural features of four-transmembrane domains and unevenly sized extracellular loops. The family includes CD20, FcεRIβ and HtM4, plus a number of relatively uncharacterised proteins / predicted proteins. MS4A proteins are discussed in relation to other protein families, such as the tetraspanins, that are also characterised by four-transmembrane domains. The aim of this study was to identify the cell and tissue distribution, subcellular localisation, and function of a newly discovered member of the MS4A family, hepatocellular carcinoma-associated antigen 112 (HCA112). At a subcellular level, HCA112 was found on the plasma membrane of transfected COS-7 cells, and also within the Golgi complex, trans-Golgi-network, and early endosomes. The molecule is orientated such that the large loop is extracellular and the Nand C-terminal domains are cytoplasmic. The presence of HCA112 associated with components of the endocytic pathway raised the question of whether some originated from the surface membrane. Antibody was used to label a HA epitope tag engineered into the large extracellular loop of HCA112, and the bound antibody was tracked through early endosomes to the recycling compartment. Here it co-localised with internalised transferrin, indicating strongly that HCA112 is internalised via clathrin-dependent mechanisms. Several endocytic sorting motifs within the intracellular domains of HCA112 were investigated for their ability to direct internalisation of HCA112. Deletion of a di-leucine motif was found to slow but not prevent endocytosis, suggesting that it is involved in endocytosis of HCA112, although not essential for the process. When HCA112 expression constructs featuring N- and C-terminal domain truncations were examined, it was found that the N-terminal tail does not affect the subcellular localisation or trafficking of HCA112, while deletion of the C-terminal intracellular domain resulted in retention of the mutant protein in the ER. HCA112 has a wide tissue distribution and is highly expressed in the lining/covering and parenchymal epithelium of some tissues, proximal renal tubules, ductal epithelium in a number of organs, endothelial cells, some steroidogenic endocrine cells, adipocytes, smooth muscle cells, follicular dendritic cells and macrophages. The expression of HCA112 by a wide range of cell types suggests that its function(s) has general importance and is not limited to any specific cell type(s). After reflection on the functions of the HCA112-expressing cells, a common theme that emerged was one of endocytic activity. This lead to speculate that one function of HCA112 might be related to uptake of macromolecules, for instance, in antigen processing and presentation. This might be a general function, such as facilitating uptake of other cell membrane proteins, or directing the traffic of endocytic vesicles. It was noted that HCA112 has a similar cell and tissue distribution to the scavenger receptor and fatty acid translocase FAT/CD36 (Zhang et al., 2003). Furthermore, in cells co-transfected with HCA112 and FAT/CD36, the two molecules co-localise in early endosomes and co-immunoprecipitate, suggesting that the molecules physically and spatially associate. Thus, HCA112 could be involved with (or complement) FAT/CD36 in its functions as a long chain fatty acid transporter and scavenger receptor. A proteomics study of proteins that co-immunoprecipitated with HCA112 detected putative interactions with a number of proteins. These included LR8, transferrin receptor, interferon induced transmembrane proteins 2 and 3, Calpain-6, stomatin, PDGF α receptor, and heat shock 70 kDa protein 8 (HSPA8, formerly known as clathrin un-coating ATPase). Of these, LR8 and the transferrin receptor were investigated in more detail. The results provide strong evidence that HCA112 forms a novel complex with LR8, and that this may be involved in macromolecule internalisation or trafficking of membrane proteins, such as FAT/CD36 or the transferrin receptor. In the case of the transferrin receptor, this traffic appears to involve the clathrin-dependent pathway, but it is possible that when HCA112 is associated with FAT/CD36, it functions within lipid raft domains. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375454 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The small envelope of hepatitis B virus (HBV) can self-assembles into virus-like particles (VLPs) and they are highly immunogenic. The use of hepatitis B surface antigen (HBsAg) as a vector to deliver foreign CTL epitopes has met with little success due to the constraints of HBsAg stability and secretion imposed by the insertion of foreign sequence into critical regions. In this study, the efficacy of the small HBsAg envelope protein to deliver foreign CTL epitopes using a protective CTL epitope of human respiratory syncytial virus (RSV) was investigated. The strategy of deleting a DNA sequence encoding HBsAg-specific CTL epitopes at different sites and replacing with DNA sequence encoding RSV CTL epitope resulted in recombinant HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and RSV protective responses in vivo when these recombinant HBsAg DNAs were used to immunised mice. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease relevant protective CTL responses. They also suggest the applicability of the approach to derive recombinant HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The use of HBsAg VLPs has been used globally as administered vaccine for hepatitis B virus infection makes it an attractive vector candidate to deliver immunogens for other diseases. Since the HBsAg DNAs we tested formed recombinant HBsAg VLPs, our results have implications for the development of vaccination strategies using either recombinant HBsAg DNA or VLP vaccines.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 5 uppsatser.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Universiẗat, Diss., 2005--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.