MOLECULAR CHARACTERIZATION OF MEMBRANE-BOUND GLYCOPROTEINS INVOLVED IN HUMAN DISEASES AND POTENTIAL TARGETS FOR NEW THERAPIES
Creators: name: DEGANI, GENNY
Publisher: name: Universit� degli Studi di Milano
Year: 2015

description: The present thesis is focused on the molecular characterization of two eukaryotic membrane glycoproteins that are promising candidates for new therapeutic approaches to human diseases. The first glycoprotein is the human Receptor for the Advanced Glycation End products (hRAGE), a member of the immunoglobulin superfamily. RAGE is a type I transmembrane glycoprotein that is beneficial in normal physiological conditions but it is also a key player in the etiology and progression of several chronic pathologies such as neurodegenerative disorders (Alzheimer), atherosclerosis, cancer and complications of metabolic diseases such as diabetes, by exacerbating the inflammatory response. A variety of ligands sharing an acidic charge, as the advanced glycation End products (AGEs), S100 proteins, HMGB1, A?-amyloids, bind to the extracellular V or VC1 domains of RAGE. These domains are N-glycosylated and stabilized by disulphide bonds. To overcome the tendency to aggregate of the V and VC1 domains expressed in bacteria, in this work V and VC1 domains were expressed as secreted proteins in the methylotrophic yeast Pichia pastoris. While VC1 was secreted into the culture medium and was functional, the V domain was retained intracellularly, providing the first in vivo indication that V requires C1 to fold into a structurally stable domain. The glycosylation pattern of VC1 reflects the glycosylation of RAGE isolated from mammalian sources. A simple procedure for the purification to homogeneity of VC1 from the medium was generated and the folded state of the purified protein was assessed by thermal shift assays. The protein showed a remarkable improved thermal stability compared to VC1 expressed in bacteria. The stability and full solubility of glycosylated VC1 may be beneficial for in vitro studies aimed at the identification of new ligands or inhibitors of RAGE. The second object of this thesis was the Phr family of Candida albicans, a dimorphic fungal pathogen responsible of life-threatening invasive infections. These glycoproteins are anchored to the plasma membrane through a GPI. Phr proteins belong to family GH72 of cell wall glucan remodelling enzymes that are unique to fungi and essential for morphogenesis, cell wall integrity and virulence. For these reasons, these enzymes are targets for inhibitors of the cell wall formation to be used in therapy, similarly to what penicillins have been for bacteria. The catalytic properties of Phr proteins were studied using a new fluorescent assay. Phr1p and Phr2p are specific for ?-1,3-glucan, the pH optimum was 5.8 for Phr1p and 3 for Phr2p and the temperature optimum was 30�C. Pga4p was inactive suggesting that it turned out into a structural cell wall protein. Finally, we studied the transcriptome of cells lacking ?-1,3-glucan remodelling (phr1? cells) after induction of growth as hypha, the invasive form of this pathogen. About 310 genes were modulated and genetic analysis showed that chitin synthesis by the Chs3p isoform is essential for viability of phr1? cells.