Although much has been unravelled with regards to the mechanisms of proteolysis of exogenously derived antigen for presentation via histocompatibility class-II (MHC-II), key questions remain unresolved. The exact role of each proteolytic enzyme in this process is not understood. The aspartic proteinase cathepsin E is hypothesised to play an important role. The aim of this study is to examine this by the use of novel aspartic proteinase inhibitors based upon the aspartic proteinase inhibitor pepstatin A. The first two inhibitors designed and synthesised included a mannose sugar molecule to improve solubility, a linker section and a single pepstatin molecule. The mannose derivative 3'-succinimidoxycarbonyl- propyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside was synthesised and linked via an amide bond to either N--butyloxycarbonyl) propane-1,3-diamine or 7V-(/-butyloxycarbonyl) cystamine, the latter of these containing a cellularly cleavable disulphide bond. After deprotection, the amines of these two molecules were further reacted with pepstatin succinimide to form the precursors to the inhibitors Af-(3-(a-D-mannopyranosyloxy) propylcarbonyl),7V' (pepstat-inyl) propane- 1,3-diamine (Mannose Pepstatin Conjugate 1, MPC1) and Ar-(3-(a-D-mannopyranosyloxy) propylcarbonyl),W-(pepstatinyl) cyst-amine (MPC2), the final products being formed by selective removal of the mannopyranosyl acetyl protecting groups. The purified inhibitors were tested in a well- characterised in vitro model of antigen processing, in which ovalbumin is processed and presented by a B-cell line A20 to ovalbumin-specific T-cells, DO11-10. Further studies aimed to address the role of cathepsin E within dendritic cells (DCs), one of the key cells involved in initiating and propagating immune response to antigen. Further inhibitors were designed with the aim of improving solubility and cellular targeting. These inhibitors contained multiple mannose sugars attached either to a protein backbone bovine serum albumin (MPC5 and 6) or a poly-lysine backbone (MPC3 and 4). As for MPC2, a cleavable linker was included in the design of MPC4 and MPC6, to facilitate intracellular release of pepstatin from the carrier. The full synthesis of MPC5 and MPC6 was achieved by the coupling of either AiodoacetylXTV'-Cpepstatinyl) propane-1,3-diamine (MPC5) or N- (iodoacetylW-Cpepstatinyl) cystamine (MPC6) to the single free sulfhydryl of mannosylated bovine serum albumin (BSA) protein. The mannosylated BSA protein was achieved by the thiourea bond formation between the multiple lysine residues contained within BSA and the amine-reactive mannose derivative 4-isothiocyanatophenyl ot-D-mannopyranoside. Biochemical tests showed that between 22 and 24 mannose units were coupled to each BSA protein. The inhibitors MPC5 and MPC6 were tested for their ability to block processing and presentation of ovalbumin by mouse bone-marrow derived DCs. Since pepstatin A inhibits not only cathepsin E but also the lysosomal proteinase cathepsin D, the inhibitors were further tested on DCs prepared from mice deficient in cathepsin D. The results suggest that cathepsin E has an important and non-redundant role in antigen processing of ovalbumin by dendritic cells. This work was supported by funds supplied by the Department of Immunology and Molecular Pathology, UCL, and the Department of Chemistry, UCL. In addition, an Interdisciplinary Research Scholarship was provided by the UCL Graduate School, and a MONBUSHO Fellowship was provided by the Japanese Science & Education Ministry / British Council.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:440515 |
| Date | January 2006 |
| Creators | Free, Paul Francis |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1444682/ |
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