| Summary: | The focus of this project is a human-specific C-type lectin with potential roles in cell signalling: blood dendritic cell antigen 2 (BDCA-2). BDCA-2, a plasmacytoid dendritic cell-specific molecular marker, has been evaluated as a therapeutic target against auto-immune disorders, because antibodies to BDCA-2 inhibit the production of type I interferon. Accordingly, key goals of the project were to identify endogenous ligands for BDCA-2, to characterise the mechanism of ligand binding and ultimately to determine how ligands stimulate signalling pathways. A combination of BDCA-2 affinity chromatography column and mass spectrometry revealed that α2 macroglobulin and immunoglobulins, IgA, IgM and IgG are potential endogenous ligands in human serum. Competition binding studies conducted to characterise the binding affinity for each glycoprotein demonstrated that IgA has the highest affinity. Strategies for biochemical development of defined glycoforms of IgG Fc domain were established. The Chinese hamster ovary cell system for expression of Fc domain and the activity of enzymes necessary for chemoenzymatic glycoengineering have been tested. BDCA2 organisation in the cell membrane was studied by development of a transfected cell system which was analysed by affinity purification of BDCA-2 followed by analysis of protein-protein interactions. The results demonstrate that it is likely that BDCA-2 assembles with Fc receptor γ-chain in a 2:2 complex.
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