Engineering Novel Angiopoietin Receptor Ligands
thesisposted on 10.01.2013, 11:17 by Andrew James Moss
Angiopoietin-1 is a multimeric glycoprotein which signals through the vascular endothelial Tie2 receptor to protect against inflammation and leakage, an effect antagonised by the selectively upregulated antagonist angiopoietin-2. In many pathologies this destabilisation by angiopoietin-2 is excessive or inappropriate, and here angiopoietin-1 has a promising role in therapeutic application. However angiopoietin-1 is difficult to purify and administer, its large multimeric structure rendering it prone to aggregation and insolubility. In this work the abilities of two small heptameric Tie2 binding peptides, VTSRGNV and NLLMAAS, multimerised using the established oligomeric scaffold cartilage oligomeric matrix protein (COMP), to bind and activate Tie2 were investigated. cDNAs for synthetic ligands were created by PCR, and protein synthesis was carried out in mammalian and bacterial expression systems. Ligands were expressed as stable, soluble pentamers and tetramers which showed similar abilities to bind Tie2 in vitro. Both ligands activated Tie2 similarly in Eahy926 and HUVEC endothelial cells. Both ligands were also able to activate two important downstream signalling mediators of Tie2 in HUVECs, namely Akt and ERK, in a dose-dependent fashion. However, the kinetics of ERK appeared different between the two ligands, implying possible differences in signalling of the two ligands through Tie2. This work is proof of principle and is among the first work to demonstrate that Tie2 binding elements other than the ang1 FRD can be used to activate Tie2. Additionally, kinetics work suggests that the two ligands, presumed to bind to different areas on Tie2, might induce slightly different patterns of receptor activation.