Regulation of Angiopoietin Signalling by Soluble Tie2 Ectodomain and Engineered Ligand Trap

Angiopoietin-1 (Angpt1) is a glycoprotein ligand important for maintaining the vascular system. It signals via a receptor tyrosine kinase expressed on the surface on endothelial cells, Tie2. This receptor can undergo regulated ectodomain cleavage that releases the ligand-binding domain (sTie2) into the circulation. The concentration of sTie2 is increased in a range of conditions, including peripheral arterial disease and myocardial infarction, where it has been suggested to bind and block Angpt1 resulting in vascular dysfunction. Here we use a joint mathematical modelling and experimental approach to assess the potential impact of sTie2 on the ability of Angpt1 to signal. We find that the concentrations of sTie2 relative to Angpt1 required to suppress signalling by the ligand are more than ten–fold higher than those ever seen in normal or disease conditions. In contrast to the endogenous sTie2, an engineered form of sTie2, which presents dimeric ligand binding sites, inhibits Angpt1 signalling at seventy-fold lower concentrations. While loss of Tie2 ectodomain can suppress Angpt1 signalling locally in the cells in which the receptor is lost, our study shows that the resulting increase in circulating sTie2 is unlikely to affect Angpt1 activity elsewhere in the body

Regulation of Angiopoietin Signalling by Soluble Tie2 Ectodomain and Engineered Ligand Trap

Angiopoietin-1 (Angpt1) is a glycoprotein ligand important for maintaining the vascular system. It signals via a receptor tyrosine kinase expressed on the surface on endothelial cells, Tie2. This receptor can undergo regulated ectodomain cleavage that releases the ligand-binding domain (sTie2) into the circulation. The concentration of sTie2 is increased in a range of conditions, including peripheral arterial disease and myocardial infarction, where it has been suggested to bind and block Angpt1 resulting in vascular dysfunction. Here we use a joint mathematical modelling and experimental approach to assess the potential impact of sTie2 on the ability of Angpt1 to signal. We find that the concentrations of sTie2 relative to Angpt1 required to suppress signalling by the ligand are more than ten–fold higher than those ever seen in normal or disease conditions. In contrast to the endogenous sTie2, an engineered form of sTie2, which presents dimeric ligand binding sites, inhibits Angpt1 signalling at seventy-fold lower concentrations. While loss of Tie2 ectodomain can suppress Angpt1 signalling locally in the cells in which the receptor is lost, our study shows that the resulting increase in circulating sTie2 is unlikely to affect Angpt1 activity elsewhere in the body

Development of an Orthogonal Tie2 Ligand Resistant to Inhibition by Ang2

Angiopoietin-1 (Ang1) is a vascular protective ligand that acts through the receptor tyrosine kinase Tie2 to enhance endothelial survival and quiescence. In sepsis, diabetic retinopathy, and a range of other diseases, Ang2, an antagonist of Tie2, increases markedly. This antagonist suppresses Ang1 protective effects leading to vascular destabilization, inflammation, and endothelial death. Administration of recombinant Ang1 can counter Ang2 antagonism and restore vascular function. However, recombinant Ang1 is needed at sufficiently high concentrations to block Ang2, and the protein is difficult to produce, requires mammalian expression systems, and is prone to aggregation. Here we present an engineered synthetic Tie2 ligand that is not antagonized by Ang2 but is easy to produce and more robust than Ang1. Using a peptide phage display, we isolated a heptameric sequence that binds Tie2-ectodomain and fused this to the coiled:coil domain of cartilage oligomeric matrix protein. This pentameric protein is 60 kDa in size, expressed in E. coli, and facile to purify. The protein, designated TSL1, binds to Tie2-ectodomain in vitro and on the cell surface. TSL1 inhibits endothelial apoptosis. Crucially, TSL1 binds at a site on Tie2 distinct from the angiopoietin-binding site and is resistant to antagonism by Ang2. This engineered ligand has several advantages over recombinant Ang1 for potential therapeutic applications. The study also highlights the value of orthogonal ligands for regulating cellular receptors without being subject to antagonism or modulation by endogenous ligands

Effects of Gln498His and Gln498Arg mutations on binding of B.1.617.1/3 and B.1.351 variant RBD to RaACE2.

Effects of Gln498His and Gln498Arg mutations on binding of B.1.617.1/3 and B.1.351 variant RBD to RaACE2.</p

Cryo-EM data collection, refinement and validation statistics.

(DOC)</p

Q498H and Q498R mutations modify binding of B.1.351 and B.1.617.1/3 variant RBD to HuACE2.

(a) Biolayer interferometry was performed with the indicated RBD in solution and HuACE2 immobilise on the sensor. Binding was measured with RBD concentrations of 12, 30, 60, 120nM, and an additional 6nM concentration in some cases. Curves were fitted and used to calculate Kon, Koff and KD. Data are shown as means and SEM for at least three independent experiments. (b) H498 and Y501 in SARS-CoV-2 RBD compete for interaction with Y41 in HuACE2 and the clash between residues is indicated by the overlapping Van der Waals surfaces. The proximity of the H498 side chain is shown with respect to the side chain of a Y501 inserted into our RBD:ACE2 complex structure. (c) R498 and Y501 side chain positions shown along with D38, Y41 and Q42 (PDB accession number 7BH9 [14]).</p

Example of multiple conformations of Q498 suggested in crystal structures.

The 2Fo-Fc electron density map (in blue, contoured at 1.5σ) and Fo-Fc difference map (contoured at -3σ (red) and green (+3σ)) are shown for the WT crystal structure of ACE2-RBD (PDB ID 6M0J [13]). The positive (green) density adjacent to the side chain of Q498 suggests this can exist in different conformations, thus weakening the interaction with neighbouring residues. Examination of the region of Q498 in PDB entries 7WQB, 7RPV, 7EFR, 7EFP, 7NXC, 7L0N, 7DMU and 6VW1 all show difference electron density adjacent to the side chain. PDB entries 7EKE, 7EKY, 7EKH, 7EKF and 6LZG all have the residue modelled in dual conformations. Only 7LO4 does not show disorder in Q498. (DOCX)</p

SARS-CoV-2 RBDs with Q498H mutations have high affinity for ACE2.

(a) Soluble forms of HuACE2, WH-RBD and mutant RBD were expressed in HEK 293 cells and purified on nickel affinity columns. Purified proteins were resolved by SDS/PAGE and detected by Coomassie staining. Molecular masses are shown in kDa. (b) Biolayer interferometry plots of kinetics of binding RBD to HuACE2. WH-RBD, or the indicated RBD, was in solution and Hu-ACE2 immobilized on sensors. Binding was measured with RBD concentrations of 12, 30, 60, 120nM for WH-RBD and 6, 12, 30, 60nM for the mutants. Fitted curves are in red. Data are shown for single experiments representative of at least three independent experiments for each RBD. (c) Kinetic binding constants for RBD binding to HuACE2 measured using biolayer interferometry. Data are shown as means and SEM for at least three independent experiments. (d) Cellular binding of RBD and S477N/Q498H-RBD. Vero-E6 cells were incubated without RBD (C) or with a range of concentrations of WH-RBD (circles) or S477N/Q498H-RBD (triangles) as indicated, at 37°C for 15 mins before washing, antibody staining of bound RBD with fluorescently conjugated antibody, and flow cytometry. Data are shown as mean fluorescence intensity vs RBD concentration (nM) for a single experiment representative of three.</p

Data processing outline.

Datasets were collected on both holey and graphene oxide support films. Each dataset present a different angular bias so the two datasets were combined. Following clean-up by 2D classification the particles were subjected to 3D classification and the single best 3D class chosen for further refinement and polishing to produce the final map. (DOCX)</p

Angular distribution and resolution.

The angular distribution of the final map is shown in (a) with an overall uniform angular distribution despite some persistent angular bias. The local resolution map is shown in (b) and the FSC of the two half maps and map vs model are shown in (c). (DOCX)</p