The CCP20 domain of SVEP1 contacts the D1-D2 headpiece domains of TIE1.

(A) Comparative PAE plots of full-chain SVEP1 displays a primary and high-confidence binding site for TIE1 (domains D1-D4) by AF3-multimer modeling, that localizes to the CCP19-21 stretch, notably encompassing CCP20 that has a sizable 45 amino acids insert loop. The PAE plot for TIE2 D1-D4 does not show an equivalent interaction signal for the same CCP19-21 region. (B) A discrete AF3 model of TIE1 bound to CCP19-21 shows that the Ig-like D1-D2 headpiece domains of TIE1 (linked by a Cys-rich D3 to an ‘open’ or flared-out D4 Ig module) are in contact with CCP20. By contrast, the AF3 model of TIE2’s N-terminal domains (that are packed in a more compact fashion, with D4 notably in a ‘closed’ position) is more loosely perched atop CCP20 with a qualitatively much lower affinity. (C) Quantitative analysis of TIE1-SVEP1 interaction using Biacore assays. In the absence of other proteins, a 70kD version of SVEP1 (depicted in diagram fashion) containing the CCP20 domain binds TIE1, but does not bind TIE2. (D) Shadowing EM shows binding of SVEP1 and TIE1. PAE: Predicted aligned error; AF3: AlphaFold 3; CCP: complement control protein (sushi repeats).

The CCP5-EGF7 domain of SVEP1 constitutes and additional binding domain for TIE1 which is sufficient, but not required for the lymphatic function of Svep1.

(A) Injection of different versions of murine Svep1 rescue constructs in svep1 -/- zebrafish zygotes at single cell stage was followed by quantifying the number of TD fragments at 5 dpf. Depicted in the confocal images, siblings show a complete TD, indicated by arrows. Non-injected svep1 mutants show a loss of the TD, indicated by asterisks, while mutants injected with the CCP5-EGF7 mRNA have significant more fragments of TD compared to the control (flt4:mCitrine transgene). For statistical analysis, the number of TD fragments of non-injected svep1 mutants was compared to the injected mutants (Mann-Whitney test). Scale bar = 100 µm. (B) Using CRISPR/Cas9 technology, an in-frame deletion of CCP5-EGF7 was generated in zebrafish. Homozygous mutants (ΔCCP5-EGF7) show no lymphatic defects (svep1ΔCCP5-EGF7 +/+ n=22, svep1ΔCCP5-EGF7 +/- n=53, svep1ΔCCP5-EGF7 -/- n=21). Therefore, the region is not required for the lymphatic function of Svep1. Scale bar =100µm. (C) The AF3 model predicts that TIE1 has a secondary binding site in the SVEP1 chain in the CCP5-7 stretch (that closely follows the PTX domain) that bridges CCP6 and (Ca2+-binding) EGFL7 modules. The same D1-D2 headpiece domains of TIE1 that recognize CCP20 are employed. In the present AF2.3 model, with the outward-facing D4 domain in TIE1 being omitted, since it does not participate in binding. (D) The binding of TIE1 domains D1-D2 with the CCP6-EGFL7 unit show a confident, but moderate affinity signal in the PAE plot, and the PISA-analyzed EGFL7 domain of SVEP1 alone is able to bind TIE1. Different versions of human SVEP1-StrepTac were immunoprecipitated and associated TIE1 was detected via Western blot analysis. In three of four replicates, the CCP5-EGFL7 bound to TIE1 much weaker in comparison to the SVEP1 proteins that contain the CCP20 domain. TD, thoracic duct; dpf, days post fertilization PAE: Predicted aligned error; AF3: AlphaFold 3; CCP: complement control protein (sushi repeats).

ANG1/2 strengthens the binding capacity of SVEP1 and TIE1.

(A) AF3-modeling of SVEP1 CCP19-21 domain and TIE1 together with ANG1/2. The affinity of TIE1 for SVEP1 is considerably heightened by the co-binding of ANG1 or ANG2 C-terminal domains to the CCP20 module. (B) Co-immunoprecipitation of human TIE1 co-transfected with either human ANG1 or ANG2 in 293T HEK cells. Human 150kDa SVEP1-StrepTac was immunoprecipitated and associated TIE1 and ANG1/2 were detected via Western blot analysis. (C) Quantification of the TIE1 levels with SVEP1 levels as a reference confirmed a significant increase in binding affinity of SVEP1 and TIE1 together with ANG2 (**p.adj = 0.008; Mann–Whitney U test; Values are presented as means ± SD; individual data points for each experiment).

Stimulation of LECs with SVEP1 and ANG2 together leads to phosphorylation of TIE1 and AKT as well as nuclear exclusion of FOXO1 in vitro.

(A) Western blot analysis of TIE1 phosphorylation at Y1007 residue in SVEP1 and ANG2 stimulated LECs after immunoprecipitation of TIE1. TIE1 is phosphorylated after stimulation with SVEP1 and ANG2 together. The antibody anti–phospho-TIE2 (pY992, AF2720 R&D Systems) detects both, TIE1 phosphorylation (at Y1007 residue) and TIE2 phosphorylation (at Y992 residue), but can be distinguished by size (Brouillard et al. 2024). (B) Western blot analysis of p-AKT and AKT in the cell lysate of in SVEP1 and ANG2 stimulated LECs. Knock-down of either TIE1 or TIE2 leads to reduced induction of phosphorylation of AKT by SVEP1-ANG2 (C) FOXO1 (red) and DAPI (blue) staining of LECs stimulated with only SVEP1 or ANG2, or in combination. FOXO1 staining is restricted to the nucleus of unstimulated (control) LECs, whereas in cells stimulated with SVEP1 and ANG2 together FOXO1 is in the cytoplasm. (D) Quantification of the pTIE1/total TIE1 ratios shown in A. (N = 4; *p = 0.0211; Mann–Whitney U test; Values are presented as means ± SD) (E) Quantification of the pAKT/total AKT ratios shown in B. Values are presented as means ± SD, N = 3/6; Mann–Whitney U test; Control vs. ANG2_SVEP1 p.adj = 0.028 *; SVEP1 vs. ANG2 p.adj = 0.022 *; ANG2 vs. ANG2_SVEP1 p.adj = 0.028 *; ANG2_SVEP1 vs. aANG2AB_SVEP1 p.adj = 0.091. (F) The ratio of nuclear FOXO1/cytoplasmic FOXO1 decreases after stimulation with either ANG2 or SVEP1. Stimulation of LECs with simultaneous SVEP1 and ANG2 leads to a further reduction of the nuclear FOXO1/cytoplasmic FOXO1 ratio. Each datapoint represents the mean value per image of the ratio of nuclear to cytoplasmic FOXO1 per cell (Mann–Whitney U test; Values are presented as means ± SD; N=4).

SVEP1, TIE1 and ANG1/2 can form 2:2:2 complexes mediated by SVEP1.

(A) Larger hexameric complexes of SVEP1-TIE1 D1-D4-ANG1/2 in a 2:2:2 stoichiometry could be confidently modeled with AF3. They show a symmetric assembly that is consistent with an interaction hub (in a dotted line box in the adjacent schematic B) of adjacent TIE1 chains that are bound to ANG1 or ANG2 by inclusion of the SVEP1 chain (as shown here, the CCP19-22 stretch). Intriguingly, the TIE1 and TIE1’ chains show domain-swapped D4 modules as the chains cross, leading to the following schematic model. (B) Model of SVEP1 enabling the multimerization of TIE1 receptors on the cell surface (with concomitant increase in cytosolic kinase signaling strength) by cross-lacing of N-terminal coiled-coil dimers (or greater assemblies) of ANG1 or ANG2. The linker chain between the C-terminus of the coiled-coil helix and the globular domain is too short to enable the cross-linking of ANG1/2 domains mounted with TIE1 on the angled SVEP1 chains, suggesting that ANG1/2 dimers bridge or glue consecutive TIE1 chains atop the cell surface.

The contrasting interactions of TIE1 and TIE2 D1-D2 domain segments against the CCP19-21 SVEP1 chain are shown by PAE plots that are unequivocally strong for TIE1, and much weaker for TIE2.

This is also seen with the nature of the respective protein interfaces (analyzed by PDBePISA at https://www.ebi.ac.uk/pdbe/pisa/) and visualized in PyMOL with the InterfaceResidues plug-in (https://pymolwiki.org/index.php/InterfaceResidues), that shows TIE1 has a larger interface footprint than TIE2, and has a series of discriminating salt bridges and hydrogen bonds that are missing in TIE2.

CCP5-EGF7 is sufficient, but not required for the lymphatic function of Svep1.

(A) Injection of different versions of murine Svep1 rescue constructs in svep1 -/- zebrafish zygotes at single cell stag was followed by quantifying the number of TD fragments at 5 dpf. For statistical analysis, the TD length of non-injected svep1 mutants was compared to the injected mutants. Various constructs containing CCP5-EGF7 and CCP20 rescue the phenotype, with the CCP5-EGF7 fragment alone also being sufficient (wtSvep1: inj. n=9; uninj. n=3, PolC: inj. n=7; uninj. n=6, ΔN-EGFL6: inj. n=10; uninj. n=6, ΔN-PTX: inj. n=12; uninj. n=14, ΔN-PTXΔCCP21: inj. n=9; uninj. n=9, ΔN-PTXΔCCP22-C: inj. n=21; uninj. n=9, ΔN-CCP21: inj. n=9; uninj. n=7, ΔN-PTXΔ7-34: inj. n=22; uninj. n=21, CCP5-EGF7: inj. n=7; uninj. n=6, ΔN-EGFL7: inj. n=15; uninj. n=19). Mann-Whitney test was applied for statistical analysis. (B) Confocal images of the lymphatic vasculature (marked by a flt4:mCitrine transgene) in the zebrafish trunk of svep1 sibling and mutants at 5 dpf,. Siblings show complete TD, indicated by arrows. Non-injected svep1 mutants show a loss of the TD, indicated by asterisks, while mutants injected with either CCP5-EGF7, ΔN-PTX, ΔN-PTXΔCCP21 or ΔN-PTXΔCCP22-C have significant more fragments of TD compared to the control. Scale bar = 100 µm. (C) Using CRISPR/Cas9 technology, an in-frame deletion of CCP5-EGF7 was generated in zebrafish. Homozygous mutants of 567 (CCP5-EGF7) show no lymphatic defects (svep1 567 +/+ n=22, svep1 567 +/- n=53, svep1 567 -/- n=21). Therefore, the region is not required for the lymphatic function of Svep1. Scale bar =100µm. TD, thoracic duct; dpf, days post fertilization

The PAE plots for the ternary assemblies of TIE1 D1-D3 bound to either ANG1 or ANG2 show equivalently strong confidence levels that improve the binding of just TIE1 to SVEP1. By contrast (not shown), TIE2 does not co-bind with ANG1/2toSVEP1.

The conformation of the TIE1-ANG1/2 heteromers in the SVEP1-bound pose resembles the solved structures of TIE2 bound to either ANG1 or ANG2. However, the weaker binding of TIE2 to CCP19-21 of SVEP1 fails to nucleate the formation of the TIE2 ternary complex with SVEP1. PDE, predicted distance error.

(A) The top view of the model hexamer shows the side-by-side, antiparallel packing of TIE1 D3 domains that produces the domain-swapping of D4 Ig domains. (B) The PAE plot of the TIE1-ANG1-SVEP1 hexamer shows the many strong and confident interactions that build the assembly.v