(A) Homology model of the isolated TSP/BR domain of human RSPO3 (residues valine 146 - isoleucine 232; UniprotKB Q9BXY4). Left panel: cartoon representation of the protein backbone, with side-chains shown for lysine (K) and arginine (R) residues. N- and C-termini are circled and disulfide bonds are labelled with roman numerals. Two positively charged HS-binding grooves, revealed by the electrostatic potential map (middle panel), are labeled Site-1 and Site-2. K and R residues lining Site-1 and Site-2 are colored purple and orange, respectively, and residues mutated to glutamic acid (E) in the RSPO3 TSP/BR (K/R→E) mutant are labeled. Middle panel: the electrostatic potential map calculated with the Adaptive Poisson-Boltzmann Solver (Jurrus et al., 2018) is displayed from −10 kT/e to +10 kT/e (blue: positively charged; red: negatively charged). Two virtually docked 8-mer heparin chains (estimated free energies are −13.9 and −14.8 kcal/mol for Site-1 and Site-2, respectively) are depicted in stick representation (red: oxygen, blue: nitrogen, gold: sulphur, green: carbon of heparin-1, yellow: carbon of heparin-2). Right panel: electrostatic potential map of the RSPO3 TSP/BR (K/R→E) mutant. The orientation of the middle and right panels is 90° rotated clockwise around the y-axis relative to the left panel. (B) Schematic representation of WT and mutant RSPO3 variants showing the domains and mutations present in these proteins. The N-terminal HA and the C-terminal Fc and 1D4 tags present in these constructs are not shown. Amino acid numbers for human RSPO3 (UniProtKB Q9BXY4) are indicated below. Green and red arrows show K→E and R→E mutations, respectively, introduced into the TSP/BR domain of the RSPO3 TSP/BR (K/R→E) mutant. Polypeptide lengths are drawn to scale. See Supplementary file 1 for the nucleotide sequences of these constructs. (C) Coomassie-stained polyacrylamide gels showing equal volumes of the three purified RSPO3 proteins used in (D) and (E). Molecular weight standards in kilodaltons (kDa) are indicated to the right. (D) and (E) Dose-response curves for the indicated purified RSPO3 variants in WT HAP1-7TGP (D) and LGR4/5/6KO (E) cells, in the presence of 1.43% WNT3A CM. Each circle represents the median WNT reporter fluorescence from 2,500 cells. Dose-response curves were fitted to the data using non-linear regression as described in Materials and methods. Where possible, half-maximal effective concentrations (EC50, all in nM) were derived from curve fits and are as follows. RSPO3 (WT): 0.048 ± 0.005 in (D) and 0.092 ± 0.007 in (E). RSPO3 ΔTSP/BR (K/R→E): 0.63 ± 0.02 in (D). RSPO3 ΔTSP/BR: 3.93 ± 1.0 in (D).