Expression and characterization of a recombinant HSulf-2. (A) Domains in HSulf-2 are schematically presented. Positions of peptides that are used to develop polyclonal antibodies are shown. A recombinant protein with a N-terminal FLAG-tag (F) (rHSulf-2) and an enzymatically inactivated protein (rΔCCHSulf-2) were designed and produced as described [25]. (B) Purified rHSulf-2 and rΔCCHSulf-2 produced in 293 cells were blotted with H2.3 anti-HSulf-2 antibody. A 72-kDa and a 74 kDa band were detected, respectively. rHSulf-2 was also blotted with a FLAG antibody. (C) Endoglucosamine-6-sulfatase activity of rHSulf-2 was measured as a function of enzyme concentration (left panel, 4 h incubation time) and reaction time (right panel, 240 ng of rHSulf-2) as described in Methods. rHSulf-2 liberated sulfate groups on C-6 position of glucosamine residues within trisulfated disaccharide units in heparin.

Sulf-2 effects on pre- and post-binding assays for VEFG and FGF-2. (A) Solid phase binding assays to measure "pre-binding effect" and "post-binding effect" are schematically shown. HSulf-2 was incubated with immobilized heparin before or after adding heparin-binding factors. Specific primary antibodies quantified the amount of factor bound to immobilized heparin. (B) Binding of ligand (VEGF₁₆₅, FGF-2 or FGF-1) to immobilized heparin-BSA as a function of ligand concentration with or without Sulf-2 treatment of heparin-BSA. Δ denotes binding of protein to untreated heparin-BSA and ● denotes binding of VEGF, FGF-2 or FGF-1 to rHSulf-2 treated heparin-BSA in pre-binding assay. (C) "Pre-binding effect" of rHSulf-2 (●) or rΔCCHSulf-2 (○) on ligand binding (VEFG or FGF-2) to heparin-BSA as a function of enzyme concentration. (D) "Post-binding effect" of rHSulf-2 (●) or rΔCCHSulf-2 (○) on ligand binding (VEFG or FGF-2) to heparin-BSA as a function of enzyme concentration. All the results shown are representative of two independent experiments.

Sulf-2 effects on pre- and post-binding assays for chemokines. (A) Binding of CXCL12 (SDF-1), CCL21 (SLC) and CXCL8 (IL8) to immobilized heparin-BSA as a function of ligand concentration with or without Sulf-2 treatment of heparin-BSA. Δ denotes binding of chemokines to untreated heparin-BSA and ● denotes binding of chemokines to rHSulf-2 treated heparin-BSA in pre-binding assay. (B) "Pre-binding effect" (left panel) and "post-binding" effect (right panel) of rHSulf-2 (●) or rΔCCHSulf-2 (○) on ligand binding (CXCL12) to heparin-BSA as a function of enzyme concentration. Values represent the means ± S.D. of triplicate determinations in the binding assays for CXCL8 and CXCL12. Statistical analysis was carried out using Student's t test. **, p < 0.01; ***, p < 0.001. The other data are representative of two independent trials.

Sulf-2 activity in MCF-7 CM. (A) Endosulfatase activity of CM on heparin as a function of CM volume (left panel, 4 h incubation time) and reaction time (right panel, 10 μl of CM). (B) Binding of ligand (VEGF₁₆₅, FGF-2 or FGF-1) to immobilized heparin-BSA as a function of ligand concentration with or without CM treatment of heparin-BSA. Δ denotes binding of ligand to untreated heparin-BSA and ● denotes binding of ligand to CM treated heparin-BSA in pre-binding assay. Means of triplicate determinations ± S.D. are shown. In those cases where the error bars are not apparent, the S.D. values are smaller than the symbols. (C) "Pre-binding effect" of CM on ligand binding (VEGF₁₆₅, black triangles; FGF-2, black squares; FGF-1, open diamonds) as a function of CM volume. These results are representative of two independent experiments (D). "Post-binding effect" of CM on ligand binding (VEGF₁₆₅, black triangles; FGF-2, black squares; FGF-1, open diamonds) as a function of CM volume. Means of triplicate determinations are shown. S.D. values for FGF-1 are indicated. The S.D. values for VEGF and FGF-2 are smaller than the symbols. (E) Immuno-depletion of HSulf-2 protein by specific antibodies. MCF-7 CM was precleared with Sulf-2 antibodies (H2.1 and H2.3) or control IgG conjugated to beads and then blotted for Sulf-2 protein (H2.3). Arrows denote specific bands detected in the untreated MCF-7 CM (~240, 135 and 72 kDa). The ~50 kDa bands indicated by the asterisk are derived from the IgG used for the precipitation (F). Immuno-depletion of HSulf-2 activity by specific antibodies. The precleared CMs in E were tested in a pre-binding assay for effects on VEGF binding to heparin-BSA. The partial depletion of activity by control IgG reflects the tendency of Sulf-2 to bind nonspecifically to Sepharose beads (not shown). Means and S.D. values are shown for triplicate determinations. Statistical analysis was carried out using Student's t test. **, p < 0.01.

The effect of Sulf-2 on FGF-1 binding to heparan sulfate-BSA. FGF-1 binding to heparan sulfate-BSA was measured after the following treatments of the conjugate: no treatment; MCF-7 CM; rHSulf-2; rΔCCHSulf-2; or a mixture of heparinases. Values represent the means ± S.D. of three separate determinations. Statistical analysis was carried out using Student's t test. **, p < 0.01.