Polymeric fluorescent heparin as one-step FRET substrate of human heparanase

Abstract

Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains and plays important roles in cellular growth and metastasis. Heparanase assays reported to-date are labor intensive, complex and/or expensive. A simpler assay is critically needed to understand the myriad roles of heparanase. We reasoned that fluorescent heparin could serve as an effective probe of heparanase levels. Following synthesis and screening, a heparin preparation labeled with DABCYL and EDANS was identified, which exhibited a characteristic increase in signal following cleavage by human heparanase. This work describes the synthesis of this heparin substrate, its kinetic and spectrofluorometric properties, optimization of the heparanase assay, use of the assay in inhibitor screening, and elucidation of the state of heparanase in different cell lines. Our FRET-based assay is much simpler and more robust than all assays reported in the literature as well as a commercially available kit.

Keywords: Enzyme assay; Enzyme inhibition; FRET; Fluorescent heparin; Heparanase; Heparin.

Figure 1.

Generic structure of heparin (R = H or SO₃⁻; R₁ = H, COCH₃ or SO₃⁻) showing typical site (↓) cleaved by human heparanase (HPSE).

Figure 2.

FRET-based assay for human heparanase. Heparin-DE (1 mg/ml) was incubated with HPSE (1 μM) at 37°C in 20 mM sodium acetate buffer, pH 5.0. Fluorescence emission spectra (λ_EX = 340 nm) at (bold line) 0 h, (dotted line) 4 h.

Figure 3.

Optimization of heparanase assay using heparin-DE as substrate. (A) Labeled heparin (1 mg/ml) was incubated 1 μM HPSE at 37 °C for indicated times followed by measurement of emission at 500 nm. (B) Labeled heparin (1 mg/ml) was incubated with varying concentrations HPSE at 37 °C for 4 h followed by fluorescence measurement. (C) Michaelis–Menten kinetics of HPSE cleavage of heparin–DE using the optimized FRET quenching assay. The cleavage reactions were performed in microplate format (100 μL) in 20 mM sodium acetate buffer, pH 5.0, containing 1 mg/mL heparin–DE and 1 μM HPSE at 37 °C for 4 h. Solid lines represent curve fitting to the standard Michaelis equation. Error bars show variation from at least 3 measurements. F and F₀ are fluorescence signals corresponding to the test sample and blank, respectively.

Figure 4.

Suramin inhibition of HPSE using the one-step FRET quenching assay. The experiments were performed in microplate format (100 μL) in 20 mM sodium acetate buffer, pH 5.0, containing 1 mg/mL heparin–DE and 1 μM HPSE at 37 °C for 4 h in the presence of varying concentrations of suramin. Solid lines represent curve fitting to standard dose-response equation.

Figure 5.

Detection of heparanase activity in the monolayer MCF7 cells expression medium of under normoxic and hypoxic conditions. The cells were cultured for 48 h in FBS-free (red bars) and 2% FBS (black bars). Results are presented as the mean±SD from at least three independent measurements.

Figure 6.

Expression of active heparanase by MCF7 cells under normoxic conditions. Inset shows the HEK cells expressed HPSE activity measurement. Results are presented as the mean±SD (n>3).