人乙酰肝素酶的一步快速FRET检测

Jyothi C. Sistla; Umesh R.  Desai

doi:10.21769/BioProtoc.3356

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Peer-reviewed

A Robust, One-step FRET Assay for Human Heparanase

人乙酰肝素酶的一步快速FRET检测

Jyothi C. Sistla email

UD Umesh R. Desai

发布: 2019年09月05日第9卷第17期 DOI: 10.21769/BioProtoc.3356 浏览次数: 3923

评审: Vivien Jane Coulson-ThomasPaul Ross Wildermantarsis ferreira

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参见作者原研究论文

The authors used this protocol in:

Cover of Carbohydrate Polymers, featuring study using the protocol.

Feb 2019

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Abstract

Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains at distinct sites and plays important biological roles including modulation of cell growth and metastasis. Although a number of different types of heparanase assays have been reported to date, most are labor intensive, complex and/or expensive to carry out. We reasoned that a simpler heparanase assay could be developed using heparin labeled with Dabcyl and EDANS as donor and acceptor fluorophores so as to generate a FRET signal. Our results show that a more robust heparanase assay could be developed based on the principle studied herein and more homogeneous preparation of heparin. Yet, the assay in its current form could be used for routine screening of potential inhibitors in a high-throughput manner as well as for studying heparanase activity expressed in tumors as well as biological fluids like plasma.

Keywords: Heparanase (乙酰肝素酶)

Enzyme assay (酶活测定)

Enzyme Inhibition (酶活抑制)

FRET (荧光能量共振转移)

Heparin (肝素)

Background

Human heparanase is an endo-β-D-glucuronidase that cleaves heparan sulfate (HS) chains present in proteoglycan form on cell surfaces and in the extracellular matrix (Fairbanks et al., 1999; Kussie et al., 1999; Toyoshima and Nakajima, 1999; Dempsey et al., 2000; Sanderson et al., 2017). Although other sequences are also likely to be targeted by heparanase (Peterson and Liu, 2013), substrate specificity studies indicate that heparanase preferably cleaves the 1→4-inter-glycosidic bond between a glucuronic acid (GlcA) and glucosamine-N,6-disulfate (GlcNS6S) residues.

The importance of heparanase as a therapeutic target has led to the development of several biochemical and biophysical assays over the past 2 decades. Despite the availability of numerous assays, no particular assay appears to have been broadly used to understand heparanase biology, pharmacology, and drug discovery. The reported assays typically suffer from the involvement of multiple steps and incubation up to 24 h (Freeman and Parish, 1997; Behzad and Brenchley, 2003; Tsuchida et al., 2004; Enomoto et al., 2006; Hammond et al., 2010; Schiemann et al., 2012; Melo et al., 2015). An easier, one-step assay would greatly help deduce inhibitors, understand substrate specificity, elucidate the mechanism of action, and clarify enzymatic or non-enzymatic role in cellular systems. A more robust heparanase assay would be easier to implement (fewer steps, faster screening time, no immobilization, no post-assay signal development), which is adaptable to microplate format, enable assaying active heparanase in cellular media, and perhaps help monitor heparanase in vivo. We hypothesized fluorescence resonance energy transfer (FRET)-based assay may offer a one-step solution that can address several attributes. This documents, method of developing a FRET-based assay for active heparanase. Based on our work, FRET-enabled heparin chain could effectively serve as a substrate of heparanase, help detect an active enzyme in media, and to screen potential inhibitors.

Materials and Reagents

1 cm path length quartz cuvette (Hellma^®, catalog number: Z801712)
96-well plates (Corning, catalog number: 3897)
Wilmad-LabGlass^TM NMR Tubes (Fisher Scientific, 16-800-554)
6-well tissue culture-treated plates (Cyto one, catalog number: CC7672-7506)
Low-Retention Microcentrifuge Tubes (Fisher Scientific, catalog number: 02-681-331; Amber, catalog number: 05-402-31)
Fisherbrand^TM Micro Stir Bars (Fisher Scientific, catalog number: 16800523)
FLEX-COLUMN^® Economy Columns (Fisher Scientific, catalog number: K420400-1030)
Spectrum^TM Spectra/Por^TM Float-A-Lyzer^TM G2 Dialysis Devices (Fisher Scientific, catalog number: 08-607-020)
Fisher brand^TM Class B Amber Glass Threaded Vials with Attached Caps (Fisher Scientific, catalog number: 14-955-332)
Pierce^TM Protein Concentrator PES, 3K MWCO, 2-6 ml (Fisher Scientific, catalog number: PI88515)
Sephadex G-15 (GE Healthcare, catalog number: 17002001)
MCF7 (ATCC^® HTB-22^TM) and HEK 293T (ATCC^® ACS-4500^TM) cell lines
Heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, catalog number: H4784-1G stored at 4 °C)
Dabcyl C2 amine (100 mg) (AnaSpec, catalog number: AS-81819, stored at -20 °C under N₂)
EDANS, sodium salt, 5- [(2-aminoethyl) amino] naphthalene-1-sulfonic acid, sodium salt (0.5 g) (AnaSpec, catalog number: AS-23886, stored at -20 °C under N₂)
N-(3-Dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC) (Sigma-Aldrich, catalog number: 03449, stored at -20 °C)
N-hydroxysuccinimide (NHS) (Sigma-Aldrich, catalog number: 130672-5G, -20 °C under N₂ )
Glacial Acetic Acid (HPLC grade) (Fisher Scientific, catalog number: A35-500)
Methyl sulfoxide, 99.7%, Extra Dry, anhydrous, SC (AcroSeal^TM, ACROS Organics^TM, catalog number: AC610971000)
Thermo Scientific^TM NERL^TM High Purity Water (Fisher Scientific, catalog number: 23-249-590)
Water (HPLC grade) (Fisher Scientific, Fisher Chemical W5-4)
Suramin (Sigma-Aldrich, catalog number: S2671)
Deuterium oxide, D₂O (Sigma-Aldrich, catalog number: 151882)
Sodium acetate (Sigma-Aldrich, catalog number: S7545)
Sodium Chloride (Fisher Scientific, catalog number: BP358-1)
HEPES (Fine White Crystals/Molecular Biology) (Fisher BioReagents, catalog number: BP310-500)
HyClone^TM Fetal Bovine Serum (FBS) (Fisher BioReagents, catalog number: SH3007102)
Dulbecco's Modified Eagle Medium (DMEM) growth media (Invitrogen, USA)
(Optional) Heparanase protein (R&D systems, catalog number: 7570-GH-005)
Antibiotic-antimycotic Solution (AA) (Fisher Scientific, catalog number: 15-240-062), antibiotic spectrum: Amphotericin B, Penicillin, Streptomycin
Assay buffer (see Recipes)

Equipment

FreeZone Plus 6 Lyophilizer (Labconco Corporation)
Peristaltic Pump P-1 (GE healthcare, catalog number: 18111091 )
Temperature controlled Centrifuge (Beckman coulter, model: Microfuge 20R; Eppendorf, model: 5804R)
Flex Station III (Molecular Devices, CA)
Shimadzu Spectrophotometer (Shimadzu Scientific Instruments, USA)
Fluorimeter PTI QM-400 (Horiba Canada)
pH meter (Denver Instrument, model: 220)
400 MH_Z topspin H¹NMR Bruker
C24 classic series Incubator (New Brunswick Scientific, NJ)
Precision Water bath (Precision Scientific, model: 25)
-20 °C (Norlake Scientific) and -80 °C freezer (Thermo scientific)
Forma Series II, water jacketed CO₂ incubator (Thermo Electron Corporation, USA)
Stirring Hotplate (Fisherbrand^TM Isotemp^TM, catalogue number: SP88850200)
Fluorescence cuvette (Hellma^®, catalog number: 101.015-QS; Cell Holder, catalog number: 013.013)