Abstract
Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D that hydrolyses lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX has been implicated in tumour progression and inflammation, and might serve as a biomarker. Here we describe the development of a fluorescent activity-based probe that covalently binds to the active site of ATX. The probe consists of a lysophospholipid-based backbone linked to a trapping moiety that becomes reactive after phosphate ester hydrolysis, and a Cy5 fluorescent dye to allow visualisation of active ATX. The probe reacts specifically with the three known isoforms of ATX, it competes with small-molecule inhibitors for binding to ATX and allows ATX activity in plasma to be determined. Our activity-based reporter will be useful for monitoring ATX activity in biological fluids and for inhibitor screening.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Catalytic Domain
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Enzyme Inhibitors / chemical synthesis
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Enzyme Inhibitors / chemistry*
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Fluorescent Dyes / chemical synthesis
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Fluorescent Dyes / chemistry*
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Humans
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Multienzyme Complexes / antagonists & inhibitors
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Multienzyme Complexes / genetics
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Multienzyme Complexes / metabolism*
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Phosphodiesterase I / antagonists & inhibitors
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Phosphodiesterase I / genetics
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Phosphodiesterase I / metabolism*
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Phosphoric Diester Hydrolases
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Protein Binding
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Protein Isoforms / antagonists & inhibitors
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Pyrophosphatases / antagonists & inhibitors
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Pyrophosphatases / genetics
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Pyrophosphatases / metabolism*
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
Substances
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Enzyme Inhibitors
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Fluorescent Dyes
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Multienzyme Complexes
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Protein Isoforms
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Recombinant Proteins
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Phosphoric Diester Hydrolases
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Phosphodiesterase I
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alkylglycerophosphoethanolamine phosphodiesterase
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Pyrophosphatases