Plant cysteine proteases that evoke itch activate protease-activated receptors

Abstract

Background: Bromelain, ficin and papain are cysteine proteases from plants that produce itch upon injection into skin. Their mechanism of action has not been considered previously.

Objectives: To determine the mechanism by which these proteases function.

Methods: The ability of these proteases to activate protease-activated receptors was determined by ratiometric calcium imaging.

Results: We show here that bromelain, ficin and papain activate protease-activated receptors 2 and 4.

Conclusions: Bromelain, ficin and papain function as signalling molecules and activate protease-activated receptors. Activation of these receptors is the likely mechanism by which these proteases evoke itch.

Fig 1

Protease-activated receptors (PARs) are activated by proteolytic cleavage. Proteases cleave PARs near the extracellular N-terminus resulting in exposure of a previously cryptic tethered ligand that folds back on to the receptor to become active.

Fig 2

Polyacrylamide gel electrophoresis of plant proteases. Extracts containing bromelain and ficin were passed over G-50 Sephadex columns to remove most contaminants. Papain was a cleaner preparation, having been obtained in crystalline form. Molecular weight and proteases are indicated. These preparations were used in the receptor activation studies.

Fig 3

Plant proteases activate human protease-activated receptors (PARs). Single-cell imaging of (a) bromelain (2 µmol L⁻¹), (b) ficin (1.5 µmol L⁻¹) and (c) papain (2.17 µmol L⁻¹) induced responses in HeLa cells transfected with either PAR2 or PAR4 as measured by ratiometric calcium imaging in cells loaded with fura-2. Labels of curves for bromelain and ficin correspond to those for papain. The responses to PAR2 and PAR4 were blocked by the protease inhibitor E64 (10 µmol L⁻¹). Other negative controls included cells transfected with either salmon sperm DNA or PAR1. Positive controls for PAR2 and PAR4 included cathepsin while thrombin served as a positive control for PAR1 and PAR4 (not shown). Proteases were added as indicated.