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Comparative Study
.2008 Apr 23;28(17):4331-5.
doi: 10.1523/JNEUROSCI.0716-08.2008.

Cowhage-evoked itch is mediated by a novel cysteine protease: a ligand of protease-activated receptors

Affiliations
Comparative Study

Cowhage-evoked itch is mediated by a novel cysteine protease: a ligand of protease-activated receptors

Vemuri B Reddy et al. J Neurosci..

Abstract

Cowhage spicules provide an important model for histamine-independent itch. We determined that the active component of cowhage, termed mucunain, is a novel cysteine protease. We isolated mucunain and demonstrate that both native and recombinant mucunain evoke the same quality of itch in humans. We also show that mucunain is a ligand for protease-activated receptors two and four. These results support and expand the relationship between proteases, protease-activated receptors, and itch.

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Figures

Figure 1.
Figure 1.
Photomicrograph ofM. pruriens spicules and biochemical analysis of mucunain.a, Photomicrograph of native spicules. Each spicule is 2–3 mm in length and typically one or a few micrometers in diameter at the tip. Scale bar, 100 μm.b, SDS-PAGE ofM. pruriens extract (lane 1) and recombinant mucunain (lane 2). The native protein may undergo posttranslational modification and thus run more slowly than the recombinant protein under reducing conditions. Molecular weight markers (kDa) are shown on the left.c, Mucunain cleaves the chromogenic substrate Z-FR-pNA, the cleavage of which was blocked by the protease inhibitor E64. Papain served as a positive control. The proteases were used at the following concentrations: native mucunain, 0.9 μm; recombinant mucunain, 2.7 μm; and papain, 4.3 μm. Experiments were performed in triplicate, and error bars represent SD.
Figure 2.
Figure 2.
Perceived intensities of sensations evoked by native spicules and spicules reconstituted with native or recombinant mucunain. For each quality of sensation, the geometric mean of the ratings of nine subjects is plotted as a function of time after spicule insertion. Mucunain evoked itch (blue) and nociceptive sensations consisting of burning (green) and/or pricking/stinging (pink). Addition of E64 during reconstitution produced spicules that did not evoke significant itch or nociceptive sensations. Autoclaved spicules elicited no sensations after insertion into the skin (data not shown). Statistical comparisons were made of the treatments (native, extract, and recombinant mucunain) and sensory qualities (itch, prick/sting, and burn). For each subject, sensory quality, and treatment, the area under the curve plotting the natural log of the original rating versus time was obtained. The log of the lowest value possible on the scale was substituted for any rating of zero. A two-way, repeated-measures ANOVA of the mean areas for sensory quality versus treatment revealed a significant effect of quality (p < 0.0001), but no significant differences resulting from treatment (p > 0.05).
Figure 3.
Figure 3.
Native and recombinant mucunain induce responses in HeLa cells transfected with PAR receptors.a, Mean peak response (mean peak 340:380 ratio) to the indicated protease of cells transfected with cDNAs encoding PAR1 (white bars), PAR2 (bars with angled lines), PAR3 (solid bars), and PAR4 (bars with horizontal lines). Mucunains were used at concentrations that yielded the greatest response: native mucunain, 0.3 μm; recombinant (Rec.) mucunain, 2 μm. Hexapeptides were all used at 1 mm. The data from 10–20 individual cells were averaged within a single experiment. The averages from at least three separate experiments were then combined and are presented ±SD.b,c, Data from single-cell imaging. The curves demonstrate the effect of native (b) and recombinant (c) mucunain on PAR2 transfected cells and PAR4 transfected cells in the absence or presence of E64. Mucunain was added as indicated and remained present throughout the experiment. Concentrations of mucunain are as noted ina. The concentration of E64 was 10 μm.
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