Papain was obtained from Sigma-Aldrich (St. Louis, MO), product number P 4762. E64 (Barrett et al., 1982
), a cysteine protease inhibitor, was also from Sigma-Aldrich, product number E 3132. Stock solutions of 430 μM papain and 1 mM E64 respectively were prepared in phosphate buffered saline and diluted in the relevant assay buffers. Hexapeptide agonists of PAR receptors were prepared at the peptide synthesis facility at Massachusetts General Hospital.
Protein extraction from spicules
Dry pods of M. pruriens were obtained from Costa Rica. The spicules were collected from the pods by shaking the pods in a closed bag. Ten grams of spicules were stirred at 4°C for 2 hours in 300 ml of a buffer containing 0.1 M NaCl and 0.01 M L-cysteine, pH 5.7. This crude extract was passed through cheesecloth and then a 0.4 μM filter. The extract was size-selected using centrifugal concentrators that had a molecular weight cut-off of 5 kDa ((Millipore, Billerica, MA). It was further purified using a P-60 column and fractions were run on SDS-PAGE gels. The fraction showing a single prominent band at 36 kDa was subjected to further analysis. This material is referred to as native mucunain and quantified using a non-interfering protein assay (Pierce, Rockford, IL). By counting the number of spicules in a small sample, the number of spicules per mg was estimated. When combined with the quantization of protein, it was deduced that each spicule contained 4 - 12 ng of protein.
The protein band running at 36 kDa was subjected to amino acid sequence analysis by tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer (Thermo Scientific, Waltham, MA). The protein was also subjected to NH2-terminal sequence analysis by Edman degradation.
Preparation of total RNA, poly-A RNA and ds cDNA
RNA was extracted from leaves and stems as it was found that spicules did not contain workable quantities of RNA. Leaves and stems of M. pruriens were ground to a fine powder in liquid nitrogen and total RNA was extracted using the Qiagen RNAeasy protocol (Valencia, CA). Poly-A RNA was isolated by Qiagen's Oligotex procedure. The poly-A RNA was used as a template to make ds cDNA using a cDNA preparation kit from Stratagene (La Jolla, CA).
Cloning of M. pruriens cysteine protease cDNA by PCR
One μg of ds cDNA was used as a template in a 50 μl PCR volume using a forward primer, TGG GGC GCC AGC TGG GGY TTC GAG GGY TAY based on the internal peptide, NSW GAS WGF EGY VR, and a reverse primer, dT28. The reaction containing pfu DNA polymerase from Stratagene was heated at 95°C for 5 minutes followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 1 minute, extension at 68°C for 2 minutes and a final extension at 68°C for 10 minutes. PCR products greater than 100 bp in length were cloned into a Zero-blunt TOPO PCR vector (Invitrogen) and sequenced. A reverse primer, AGG ACA AAT TTA AGC ATT GCT GCA TTA TCT ACC (based on the C-terminal sequence near the poly-A tail) and a forward primer, GAT AAC TTG CCG GAA TCT GTT GAT TGG AG (based on the NH2 terminal peptide, DNL PES VDW RNE GAV LPC KS) were used to isolate the entire mature protein coding sequence of 1300 bp. The entire cDNA of 1600 bp was later isolated using a forward primer, CAC GTG GCG GAG CGA CGA GGA GGT GAT GTC, based on the sequence containing the signal peptide, and the reverse primer at the C-terminus, AGG ACA AAT TTA AGC ATT GCT GCA TTA TCT ACC.
Expression of M. pruriens cysteine protease in E. coli
The cDNA (1300 bp) coding for the pro region and the mature portion of the protease was cloned into the expression vector pTWIN1 (New England Biolabs, Beverly, MA) containing the T7 promoter and lac I repressor. The vector, pTWIN1MUC1300, was transformed into ER2566. The cells growing in log phase were induced with 0.3 mM IPTG. The E. coli were harvested, suspended in 30 ml of lysis buffer (0.5 M NaCl, 0.02 M Tris, 1 mM EDTA, pH8.5) and lysed in a French press. The protein, present predominately in the insoluble fraction as inclusion bodies, was solubilized with 8 M urea and refolded in the presence of 0.5 M NaCl, 0.05 M Tris, pH 8.5, 0.7 M L-arginine, 0.01 M glutathione, 0.001 M glutathione disulfide, and 0.005 M EDTA as described. The refolded pro-protease, or zymogen, was activated in the presence of 0.2 M sodium acetate, pH 4.0, 0.005 M DTT and 0.005 M EDTA, and incubated at 37°C for 30 minutes. The recombinant protein was purified by Centricon Plus-70 concentrators with 30 kDa molecular weight cutoffs and dialyzed extensively against 0.1 M NaCl and 0.01 M L-cysteine, pH 5.7. In the chromogenic and calcium studies below, higher concentrations of recombinant as compared to native mucunain were used to achieve similar effects. In contrast, similar concentrations of recombinant and native mucunain used in the sensory studies achieved similar effects. A possible explanation for this discrepancy is that the recombinant protein folds more effectively in vivo than in vitro.
Chromogenic substrate assay
Native and recombinant mucunain protease activity were measured in a buffer containing 50 mM sodium acetate (pH 5.5), 2.5 mM EDTA, 2.5 mM DTT, and 250 μg/ml Z-Phe-Arg-pNA (Bachem, King of Prussia, PA) as the chromogenic substrate. After adding protease, or protease and E64, the reaction was incubated at room temperature for 1 hr. Generation of the yellow product, p-nitroaniline, was assayed by measuring the absorbance at 405 nm. Papain served as a control cysteine protease. E64 was used at a concentration of 10 μM. Experiments were performed in triplicate and the error bars represent standard deviation.
Reconstitution of spicules
Native spicules were inactivated by autoclaving for 1 hour and reconstituted with native and recombinant mucunains in the presence or absence of the cysteine protease inhibitor, E64. Typically, 50 mg of inactivated spicules were mixed with 0.4 ml of 1 mg of native or recombinant mucunain in 0.1M NaCl with 0.01 M L-cysteine in a 2 ml microfuge tube, vortexed, dried in a Speed-vac for 2 hours at room temperature, vortexed again and transferred to a new tube. This second vortex step allowed for precipitated salt to be separated from the spicules. These spicules were then considered as ‘reconstituted’ with the protease alone. Spicules prepared in a similar manner, but with E64 diluted from the 1 mM stock solution to achieve the same concentration as mucunain, were considered ‘reconstituted’ with the protease and the inhibitor. The use of 1 mg of mucunain for reconstitution was determined following a series of reconstitutions using a range of quantities of mucunain and then re-extraction. 1 mg of mucunain resulted in 4 ng of mucunain per spicule, approximating the natural situation, although the distribution of protein on the spicule would not necessarily match the native ones.
Psychophysical studies in humans
Protocols were approved by the Yale University Human Investigation Committee. A row of 10 cowhage spicules were lightly glued to the flattened end of a Weck-Cel (Medtronic, Jacksonville, FL) surgical spear, spaced at 1 mm, and applied parallel to the surface of the skin at the mid-volar forearm. This resulted in an average of 7 spicules superficially inserted approximately 0.2 mm into skin. Every 30 sec, subjects rated the magnitude of each of three qualities of sensation: Itch, pricking/stinging, and burning using a scale that employs appropriately placed intensity descriptors of “none”, “barely detectable”, “weak”, “moderate”, “strong”, “very strong” and, at the top of the scale, “the strongest sensation imaginable” (Bartoshuk et al, 2004
). Voltages derived from each subject's movements of a computer mouse that controlled a cursor on a visual representation of this scale were converted to numbers between 0 and 100 and plotted for each quality at successive intervals of 30s up to 20 min after the insertion of cowhage. Each of nine subjects received, in separate tests, five types of cowhage spicules: native cowhage spicules, heat-inactivated spicules that had been soaked in a solution of cowhage extract containing E64, or the same but without E64, and heat-inactivated spicules that had been previously soaked in a solution of recombinant mucunain containing E64, or the same but without E64. The subject and experimenter were blinded as to the type of spicule administered.
PAR activation observed by cytoplasmic calcium release
The four human PAR cDNAs were obtained from commercial sources and cloned into the pcDNA3.1(-) vector (Invitrogen, Carlsbad, CA). HeLa cells were transfected with 10 μg of vector cDNA using Lipofectamine 2000 (Invitrogen) and cultured for two days in 96-well glass bottom plates at 30,000 cells/well. Cells were loaded with Fura-2 as follows. The medium was aspirated and replaced with 100 μl of complete DMEM containing 2 μM Fura-2 (Invitrogen) and kept at room temperature in the dark for 1 hr. The medium was again aspirated and each well received 90 μl of HEPES buffered saline (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 0.8 mM MgCl2, 13.8 mM glucose, pH 7.4). Plates were observed using a Zeiss (Peabody, MA) Axiovert 200M microscope equipped with a filter wheel for monitoring excitation at 340 and 380 nm. Responses to test agents, analyzed using Axiovision software, version 4.6, were expressed as emission ratios of 340:380 which are proportionate to intracellular calcium. Ten μl of protease or hexapeptide were applied approximately 20 seconds after the start of excitation. For experiments that included E64, this inhibitor and mucunain were incubated together on ice for five minutes prior to application to the cells. Images were taken every 5 seconds, beginning at time zero, for 3 minutes. The software was used to analyze the 37 images taken during the 3-minute period.