BmK-YA, an Enkephalin-Like Peptide in Scorpion Venom

By screening extracts of venom from the Asian scorpion Buthus martensii Karsch (BmK) for their abilities to activate opioid receptors, we have identified BmK-YA, an amidated peptide containing an enkephalin-like sequence. BmK-YA is encoded by a precursor that displays a signal sequence and contains four copies of BmK-YA sequences and four of His4-BmK-YA, all flanked by single amino acid residues. BmK-YA and His4-BmK-YA are amidated and thus fulfill the characteristics expected of bioactive peptides. BmK-YA can activate mammalian opioid receptors with selectivity for the δ subtype while His4-BmK-YA is inactive at opioid receptors. The discovery of BmK-YA suggests that scorpion venom may represent a novel source of bioactive molecules targeting G protein-coupled receptors (GPCRs) and reveal additional insights on the evolution of the opioid precursors.


Introduction
Animal venoms constitute a vast library of biologically active peptides that are directed at a variety of membrane proteins. It is estimated that more than 10 million peptide toxins exist in 1400 species of scorpions, 400 species of snakes, 600 species of sea cone snails and 35000 species of spiders [1]. However, only a very small portion (less than 0.02%) of the estimated natural bank has been identified. It is becoming clear that venom peptides have diverse pharmacological properties and that several of these peptide toxins are useful as molecular tools for the study of their receptors [2], and may represent a unique source of leads and structure templates for the development of novel therapeutic molecules and insecticides [3]. Venoms can be used as the natural equivalents of large combinatorial libraries in drug discovery. Indeed a number of peptide toxins have been used in vivo for proofof-concept studies, and several have undergone preclinical or clinical development for the treatment of pain, diabetes, multiple sclerosis and cardiovascular diseases [4][5][6][7][8][9][10]. Most venoms are known to contain peptide toxins that act on ion channels. Only a few are known to act on GPCRs. Indeed, among the 1800 toxins described in 2006 [11], less than 30 are known to be active on GPCRs [12].
The Asian scorpion BmK is widely distributed in Mongolia, Korea and China where it has been used in Traditional Chinese Medicines (TCMs) for thousands of years as a source of pain relieving drugs. Animal study has shown that scorpion venom do not elicit dependence [13]. This suggests that further exploration of BmK might provide a potential analgesic medicine without addictive properties. In recent decades, BmK venom has been extensively studied and has led to the discovery of more than a hundred peptides through biochemical purification or deduced from gene cloning [14][15][16][17][18][19][20][21]. These peptides exhibit a wide range of physiological and pharmacological activities and have been developed as biopesticides, vaccines, cancer treatments, and protein engineering scaffolds [22,23]. These scorpion peptides are mainly interacting with ion channels [15], but very little is known about their capacity to modulate GPCR activity.
In this study, we have used BmK scorpion venom as a source of new ligands for opioid receptors. The opioid receptors are GPCRs that are activated by endogenously produced opioid peptides that contain an enkephalin sequence at their N-termini and also by exogenously administered opiates, such as morphine, a well known analgesic drug. Pharmacological and biochemical evidence supports the existence of three major subtypes of opioid receptors, m, d and k [24]. Herein, we described the purification and biochemical characterization of a novel peptide, BmK-YA that displays a sequence related to the enkephalin sequence and is encoded by a precursor that contains not only four sequences of BmK-YA but also four of His 4 -BmK-YA. BmK-YA can activate mammalian opioid receptors. These data indicate that BmK-YA and His 4 -BmK-YA are bioactive peptides and reveal additional insights on the evolution of opioid precursors.

Materials
The crude venom of BmK was collected by electrical stimulation of the telson of the scorpion and lyophilized in Luoyang city, Henan Province, China. HPLC-grade water was purified with a Milli-Q system (Millipore, Bedford, MA). Acetonitrile (ACN) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Trifluoroacetic acid (TFA) and ortho-phosphoric acid was from Tedia, USA. Formic acid was obtained from Acros (Geel, Belgium). Triethylamine was obtained from Sigma-Aldrich (St.Louis, Mo, USA). Fluo-4 AM was purchased from Molecular Probes (Eugene, OR). Lipofectamine was purchased from Invitrogen (Carlsbad, CA). All other reagents were analytical grade and used without purification.
Venom pre-treatment and Purification procedures Solid phase extraction (SPE) cartridges packed with octadecyl sorbent (30 g sorbent/cartridge) were washed with methanol, 60% ACN/0.1% TFA and 5% ACN/0.1% TFA sequentially. The lyophilized crude venom (ca. 4g) was dissolved in 5% ACN/0.1% TFA aqueous solution and loaded onto the cartridges in batches. Each cartridge was rinsed with 100 mL 5% ACN/0.1% TFA aqueous solution to remove some polar substances, and then the peptides were eluted with 100 mL 60%ACN/0.1%TFA aqueous solution. In total, 1.3 L eluate was collected, pooled and lyophilized by the Refrigerated CentriVap Centrifugal Concentrator (Labconco, Kansas, USA). Finally, about 1.2 g lyophilized sample was generated.
BmK-YA was purified by an offline two-dimensional chromatographic strategy,in which reverse phase liquid chromatography (RPLC) was combined with hydrophilic interaction chromatography (HILIC). Briefly, the SPE treated venom was redissolved in 5% ACN/0.05% TFA aqueous solution and fractionated by a preparative C18 column (XTerra MS C18,100619 mm i.d., 5 mm particle size, 120 Å pore size, Waters). The sample was loaded on the column at 100 mg per run. The mobile phase was composed of 0.05% (v/v) TFA aqueous solution (mobile phase A) and ACN with 0.05% (v/v) TFA (mobile phase B). The gradient was run from 5% to 35% mobile phase B over 50 min. The flow rate was 17 mL/min and the elution was monitored by MS (Micromass ZQ2000). The passive splitter was about 1/3000. Mass Scans were acquired in positive ion mode from m/z 300-2000.
Fractions were collected automatically at 1 minute interval and denoted as Fraction 1 to Fraction 50. The fraction with the same elution time from each round of HPLC was pooled and evaporated to dryness in the Centrifugal Concentrator.
The fraction of interest (Fraction 17) was further purified on a homemade Click Maltose column (15064.6 mm, 5 mm). This stationary phase was prepared through click chemistry as described earlier [25]. The mobile phase was composed of water (A), ACN (B) and 100 mM triethylamine phosphate (TEAP) buffer (pH 2.3) (C). The gradient was from 10%A/85%B/5%C to 45% A/50%B/ 5%C in 40 min, then from 45% A/50%B/5%C to 50% A/45%B/ 5%C in 20 min. The absorbance was measured at 214 nm.

Peptide de novo sequencing and synthesis
ESI MS and ESI MS/MS were performed using a nano-LC-MS/MS system (nano-Acquity TM coupled to a Q-TOF premier TM , Waters, Manchester, UK). Scans were acquired in positive ion mode from m/z 500-1500 Da for MS analysis and m/z 50-1000 Da for MS/MS analysis with nanospray voltage at 2.0kV. The source temperature was maintained at 80uC and the cone voltage was set to 35 kV. The collision energy for MS/MS analysis of BmK-YA was set at 27 eV. The amino acid sequence of BmK-YA was analyzed by MassLynx 4.1 software incorporating the MaxEnt3 deconvolution algorithm and PepSeq tools (Waters, Manchester, UK).
The BmK-YA peptide was synthesized through solid-phase synthesis (ChinaPeptides Co., Ltd, Shanghai, China) on an Applied Biosystems 433A system as described previously [26]. Briefly, the peptide was synthesized in a small reactor, on the 2chlorotrityl chloride resin preloaded with Fmoc-L-Ala-OH. Side chain protecting groups used for tri-functional residues were tertbutyl for Tyr and trityl (Trt) for Asn. After full assembly was completed, the resin was treated with TFA/water/ethanedithiol/ thioanisol (94%/2.5%/2.5%/1%) for 120 min. The crude peptide was extracted with cold diethyl ether six times and dried under a flow of nitrogen. Finally, the extracted peptide was purified to homogeneity through a Kromasil 100-5 C18 column (25064.6 mm, 5 mm) with a linear gradient from 5% to 35% ACN/0.1%TFA in 30 min.

Plasmid construction and stable cell lines
All GPCRs used in this study were amplified from human cDNA library (Clontech, Palo Alto, CA) and cloned into pcDNA 3.1 (-) (Invitrogen, Carlsbad, CA). The sequences were confirmed by sequencing from both ends and with internal primers by Laragen (Los Angeles, CA). Human embryonic kidney-293 T cells (HEK293T) were cultured in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal calf serum (FBS). The stable cell lines expressing human opioid receptors m, d or k individually were created as previously reported [27]. The individual human opioid receptors m, d or k DNA plasmid were cotransfected with Gqai3, a chimeric G protein with which opioid receptors can be redirected to mediate intracellular calcium mobilization upon stimulation. Transfection was carried out with lipofectamine using the protocol provided by the supplier. Stable cell clones were selected in the presence of 200 mg/mL G418, 200 mg/mL hygromycin and 200 mg/mL zeocin.

Identification of a cDNA clone encoding BmK-YA
Total RNA was extracted from Asian scorpion BmK using Trizol Reagent (Invitrogen, CA). Messenger RNA was purified with Qiagen Oligotex mRNA Kit. Based on the information obtained from direct peptide sequencing, RACE-ready cDNA and subsequent amplification of 59 and 39 ends were performed using Smart RACE cDNA kit from Clontech. RACE primers are:59-GTTTTCACCGCTTTAATTTATCTACATAGAATG-39and 59-CATTCTATGT-AGATAAATTAAAGCGGTGAAAAC-39. PCR products were cloned into pcDNA3.1/V5-His-TOPO vector and sequenced by Laragen (Los Angelus, CA).

Ca 2+ response monitored by Fluorometric Imaging Plate Reader Assay (FLIPR)
The assay was performed as reported earlier [28]. Briefly, the stable cells were seeded into poly-D-lysine-coated black wall, clearbottom 96-well plates at a density of 80,000 cells per well. Twentyfour hours later the medium was removed and replaced with 100 mL of dye loading solution (2 mM Fluo-4 AM dissolved in FLIPR buffer, which consists of 0.2 mg/mL pluronic acid in 16Hank's buffer supplemented with 20 mM HEPES, pH 7.4) for 1 h at 37uC. The cells were then washed 3 times with FLIPR buffer prior being assayed. The samples, which were re-dissolved in dimethyl sulphoxide (DMSO) and stored in 96-well drug plates, were diluted with FLIPR buffer and then added into the cells within 4 sec automatically. The intracellular Ca 2+ concentration was monitored at 520 nm with excitation wavelength at 488 nm over a period of 4 min.

Data processing
EC 50 values and curve fitting were determined using Graphpad Prism (GraphPad Software, Inc., San Diego, CA). The maximal stimulation was determined by the response of the selective ligands endomorphin-1, deltrophin, and dynorphin A in m-, dand kexpressing stable cell lines, respectively. Data from each dose response curve were normalized to the maximal stimulation of each cell line. Potency is determined by EC 50 . Efficacy is determined by Emax, which is the ratio of the maximal response of each peptides and the maximal stimulation induced by selective ligands in corresponding opioid receptor-expressing cells. When the Emax reaches 100%, the peptide is considered a full agonist.

Results
Identification and characterization of a novel enkephalinlike peptide from venom Scorpion venom was fractionated by a preparative C18 column into 50 fractions. These fractions were screened against three individual opioid receptors-expressing cell lines and one vectorexpressing HEK293T cell line. Intracellular Ca 2+ changes were monitored using the FLIPR system. A reproducible and robust change in Ca 2+ concentration in d-expressing cells but not in vectorexpressing cells was observed in Fraction 17 (labeled with asterisk in Fig. 1). Fraction 17 was further purified in an analytical scale Click Maltose column ( Fig. 2A, B), yielding the component of interest ( Fig. 2A, peak labeled with asterisk). HPLC and Mass spectrometry analysis of this peak revealed a single peptide with 870.3 atomic mass unit (Fig 2B, C). The amino acid sequence Tyr-Gly-Gly-Tyr-Met-Asn-Pro-Ala-NH 2 (YGGYMNPA) was obtained by nanoLC-Q-TOF-MS/MS and PepSeq software (Fig. 2 D). Included in the sequence is a C-terminal amidation, a typical post-translation in scorpion venom. We have termed this peptide BmK-YA, based on the genus of the scorpion and its first and last amino acid. By searching NCBI data bank it is revealed that BmK-YA is a fragment of a large predicted protein (accession number: AAD39510).

Chemical synthesis of BmK-YA
BmK-YA was synthesized chemically. The synthesized material was found to have the same retention time as the native peptide (Fig. 2B) and the same monoisotopic mass [M+H] (MW 871.3032). The synthesized peptide was tested in d-expressing cells and shown to exhibit a reproducible d receptor agonist response indicating that the synthesized peptide is the same as the native one found in scorpion venom. Because of this, the synthesized peptide was used for further pharmacological characterization.

cDNA Cloning and sequencing
Degenerate oligonucleotides synthesized according to the amino acid sequence of BmK-YA were used to screen a cDNA library. This procedure allowed the identification of a long DNA segment corresponding to the majority of the BmK-YA gene. The full DNA sequence was obtained using the nucleotides indicated in Material and Methods. Fig. 3A shows that ther BmK-YA cDNA precursor encodes a 200-residue protein containing a 23-residue signal peptide, followed by 177-residues. Aspartic acid cleavage at Cterminal has been reported in murine [29,30]. The single arginine cleavage found at N-terminal of the BmK-YA occurs in a number of other mammalian neuropeptide presursors. The cDNA encoding the mature peptides contained 4 copies of the repeated sequence ''YGGYMNPA'', the BmK-YA, and 4 copies of the sequence ''YGGHMNPA'', predicted to be a novel peptide named His 4 -BmK-YA. The glycine residues at the C-terminals of BmK-YA or His 4 -BmK-YA are expected to be coverted into amide groups. The sequences of the cDNA and the purified peptide demonstrate that BmK-YA is a natural peptide in scorpion.

Pharmacological characterization of BmK-YA
BmK-YA acted as a full agonist when tested in d-expressing cells as shown in Fig. 4A. No response was observed in vectorexpressing cells (data not shown). BmK-YA has a similar efficacy but exhibited lower potency (2.5 mM) when compared with deltorphin, a selective d opioid receptor agonist, (potency 3.1 nM [27]). We also tested the effects of BmK-YA on m-and k-expressing cells. As shown in Fig. 4A, at concentrations up to 100 mM, BmK-YA does not reach a maximal effect and exhibit lower potencies (approximately17 mM and 30 mM, respectively).
The d-activity of BmK-YA was inhibited by naloxone, in a dosedependent manner (Fig. 4A, insert) (IC 50 539 nM), providing additional evidence that BmK-YA can interact with opioid receptors.

Synthesis and activities of His 4 -BmK-YA and Phe 4 -BmK-YA
His 4 -BmK-YA was synthesized and tested for its ability to activate opioid receptors. As shown in Fig. 4B, no activity was detected in the concentration range from 1 nM to 50 mM.
According to the typical opioid peptide sequence, Phe 4 -BmK-YA was synthesized in which phenylalanine (F) was substituted for tyrosine in the fourth position. As shown in Fig. 4B, this substitution dramatically increased the activity of the peptide which then has potencies of 157 nM, 60 nM and 77 nM, in m-, d-, and k-expressing cells, respectively.

Discussion
The toxins found in venomous animals have been optimized over time to aid in prey capture and digestion and also to help the animals defend themselves. While venomous animals receive their fair share of notoriety for the painful (and often deadly) effects of their bites and stings, their venoms have been harnessed for the treatment of human diseases for thousands of years. In recent years, venoms have been subjected to more rigorous scientific  investigation as a potential source of new therapeutic entities. Currently, five venom-derived peptide drugs are on the market, and many more are in pre-clinical or clinical development for indications such as cancer, pain, heart disease, stroke, and diabetes [31]. Animal venoms represent a valuable source of untested bioactive molecules, as the venoms of only a few hundred species have been studied to date.
Animal venom peptides that are active at GPCRs can be divided into two families [12]. The members of the first family mimic the natural agonist at the target receptor. Peptides belonging to this family are snake sarafotoxins, which are functional analogs of the endogenous endothelins [32], the cone snail toxin conopressin, which is similar to the arginine-vasopressin peptide [33], and the cone snail toxin contulakin-G, which is similar to the neurotensin peptide [34]. The second family of GPCR toxins consists of highly reticulated peptides with folds unrelated to those of natural ligands [1,[35][36][37][38][39][40]. We have discovered BmK-YA, the first scorpion venom peptide that displays a primary structure resembling that of the enkephalin-like peptides. BmK-YA thus belongs to the family of venom peptides that mimic the natural agonists and suggests that scorpion venom may represent a novel source of GPCRs ligands.
By analyzing the sequence of the protein encoding BmK-YA, we found a polyprotein containing four BmK-YA (YGGYMNPA) and four His 4 -BmK-YA (YGGHMNPA). This polyprotein contains a typical signal sequence, which indicates that it is secreted. Furthermore BmK-YA and His 4 -BmK-YA can be amidated suggesting that they are bioactive. The organization of this precursor is reminiscent to that of the mammalian opioid peptide precursors where multiple sequence-related peptides within a single genomic transcript. Excluding the endomorphins, the classical opioid peptides are derived from three larger precursors: proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN), which encodes for one, seven and three enkephalin-containing sequences (YGGFM or YGGFL). Compared to these, the precursor encoding BmK-YA, contains eight copies of enkephalin-like sequences (YGGYM or YGGHM). Interestingly the core BmK-YA enkephalin-like sequences are followed by four conserved residues (NPAG), of which the glycine residue serves as amide donor. This amidation is thought to be mediated by a specific amidation enzyme [41,42]. Indeed, two amidated enkephalin-like peptides, amidorphin and metorphamide, have been reported in mammals [43,44]. A data base search did not yield any significant hits in mammalian genomes although eight repeats of the sequence (…RGGYVNPAG…) are found as part of the TBC1 domain family member 14 protein. This is however a non secreted protein.
We show that BmK-YA in vitro interacts with the three subtypes of opioid receptors, m, d and k, but with preference to the d subtype. Its selectivity to the d-subtype is 6.8 times higher than that to the m, and 12 times higher than that to the k subtype. It therefore displays a pharmacological profile that is different from morphine. BmK-YA is a full agonist at the d receptor with an EC 50 of 2.5 mM while morphine is only a partial agonist with an EC 50 of 15 mM [27]. Although both molecules can activate d receptors with low potency, morphine cannot stimulate d receptors as effectively as BmK-YA at high concentrations. On the other hand, morphine is a full agonist at the m receptor with an EC 50 of 180 nM [27], while BmK-YA is only a partial agonist with an EC 50 of 17 mM. Thus BmK-YA might induce fewer of the side effects associated with m receptors. This may serve as a starting point for structure-function relationship studies leading to design specific antinociceptive drugs.
Whether BmK-YA acts at opioid receptors in the scorpion is not known but not expected. The fact that BmK-YA is encoded in a precursor that also contains His 4 -BmK-YA leads us to hypothesize that both peptides should act at the same receptors. However, His 4 -BmK-YA is inactive at the opiod receptors. Indeed, it is the specific His 4 substitution that is responsible for the lack of activity since Phe 4 -BmK-YA (YGGFMNPA), which contains a copy of Met-enkephalin, exhibits high affinity for the opioid receptors. Several lines of evidence indicate that BmK-YA is the first member of a new bioactive peptide family in scorpions. First, BmK-YA is encoded by a precursor that can be secreted. Second, the organization of this precursor is similar to that of the mammalian opioid peptide precursors with multiple sequencerelated peptides within a single genomic transcript. Third, BmK-YA and His 4 -BmK-YA are flanked by processing cleavage sites and can be amidated. Fourth, the NH2-tripeptide YGG sequence of BmK-YA and His 4 -BmK-YA is identical to the core sequence YGGF of the opioid peptides (Fig. 3B) and thus suggest evolutionary conservation. Whether they act as bioactive peptides in vivo will however await the identification of their receptor(s).
The discovery of BmK-YA and its identification as an enkephalin-like peptide demonstrates that relatively ''primitive'' organisms may possess opioid-like systems. The present study supports previous work that have characterized opioid peptides (enkephalin-containing) in invertebrate, for example, the mussel Mytilus edulis [45] and the digestive system of the scallop Chalmys farreri [46]. It has also been reported on the basis of binding and immunocytochemical analyses that d opioid receptors subtypes may exist in invertebrates [47,48]. BmK-YA is the first invertebrate peptide that displays a similar but not identical enkephalin sequence. Because enkephalin sequences are found in invertebrates and vertebrates while the BmK sequence is not, it is reasonable to assume that enkephalins served as templates for BmK-YA. Since the BmK-YA gene is not found in other species by database bank search it may be unique to the scorpion. The final appearance of BmK-YA gene may be the result of a genome duplication [49] which started with PENK. Over time one copy of a duplicated PENK gene may retain the PENK organization scheme and the function of the ancestral gene, while the other copy would accumulate amino acid substitution and diverge into a unique member of the BmK-YA gene family. Because we did not, in our purification, identify other enkephalin-containing peptides such as true opioid peptides (N terminus:YGGF), we expect that they do not exist in scorpion. Thus BmK-YA might have evolved to carry role(s) distinct from classical opioid function. This is reinforced by the coexistence of His 4 -BmK-YA, which does not exhibit activity at mammalian opioid receptors. Also, the presence of these peptides in the venom of the scorpion is counterintuitive to them displaying an analgesic activity. Consequently we propose that these peptides must interact with receptors that are divergent of the mammalian opioid receptors and that, in the venom, BmK-YA and His 4 -BmK-YA may have evolved for specialized use, such as prey capture, defense or immune response.