Ruiming Zhao

Extreme diversity of scorpion venom peptides and proteins revealed by transcriptomic analysis: Implication for proteome evolution of scorpion venom arsenal

Venom is an important genetic development crucial to the survival of scorpions for over 400 million years. We studied the evolution of the scorpion venom arsenal by means of comparative transcriptome analysis of venom glands and phylogenetic analysis of shared types of venom peptides and proteins between buthids and euscorpiids. Fifteen types of venom peptides and proteins were sequenced during the venom gland transcriptome analyses of two Buthidae species (Lychas mucronatus and Isometrus maculatus) and one Euscorpiidae species (Scorpiops margerisonae). Great diversity has been observed in translated amino acid sequences of these transcripts for venom peptides and proteins. Seven types of venom peptides and proteins were shared between buthids and euscorpiids. Molecular phylogenetic analysis revealed that at least five of the seven common types of venom peptides and proteins were likely recruited into the scorpion venom proteome before the lineage split between Buthidae and Euscorpiidae with their corresponding genes undergoing individual or multiple gene duplication events. These are α-KTxs, βKSPNs (β-KTxs and scorpines), anionic peptides, La1-like peptides, and SPSVs (serine proteases from scorpion venom). Multiple types of venom peptides and proteins were demonstrated to be continuously recruited into the venom proteome during the evolution process of individual scorpion lineages. Our results provide an insight into the recruitment pattern of the scorpion venom arsenal for the first time.

Hg1, Novel Peptide Inhibitor Specific for Kv1.3 Channels from First Scorpion Kunitz-type Potassium Channel Toxin Family

Background: The potassium channel inhibitory activity of scorpion Kunitz-type toxins has not yet been determined. Results: We identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. Conclusion: A novel peptide, Hg1, specific for Kv1.3 channel, was found. Significance: Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases. The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nm for recombinant LmKTT-1a, 160 nm for LmKTT-1b, 124 nm for LmKTT-1c, 136 nm for BmKTT-1, 420 nm for BmKTT-2, 760 nm for BmKTT-3, and 107 nm for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ∼50–80% of Kv1.3 channel currents at a concentration of 1 μm. The exception was rBmKTT-3, which had weak activity. The IC50 values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ∼129.7, 371.3, and 6.2 nm, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

Molecular diversity of toxic components from the scorpion Heterometrus petersii venom revealed by proteomic and transcriptome analysis.

Scorpion venoms contain a vast untapped reservoir of natural products, which have the potential for medicinal value in drug discovery. In this study, toxin components from the scorpion Heterometrus petersii venom were evaluated by transcriptome and proteome analysis. Ten known families of venom peptides and proteins were identified, which include: two families of potassium channel toxins, four families of antimicrobial and cytolytic peptides, and one family from each of the calcium channel toxins, La1‐like peptides, phospholipase A2, and the serine proteases. In addition, we also identified 12 atypical families, which include the acid phosphatases, diuretic peptides, and ten orphan families. From the data presented here, the extreme diversity and convergence of toxic components in scorpion venom was uncovered. Our work demonstrates the power of combining transcriptomic and proteomic approaches in the study of animal venoms.

Imcroporin, a new cationic antimicrobial peptide from the venom of the scorpion Isometrus maculates.

ABSTRACT The pace of resistance against antibiotics almost exceeds that of the development of new drugs. As many bacteria have become resistant to conventional antibiotics, new drugs or drug resources are badly needed to combat antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial peptides, rich sources existing in nature, are able to effectively kill multidrug-resistant pathogens. Here, imcroporin, a new antimicrobial peptide, was screened and isolated from the cDNA library of the venomous gland of Isometrus maculates. The MIC of imcroporin against MRSA was 50 μg/ml, 8-fold lower than that of cefotaxime and 40-fold lower than that of penicillin. Imcroporin killed bacteria rapidly in vitro, inhibited bacterial growth, and cured infected mice. These results revealed that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.

Mucroporin, the First Cationic Host Defense Peptide from the Venom of Lychas mucronatus

ABSTRACT The misuse of antibiotics has led our age to a dangerous edge, as antibiotic-resistant pathogens appear to evolve more quickly than antibiotics are invented. Thus, new agents to treat bacterial infection are badly needed. Cationic host defense peptides are on the first line of a host defense system and are thought to be good candidates for treating bacterial infection. Here, a novel cationic host defense peptide, mucroporin, was cloned and characterized from the venom of Lychas mucronatus. The MIC for Staphylococcus aureus was 25 μg/ml, including antibiotic-resistant pathogens. Based on the molecular template of mucroporin, mucroporin-M1 was designed by amino acid substitution. The MIC for S. aureus was 5 μg/ml, including the antibiotic-resistant pathogens methicillin-resistant S. aureus, methicillin-resistant coagulase-negative Staphylococcus, penicillin-resistant S. aureus, and penicillin-resistant S. epidermidis. Moreover, mucroporin-M1 also inhibited gram-negative bacteria. The modes of action of mucroporin and mucroporin-M1 were both rapid killing by disrupting the cell membrane of bacteria, and the number of surviving bacteria was reduced by about 4 to 5 orders of magnitude immediately after peptide delivery. These results showed that mucroporin could be considered a potential anti-infective drug, especially for treating antibiotic-resistant pathogens.

Molecular diversity of Chaerilidae venom peptides reveals the dynamic evolution of scorpion venom components from Buthidae to non-Buthidae

UNLABELLED The scorpion family Chaerilidae is phylogenetically differentiated from Buthidae. Their venom components are not known, and the evolution of the venom components is not well understood. Here, we performed a transcriptome analysis of the venom glands from two scorpion species, Chaerilus tricostatus and Chaerilus tryznai. Fourteen types of venom peptides were discovered from two species, 10 of which were shared by both C. tricostatus and C. tryznai. Notably, the venom components of Chaerilidae were also found to contain four toxin types (NaTx, β-KTx, Scamp and bpp-like peptides), previously considered to be specific to Buthidae. Moreover, cytolytic peptides were the most abundant toxin type in C. tricostatus, C. tryznai and the family Euscorpiidae. Furthermore, 39 and 35 novel atypical venom molecules were identified from C. tricostatus and C. tryznai, respectively. Finally, the evolutionary analysis showed that the NaTx, β-KTx, and bpp-like toxin types were recruited into the venom before the lineage split between Buthidae and non-Buthidae families. This study provides an integrated understanding of the venom components of the scorpion family Chaerilidae. The family Chaerilidae has a specific venom arsenal that is intermediate between Buthidae and non-Buthidae, which suggests the dynamic evolution of scorpion venom components from Buthidae to non-Buthidae species. BIOLOGICAL SIGNIFICANCE This work gave a first overview of the venom components of Chaerilidae scorpions, and discovered large numbers of new toxin molecules, which significantly enriches the molecular diversity of scorpion venom peptides/proteins components. Based on phylogenetic analysis we speculated that the NaTx, β-KTx and bpp-like toxin type genes were recruited into venom before the lineage split between Buthidae and non-Buthidae. By Comparing the toxin types and abundance of the Buthidae, Chaerilidae and non-Buthidae families, we found that the family Chaerilidae has a specific venom arsenal that is intermediate Buthidae and non-Buthidae, which suggests the dynamic evolution of scorpion venom components from Buthidae to non-Buthidae species.

SdPI, The First Functionally Characterized Kunitz-Type Trypsin Inhibitor from Scorpion Venom

Background Kunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized. Principal Findings A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (Ki = 1.6×10−7 M) and thermostability. Conclusions The results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitz-type venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derived from scorpion venom, and it represents a new class of Kunitz-type venom peptides.

Genomic and Structural Characterization of Kunitz-Type Peptide LmKTT-1a Highlights Diversity and Evolution of Scorpion Potassium Channel Toxins

Background Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported. Principal Findings Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two β-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, β-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/β fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx. Conclusions/Significance Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.

Cloning and functional characterization of a new antimicrobial peptide gene StCT1 from the venom of the scorpion Scorpiops tibetanus

Scorpion has an innovative venom gland, which is an important determinant in contributing to its successful survival for more than 400 million years. Scorpion venom contains a diversity of bioactive peptides, which represent a tremendous hitherto unexplored resource for use in drug design and development. Here, StCT1, a new antimicrobial peptide gene, was screened and isolated from the venomous gland cDNA library of the scorpion Scorpiops tibetanus. The full-length cDNA of StCT1 is 369 nucleotides encoding the precursor that contains a putative 24-residue signal peptide, a presumed 14-residue mature peptide, and an uncommon 37-residue acidic propeptide at the C-terminus. The minimal inhibitory concentrations (MICs) of the synthetic StCT1 peptide against Staphylococcus aureus and Micrococcus luteus were 12.5microg/ml and 100microg/ml, respectively. The MICs of StCT1 against clinical antibiotics-resistant bacterial strains, were 50-250microg/ml, 2-40 folds lower than those of penicillin. These results show that the antimicrobial peptide encoded by StCT1 gene from the venom of the scorpion S. tibetanus is a potential anti-infective polypeptide or lead compound, especially for treating antibiotics-resistant pathogens.

Molecular cloning and electrophysiological studies on the first K(+) channel toxin (LmKTx8) derived from scorpion Lychas mucronatus.

LmKTx8, the first toxic gene isolated from the venom of scorpion Lychas mucronatus by constructing cDNA library method, was expressed and characterized physiologically. The mature peptide has 40 residues including six conserved cysteines, and is classified as one of alpha-KTx11 subfamily. Using patch-clamp recording, the recombinant LmKTx8 (rLmKTx8) was used to test the effect on voltage-gated K(+) channels (Kv1.3) stably expressed in COS7 cells and large conductance-Ca(2+)-activated K(+) (BK) channels expressed in HEK293. The results of electrophysiological experiments showed that the rLmKTx8 was a potent inhibitor of Kv1.3 channels with an IC(50)=26.40+/-1.62nM, but 100nM rLmKTx8 did not block the BK currents. LmKTx8 or its analogs might serve as a potential candidate for the development of new drugs for autoimmune diseases.