Proteomic Characterization of Venom from Scorpio fuscus: Insights for Therapeutic Applications

Highlights

• This is the first proteomic profiling of Scorpio fuscus venom using gel-based decomplexation and LC-MS/MS.

• A total of 84 proteins were identified, including antimicrobial peptides with toxin-like characteristics.

• Identified venom proteins include phospholipases and ion channel inhibitors targeting calcium and potassium channels.

• UniProtKB annotation suggests 13 proteins are associated with lipid metabolism, with possible implications for angiogenesis.

• Identification of hemocyanins and unidentified proteins suggests venom is complex and needs more functional research.

  
  

Graphical Abstract

Introduction

Scorpions belong to the class Arachnida and order Scorpiones. They inhabit tropical and subtropical regions worldwide, excluding New Zealand (Nenilin and Fet, 1992). These ancient arthropods adapted to terrestrial life 437 million years ago during the late Silurian period. They are often considered "living fossils" due to their preserved morphology (Dunlop and Webster, 1999; Lourenco, 2018; Wendruff et al., 2020).

With 2,772 identified species across 23 families, scorpions exhibit a distinctive body structure. This structure comprises the Prosoma and Opisthosoma, which is further split into Mesosoma and Metasoma. The Metasoma has five segments that culminate in the telson housing venom glands (Hjelle, 1990; Rein, 2023). Notably, Buthidae species are characterized by neurotoxic venom, while other families, such as Scorpionidae, possess cytotoxic venom (Quintero-Hernandez et al., 2013).

Scorpionidae comprises 18 genera and 193 species, with the genus Scorpio being the first established in the order Scorpiones by Fet et al. (2002). Although previously considered subspecies of Scorpio maurus, many Scorpio populations have been elevated to species level (Lourenco, 2011; Talal et al., 2015).

Scorpio fuscus, a member of Scorpionidae, is a small to medium-sized scorpion found in Iraq, Israel, Jordan, Lebanon, Saudi Arabia, Syria, and Turkiye (Fet, 2000). Initially described as Buthus (Heterometrus) palmatus fuscus, it has undergone taxonomic revisions. It was classified as a subspecies and later reinstated as a species by Talal et al. (2015). In Turkiye, it is documented in provinces such as Adana, Adıyaman, Diyarbakır, Gaziantep, Hatay, Mardin, Mersin, and Şanlıurfa (Birula, 1898; Crucitti and Malori, 1998; Crucitti and Vignoli, 2002; Levy and Amitai, 1980).

Birula (1910) reviewed Scorpio maurus populations and Scorpio maurus fuscus records from Mersin and Adana provinces, where specimen collection was made in the present study. A close correlation has been observed between body size and chelae size, along with the toxicity of the scorpion venom (Xia et al., 2023).

Scorpion venom is a complex concoction. It encompasses lipids, nucleotides, amines, polypeptides, and uncharacterized constituents (Heinen and da Veiga, 2011). The peptide/protein content is rigorously limited to a maximum of 5% of the total dried weight. This represents a heterogeneous pool of peptides/proteins with distinct structures, target sites, functions, and toxicities against diverse organisms (Hmed et al., 2013).

A noteworthy aspect lies in the specific affinity of scorpion venom peptides for cancer cells. This accentuates the potential translational implications of these molecules (Aroui et al., 2009; Heinen and da Veiga, 2011; Xia et al., 2023). Consequently, scorpion venom emerges as a compelling subject for scientific inquiry. It offers avenues for nuanced drug design, developmental exploration, and clinical utility (Uzair et al., 2018).

Harnessing the formidable toxicity of scorpion venom, it has been employed historically for medicinal purposes in various regions. This has fostered an ongoing quest to unveil therapeutic constituents within this potent venom. The therapeutic attributes of scorpion venom stem predominantly from a class of small-molecule peptides, commonly referred to as scorpion toxins. These toxins consist of 20–90 amino acids and exhibit a range of diverse biological activities (Hmed et al., 2013).

Scorpion venom peptides can be broadly categorized into disulfide-bridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs). DBPs (13–70 amino acids) contain three or four disulfide bridges. They include sodium (NaTx), potassium (KTx), calcium (CaTx), chloride (ClTx), and TRP channel toxins, which modulate ion channel permeability (Cid-Uribe et al., 2020; Peter Muiruri et al., 2023).

While specific studies on Scorpio fuscus are lacking, related species show pharmacologically valuable venoms. For example, Scorpio maurus phospholipases interfere with integrins, inhibiting angiogenesis and tumor cell invasion (Krayem et al., 2018a, 2018b). Additionally, Abdel-Rahman et al. (2013) identified 65 peptides/proteins (413–14,009 Da) in Scorpio palmatus venom. They found toxin-like and antimicrobial sequences, highlighting therapeutic potential (Abdel-Rahman et al., 2013).

Considering the taxonomic ambiguity surrounding Scorpio fuscus, which was initially treated as a subspecies of both Buthus palmatus and Scorpio maurus, its current recognition as a distinct species is primarily based on recent morphological, ecological, and behavioral assessments.

Thus, the aim of the present study is to identify the proteins and peptides found in the venom of Scorpio fuscus (Scorpiones: Scorpionidae) using a gel-based proteomics approach. This will provide insight into toxin sequences, enhance understanding of venom function, and reveal its molecular diversity from a proteomics perspective. This study expands understanding of scorpion venom's pharmacological implications, offering avenues for drug development and clinical exploration.

Collection of Scorpion Specimens, Obtaining and Storage of Scorpion Venoms

Scorpio fuscus specimens for milking of venom were collected from Kozan District of Adana Province (Eski Mantaş Village, 37°31′30″N, 35°54′22″E, 434 m and Eski Kabasakal Village, 37°26′09″N, 35°51′23″E, 158 m) from July 8 to 14, 2021, and July 7 to 16, 2022. Scorpions were kept in 5 L containers, including cocopeat soil and a hiding container. They were fed crickets, grasshoppers, and larvae of Tenebrio molitor. Specimens were milked after carbon dioxide anesthesia, using 12V/25A electrical stimulation.

Results and Discussion

The venom proteins of Scorpio fuscus (Scorpiones: Scorpionidae) were identified using de novo protein characterization with LC-MS/MS. Following one-dimensional polyacrylamide gel electrophoresis (8, 15, and 20%) (Fig. 1), ten bands were excised and subjected to in-gel trypsin digestion and LC-MS/MS (Fig. 2). Using these ten bands, 84 proteins and peptides were identified from seven bands using Peaks Studio (version 10.5) for DENOVO, PEAKS, and SPIDER search (Table 1 and Supplementary Table S1).

Conclusion

In conclusion, this comprehensive proteomic analysis of Scorpio fuscus venom provides valuable insights into the potential therapeutic applications of venom proteins. Using SDS-PAGE-based decomplexation prior to LC-MS/MS analysis is a reliable and widely applied strategy. It reduces sample complexity and achieves robust protein identification. While the current approach allowed for effective protein identification, we acknowledge that complementary techniques such as RP-HPLC-based fractionation could enhance the results further.

CRediT Authorship Contribution Statement

Hasan Ufuk Celebioglu: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization.

Sinan Kandir: Writing – original draft, Methodology, Data curation.

Cigdem Gokcek-Sarac: Writing – original draft, Methodology, Investigation.

Ersen Aydin Yagmur: Writing – original draft, Methodology, Investigation.

Birte Svensson: Writing – review & editing, Methodology.

Serdar Karakurt: Writing – review & editing, Writing – original draft, Funding.

Data Availability Statement

The mass spectrometry proteomics data have been deposited to the MassIVE Database (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp) via partner repository with the data set identifier MSV000095748 (FTP Download Link; ftp://MSV000095748@massive.ucsd.edu).

Ethical Statement

Authors declare that international ethical guidelines for scientific publications were followed in the preparation of this manuscript.

Funding Sources

This study was supported by the Scientific and Technological Research Council of Turkiye (TUBITAK; Grant No. 120Z942) and Selcuk University Research Foundation, Turkiye (BAP, Grant No. 21401070).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors cordially thank Dr. Marie Vestergaard Lukassen, DTU Bioengineering Proteomics Core, for assistance with mass spectrometry analyses. We want to express our gratitude to Ozgun Sipahioglu for his contributions to scorpion collection efforts in the specified regions.

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