Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from Brown spider (Loxosceles intermedia) venom: From cloning to functional characterization

Silveira, Rafael Bertoni da; Pigozzo, Romine Bachmann; Chaim, Olga Meiri; Appel, Márcia Helena; Trevisan-Silva, Dilza; Dreyfuss, Juliana Luporini; Toma, Leny; Dietrich, Carl P.; Nader, Helena B.; Veiga, Sílvio Sanches; Gremski, Waldemiro

doi:10.1016/j.biochi.2006.12.002

Cited by 70 publications

(67 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Surprisingly, La_␤ID1 also slightly preferred choline-containing substrates, despite belonging to the ␤I clade like St_␤IB1i. The observation of robust SMase D activity in La_␤ID1 is intriguing given that it shares 84% sequence identity with LiRecDT5 (␤ID1) from L. intermedia, an enzyme with very limited SMase D activity compared with ␣ clade toxins from the same species (9). Direct experimental comparison of La_␤ID1 and LiRecDT5 could further illuminate whether these two enzymes differ in SMase D activity and/or substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

“…The fulllength clone was then amplified from the venom gland cDNA pool using two primers designed from the initial transcript sequence, TOPO cloned (Invitrogen) and sequenced. The predicted amino acid sequence of locus 1239 shares 85% sequence identity with LiRecDT5 (LiSicTox-␤ID1) from L. intermedia (9), another SicTox PLD reported to have diminished SMase D activity compared with ␣ clade isoforms from the same species. Because of this relatedness (Fig.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Lajoie

Roberts

Zobel-Thropp

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Phospholipase D toxins from brown spider venoms can cause disease in humans. Results: Different toxin family members show specificity for lipid substrates with choline or ethanolamine headgroups or can be ambiguous. Conclusion: Spider phospholipase D toxins have evolved diverse substrate preferences. Significance: The diverse substrate preference may be significant for predation and the mammalian toxicity of venom.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Lajoie

Roberts

Zobel-Thropp

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The dermonecrotic toxins are major molecules in the crude venom of brown spiders, being responsible for many clinical complications and deaths in humans. These dermonecrotic toxins were characterized as sphingomyelinase D (SMase D) in previous studies [18,19,20,21]. The toxins are able to hydrolyze sphingomyelin (SM), the major component of the outer leaflet of plasma membranes, into choline and ceramide 1-phosphate [18,19,20,21].…”

Section: Introductionmentioning

confidence: 99%

Characteristics and Lethality of a Novel Recombinant Dermonecrotic Venom Phospholipase D from Hemiscorpius lepturus

Torabi

Behdani

Chafi

et al. 2017

Toxins

View full text Add to dashboard Cite

Hemoscorpius lepturus is the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation are remarkably similar to those reported for brown spiders, including dermonecrosis, hematuria, renal failure and even death. The lethality and toxicity of brown spiders’ venom have been attributed to its phospholipase D activity. This study aims to identify a phospholipase D with possible lethality and dermonecrotic activity in H. lepturus venom. In this study, a cDNA library of the venom glands was generated by Illumina RNA sequencing. Phospholipase D (PLD) from H. lepturus was characterized according to its significant similarity with PLDs from brown spiders. The main chain designated as Hl-RecPLD1 (the first recombinant isoform of H. lepturus PLD) was cloned, expressed and purified. Sphingomyelinase, dermonecrotic and lethal activities were examined. Hl-PLD1 showed remarkable sequence similarity and structural homology with PLDs of brown spiders. The conformation of Hl-PLD1 was predicted as a “TIM beta/alpha-barrel”. The lethal dose 50 (LD50) and dermonecrotic activities of Hl-RecPLD1 were determined as 3.1 µg/mouse and 0.7 cm2 at 1 µg respectively. It is the first report indicating that a similar molecular evolutionary mechanism has occurred in both American brown spiders and this Iranian scorpion. In conclusion, Hl-RecPLD1 is a highly active phospholipase D, which would be considered as the lethal dermonecrotic toxin in H. lepturus venom.

show abstract

“…In these experiments, LgRec2 was assayed along with LgRec1 to evaluate possible activity variations between both PLDs, as studies comparing recombinant PLD isoforms from Loxosceles have shown differences in the intensity of their effects [30,31,37,63]. In all experiments, LgRec2 showed similar activities to LgRec1, except for platelet aggregation for which LgRec2 was slightly more active.…”

Section: Discussionmentioning

confidence: 99%

“…[27]. Despite the cloning of several PLD isoforms from L. intermedia [28,29,30,31,32,33] and L. laeta [34,35,36,37] into E. coli , LgRec2 is only the second PLD cloned and expressed from L. gaucho .…”

Section: Introductionmentioning

confidence: 99%

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Shimokawa-Falcão

Caporrino

Barbaro

et al. 2017

Toxins

View full text Add to dashboard Cite

Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.

show abstract

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from Brown spider (Loxosceles intermedia) venom: From cloning to functional characterization

Cited by 70 publications

References 33 publications

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Characteristics and Lethality of a Novel Recombinant Dermonecrotic Venom Phospholipase D from Hemiscorpius lepturus

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Contact Info

Product

Resources

About