Characterization of Neuwiedin, a new disintegrin from Bothrops neuwiedi venom gland with distinct cysteine pattern

Lima-dos-Santos, I.; Della-Casa, Maisa S.; Portes-Junior, José Antônio; Calabria, Paula A L; Magalhães, Geraldo Santana; Moura-da-Silva, Ana Maria

doi:10.1016/j.toxicon.2015.08.006

Cited by 9 publications

(5 citation statements)

References 60 publications

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“…In our experience, SUMO were very effective at expressing a single chain variable fragment (scFv) antibody against a venom metalloproteinase BaP1 from the viper Bothrops asper [56] and a disintegrin from the viper Bothrops neuwiedi [57], both of which were previously obtained only as inclusion bodies. However, the process of removing SUMO is laborious and time-consuming.…”

Section: Discussionmentioning

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

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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.

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Section: Discussionmentioning

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

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“…Several studies have cloned and expressed the disintegrin domain of P-II SVMPs. For instance, Dis Ba -01 ( Ramos et al, 2008 ), bothrostatin ( Fernandez et al, 2005 ), BnMPIIx (neuwiedin) ( Lima-dos-Santos et al, 2015 ) and CamVMPII (r-cam-dis) ( Suntravat et al, 2013 ) were identified from cDNA libraries constructed from the venom transcriptome of four different snake species: Bothrops alternatus, Bothrops jararaca, Bothrops neuwiedi and Crotalus adamanteus , respectively. SDS-PAGE analyses showed that bothrostatin, CamVMPII and Dis Ba -01 are proteins of approximately 8 kDa ( Ramos et al, 2008 ).…”

Section: Snake Venom Metalloproteinases (Svmps)mentioning

confidence: 99%

“…SDS-PAGE analyses showed that bothrostatin, CamVMPII and Dis Ba -01 are proteins of approximately 8 kDa ( Ramos et al, 2008 ). Although MALDI-TOF analysis showed that BnMPIIx has a molecular mass of 8 kDa, data from SDS-PAGE fingerprint correlated this protein to a molecular mass of 14 kDa ( Lima-dos-Santos et al, 2015 ). It was demonstrated that BnMPIIx inhibited ADP-induced platelet aggregation whereas bothrostatin inhibited collagen-induced platelet aggregation.…”

Section: Snake Venom Metalloproteinases (Svmps)mentioning

confidence: 99%

“… BlatH1 84 Dimeric; each monomer contains metalloproteinsase, and disintegrin Bothriechis lateralis RGD Inhibition of both ADP- and collagen-induced platelet aggregation; hemorrhagic, fibrinogenolytic, gelatinolytic and azocaseinolytic Camacho et al (2014) 1 8 . BnMPIIx (neuwiedin) 8.18 Disintegrin Bothrops neuwiedi RGD Inhibition of ADP-induced platelet aggregation Lima-dos-Santos et al (2015) 19 0. CcMP-II Boukhalfa- 35 Metalloproteinsase, and disintegrin Cerastes Hemorrhagic and α-fibrinogenase activity Boukhalfa-Abib and Laraba-Djebari (2015) …”

Section: Snake Venom Metalloproteinases (Svmps)mentioning

confidence: 99%

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Snake Venom Metalloproteinases (SVMPs): A structure-function update

et al. 2020

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Snake venom metalloproteinases (SVMPs) represent a diverse group of multi-domain proteins with several biological activities such as the ability to induce hemorrhage, proteolytic degradation of fibrinogen and fibrin, induction of apoptosis and inhibition of platelet aggregation. Due to these activities, SVMPs are responsible for many of the well-known pathological phenotypes in snake envenomations caused particularly by species from the Viperidae family and the Crotalinae subfamily. These proteins have been classified based on their size and domain structure into P–I, P-II and P-III classes. Comparatively, members of the P–I SVMPs possess the simplest structures, formed by the catalytic metalloproteinase domain only; the P-II SVMPs are moderately more complex, having the canonical disintegrin domain in addition to the metalloproteinase domain; members of the P-III class are more structurally varied, comprising the metalloproteinase, disintegrin-like, and cysteine-rich domains. Proteolytic cleavage, repeated domain loss and presence of other ancillary domains are responsible for structural diversities in the P-III class. However, studies continue to unveil the relationship between the structure and function of these proteins. In this review, we recovered evidences from literature on the structural peculiarities and functional classification of Snake Venom Metalloproteinases. In addition, we reflect on diversities that exist among each class while taking into account specific and up-to-date class-based activities.

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“…For example, there are sequences that are specific for enterokinase or thrombin. Incorporating SUMO as a fusion protein allows the use of SUMO protease, which does not require extra amino acids to be expressed since it recognizes the tertiary structure of SUMO protein [ 61 , 80 , 93 , 122 ].…”

Section: Recombinant Disintegrinsmentioning

confidence: 99%

Recombinant and Chimeric Disintegrins in Preclinical Research

David

Succar

Moraes

et al. 2018

Toxins

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Disintegrins are a family of small cysteine-rich peptides, found in a wide variety of snake venoms of different phylogenetic origin. These peptides selectively bind to integrins, which are heterodimeric adhesion receptors that play a fundamental role in the regulation of many physiological and pathological processes, such as hemostasis and tumor metastasis. Most disintegrins interact with integrins through the RGD (Arg-Gly-Asp) sequence loop, resulting in an active site that modulates the integrin activity. Some variations in the tripeptide sequence and the variability in its neighborhood result in a different specificity or affinity toward integrin receptors from platelets, tumor cells or neutrophils. Recombinant forms of these proteins are obtained mainly through Escherichia coli, which is the most common host used for heterologous expression. Advances in the study of the structure-activity relationship and importance of some regions of the molecule, especially the hairpin loop and the C-terminus, rely on approaches such as site-directed mutagenesis and the design and expression of chimeric peptides. This review provides highlights of the biological relevance and contribution of recombinant disintegrins to the understanding of their binding specificity, biological activities and therapeutic potential. The biological and pharmacological relevance on the newest discoveries about this family of integrin-binding proteins are discussed.

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Characterization of Neuwiedin, a new disintegrin from Bothrops neuwiedi venom gland with distinct cysteine pattern

Cited by 9 publications

References 60 publications

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

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

Snake Venom Metalloproteinases (SVMPs): A structure-function update

Recombinant and Chimeric Disintegrins in Preclinical Research

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