Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Lajoie, Daniel M.; Roberts, S.A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew

doi:10.1074/jbc.m115.636951

Cited by 41 publications

(50 citation statements)

References 77 publications

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“…Further studies confirmed these data by showing that recombinant PLD toxins from L. intermedia venom cleave sphingomyelin, lysophosphatidylcholine, and lyso-PAF [28,29], and that a recombinant PLD toxin from L. arizonica venom presented activity on sphingomyelin, lysophosphatidylcholine and lysophosphatidylethanolamine [30]. Since these enzymes from different species of Loxosceles have the ability to hydrolyze a wide range of different phospholipids, they are now classified as Phospholipases-D [5,30]. In an attempt to standardize the nomenclature of these toxins, a classification was proposed based on the amino acid sequence alignment of the various PLDs present in the Loxosceles intermedia venom deduced from cDNA sequences, phylogenetic studies and also on the biochemical and biological properties of these molecules.…”

Section: Nomenclatures and Biochemical Classification Of Brown Spidermentioning

confidence: 63%

“…The authors argued that because of this broad phospholipase activity exhibited, the term sphingomyelinase-D was very limited, and proposed the term Phospholipase-D [27]. Further studies confirmed these data by showing that recombinant PLD toxins from L. intermedia venom cleave sphingomyelin, lysophosphatidylcholine, and lyso-PAF [28,29], and that a recombinant PLD toxin from L. arizonica venom presented activity on sphingomyelin, lysophosphatidylcholine and lysophosphatidylethanolamine [30]. Since these enzymes from different species of Loxosceles have the ability to hydrolyze a wide range of different phospholipids, they are now classified as Phospholipases-D [5,30].…”

Section: Nomenclatures and Biochemical Classification Of Brown Spidermentioning

confidence: 99%

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Forty Years of the Description of Brown Spider Venom Phospholipases-D

Gremski

Justa

Silva

et al. 2020

Toxins

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Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field.

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Section: Nomenclatures and Biochemical Classification Of Brown Spidermentioning

confidence: 63%

Section: Nomenclatures and Biochemical Classification Of Brown Spidermentioning

confidence: 99%

Forty Years of the Description of Brown Spider Venom Phospholipases-D

Gremski

Justa

Silva

et al. 2020

Toxins

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“…1) crucially also conserved in menorin and FAM151A and B. These different enzymes hydrolyse phosphodiester bonds of a large number of different substrates, ranging from Glycosylphosphatidylinositol (GPI) protein anchors (in GDPD family) to sphingolipid and lysolipid (in Phospholipase D spider toxins [8][9][10][11] .…”

Section: Resultsmentioning

confidence: 99%

Fam151b, the mouse homologue of C.elegans menorin gene, is essential for retinal function

Findlay

McKie

Keighren

et al. 2020

Sci Rep

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Fam151b is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching. the international Mouse phenotyping consortium (iMpc) aims to knock out every gene in the mouse and comprehensively phenotype the mutant animals. This project identified Fam151b homozygous knock-out mice as having retinal degeneration. We show they have no photoreceptor function from eye opening, as demonstrated by a lack of electroretinograph (eRG) response. Histological analysis shows that during development of the eye the correct number of cells are produced and that the layers of the retina differentiate normally. However, after eye opening at P14, Fam151b mutant eyes exhibit signs of retinal stress and rapidly lose photoreceptor cells. We have mutated the second mammalian menorin homologue, Fam151a, and homozygous mutant mice have no discernible phenotype. Sequence analysis indicates that the FAM151 proteins are members of the pLc-like phosphodiesterase superfamily. However, the substrates and function of the proteins remains unknown.The retina is the light sensitive tissue located in the posterior eye. Photoreceptor cells within the retina are responsible for converting light signals into electrical signals, and loss of these cells is a leading cause of blindness in humans. In the course of diseases such as retinitis pigmentosa (RP), rod and cone photoreceptor cells are slowly lost, beginning at the peripheral retina and moving centrally 1 . Multiple mutant genes in humans can cause RP, and many of these have counterpart mouse models 2 . In this paper we describe a novel mouse model of retinal degeneration which has early and rapid loss of photoreceptor cells due to loss of function of a largely uncharacterised gene. The International Mouse Phenotyping Consortium (IMPC) is a worldwide collaborative effort to generate lines of mice, each with a loss of function mutation in a single gene, and to comprehensively phenotype the lines 3,4 . The ultimate aim is to catalogue the function of every gene in the mouse genome in order to contribute to the understanding of fundamental mammalian biology and to generate models for human disease. Many institutions participate and follow an agreed pipeline for the phenotypic analysis of the mice, known as IMPReSS (International Mouse Phenotyping Resource of Standardised Screen). The analyses include growth, morphological, behavioural, metabolic and sensory phenotypes over a 15 week period, and followed post-mortem by examination of haematology, immunology, clinical chemistry and gross pathology. At 15 weeks of age mutant mice undergo slit lamp examination and ophthalmoscopy to investigate the gross morphology of the eye.As part of this study a novel gene, Fam151b, was mutated and the mice analysed through the IMPC pipeline. Fam151b knockout mice were found to have abnormal retina morphology and no other observed phenotype.Here we follow up these findings with a detailed histological and functional analysis of this disease model. In addition we have mutated the paralogous...

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“…The analysis showed that while LgRec1 exhibited a small decrease in platelet aggregation when concentration was decreased by half, LgRec2 still maintained its activity at both concentrations (Figure 5A). Next, LgRec2 was assayed for its ability to cleave sphingomyelin, as this is also an intrinsic characteristic of this class of toxins [47]. As shown in Figure 5B, both LgRec2 and LgRec1 exhibited concentration-dependent activity.…”

Section: Resultsmentioning

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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Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders

Cited by 41 publications

References 77 publications

Forty Years of the Description of Brown Spider Venom Phospholipases-D

Forty Years of the Description of Brown Spider Venom Phospholipases-D

Fam151b, the mouse homologue of C.elegans menorin gene, is essential for retinal function

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

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