ß-Hydroxy-ß-methylbutyrate (HMB) leads to phospholipase D2 (PLD2) activation and alters circadian rhythms in myotubes.

ß-Hydroxy-ß-methylbutyrate (HMB) is a breakdown product of leucine, which promotes muscle growth. Although some studies indicate that HMB activates AKT and mTOR, others show activation of the downstream effectors, P70S6K and S6, independent of mTOR. Our aim was to study the metabolic effect of HMB around the circadian clock in order to determine more accurately the signaling pathway involved. C2C12 myotubes were treated with HMB and clock, metabolic and myogenic markers were measured around the clock. HMB-treated C2C12 myotubes showed no activation of AKT and mTOR, but did show activation of P70S6K and S6. Activation of P70S6K and S6 was also found when myotubes were treated with HMB combined with metformin, an indirect mTOR inhibitor, or rapamycin, a direct mTOR inhibitor. The activation of the P70S6K and S6 independent of AKT and mTOR, was accompanied by increased activation of phospholipase D2 (PLD). In addition, HMB led to high amplitude and advanced circadian rhythms. In conclusion, HMB induces myogenesis in C2C12 by activating P70S6K and S6 via PLD2, rather than AKT and mTOR, leading to high amplitude advanced rhythms.

Phospholipase D regulates ferroptosis signal transduction in mouse spleen hypoxia response.

High-altitude hypoxia exposure can lead to phospholipase D-mediated lipid metabolism disorder in spleen tissues and induce ferroptosis. Nonetheless, the key genes underlying hypoxia-induced splenic phospholipase D and the ferroptosis pathway remain unclear. This study aimed to establish a hypoxia animal model. Combined transcriptomic and proteomic analyses showed that 95 predicted target genes (proteins) were significantly differentially expressed under hypoxic conditions. Key genes in phospholipase D and ferroptosis pathways under hypoxic exposure were identified by combining Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis techniques. Gene set enrichment analysis (GSEA) showed that the differential gene sets of the phospholipase D and ferroptosis signaling pathways were upregulated in the high-altitude hypoxia group. The genes in the phospholipase D signalling pathway were verified, and the expression levels of KIT and DGKG were upregulated in spleen tissues under hypoxic exposure. Subsequently, the mRNA and protein expression levels of genes from the exogenous pathway such as TFRC, SLC40A1, SLC7A11, TRP53, and FTH1 and those from the endogenous pathway such as GPX4, HMOX1, and ALOX15 differentials in the ferroptosis signalling pathway were verified, and the results indicated significant differential expression. In summary, exposure to high-altitude hypoxia mediated phospholipid metabolism disturbance through the phospholipase D signalling pathway and further induced ferroptosis, leading to splenic injury.

Phospholipase D, a Novel Therapeutic Target Contributes to the Pathogenesis of Neurodegenerative and Neuroimmune Diseases.

Phospholipase D (PLD) is an enzyme that consists of six isoforms (PLD1-PLD6) and has been discovered in different organisms including bacteria, viruses, plants, and mammals. PLD is involved in regulating a wide range of nerve cells' physiological processes, such as cytoskeleton modulation, proliferation/growth, vesicle trafficking, morphogenesis, and development. Simultaneously, PLD, which also plays an essential role in the pathogenesis of neurodegenerative and neuroimmune diseases. In this review, family members, characterizations, structure, functions and related signaling pathways, and therapeutic values of PLD was summarized, then five representative diseases including Alzheimer disease (AD), Parkinson's disease (PD), etc. were selected as examples to tell the involvement of PLD in these neurological diseases. Notably, recent advances in the development of tools for studying PLD therapy envisaged novel therapeutic interventions. Furthermore, the limitations of PLD based therapy were also analyzed and discussed. The content of this review provided a thorough and reasonable basis for further studies to exploit the potential of PLD in the treatment of neurodegenerative and neuroimmune diseases.

Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

BACKGROUND: Hypoxia in solid tumors is an important source of chemoresistance that can determine poor patient prognosis. Such chemoresistance relies on the presence of cancer stem cells (CSCs), and hypoxia promotes their generation through transcriptional activation by HIF transcription factors. METHODS: We used ovarian cancer (OC) cell lines, xenograft models, OC patient samples, transcriptional databases, induced pluripotent stem cells (iPSCs) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). RESULTS: Here, we show that hypoxia induces CSC formation and chemoresistance in ovarian cancer through transcriptional activation of the PLD2 gene. Mechanistically, HIF-1α activates PLD2 transcription through hypoxia response elements, and both hypoxia and PLD2 overexpression lead to increased accessibility around stemness genes, detected by ATAC-seq, at sites bound by AP-1 transcription factors. This in turn provokes a rewiring of stemness genes, including the overexpression of SOX2, SOX9 or NOTCH1. PLD2 overexpression also leads to decreased patient survival, enhanced tumor growth and CSC formation, and increased iPSCs reprograming, confirming its role in dedifferentiation to a stem-like phenotype. Importantly, hypoxia-induced stemness is dependent on PLD2 expression, demonstrating that PLD2 is a major determinant of de-differentiation of ovarian cancer cells to stem-like cells in hypoxic conditions. Finally, we demonstrate that high PLD2 expression increases chemoresistance to cisplatin and carboplatin treatments, both in vitro and in vivo, while its pharmacological inhibition restores sensitivity. CONCLUSIONS: Altogether, our work highlights the importance of the HIF-1α-PLD2 axis for CSC generation and chemoresistance in OC and proposes an alternative treatment for patients with high PLD2 expression.

Phospholipase D Mediates Glutamine-Induced mTORC1 Activation to Promote Porcine Intestinal Epithelial Cell Proliferation.

BACKGROUND: The intestinal epithelium is one of the fastest self-renewal tissues in the body, and glutamine plays a crucial role in providing carbon and nitrogen for biosynthesis. In intestinal homeostasis, phosphorylation-mediated signaling networks that cause altered cell proliferation, differentiation, and metabolic regulation have been observed. However, our understanding of how glutamine affects protein phosphorylation in the intestinal epithelium is limited, and identifying the essential signaling pathways involved in regulating intestinal epithelial cell growth is particularly challenging. OBJECTIVES: This study aimed to identify the essential proteins and signaling pathways involved in glutamine's promotion of porcine intestinal epithelial cell proliferation. METHODS: Phosphoproteomics was applied to describe the protein phosphorylation landscape under glutamine treatment. Kinase-substrate enrichment analysis was subjected to predict kinase activity and validated by qRT-PCR and Western blotting. Cell Counting Kit-8, glutamine rescue experiment, chloroquine treatment, and 5-fluoro-2-indolyl deschlorohalopemide inhibition assay revealed the possible underlying mechanism of glutamine promoting porcine intestinal epithelial cell proliferation. RESULTS: In this study, glutamine starvation was found to significantly suppress the proliferation of intestinal epithelial cells and change phosphoproteomic profiles with 575 downregulated sites and 321 upregulated sites. Interestingly, phosphorylation of eukaryotic initiation factor 4E-binding protein 1 at position Threonine70 was decreased, which is a crucial downstream of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Further studies showed that glutamine supplementation rescued cell proliferation and mTORC1 activity, dependent on lysosomal function and phospholipase D activation. CONCLUSION: In conclusion, glutamine activates mTORC1 signaling dependent on phospholipase D and a functional lysosome to promote intestinal epithelial cell proliferation. This discovery provides new insight into regulating the homeostasis of the intestinal epithelium, particularly in pig production.

High-Yield Synthesis of Phosphatidylserine in a Well-Designed Mixed Micellar System.

A sustainable enzymatic system is essential for efficient phosphatidylserine (PS) synthesis in industrial production. Conventional biphasic systems face challenges such as excessive organic solvent usage, enzyme-intensive processes, and increased costs. This study introduces a novel approach using chitin nanofibrils (ChNFs) as an immobilization material for phospholipase D (PLD) in a mixed micellar system stabilized by the food-grade emulsifier sodium deoxycholate (SDC). The immobilized enzyme, ChNF-chiA1, was quickly prepared in a one-step process, eliminating the need for purification. By optimizing the reaction conditions, including l-Ser concentration (1.0 M), SDC concentration (10 mM), reaction time (8 h), and enzyme dosage (1.0 U), a remarkable PS yield of 96.74% was achieved in the solvent-free mixed micellar system. The catalytic efficiency of ChNF-chiA1 surpassed that of the free PLD-chiA1 biphasic system by 6.0-fold. This innovative and green biocatalytic technology offers a reusable solution for the high-value enzymatic synthesis of phospholipids, providing a promising avenue for industrial applications.

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

The role and regulation of phospholipase D in metabolic disorders.

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and free choline. In mammals, PLD exists in two well-characterized isoforms, PLD1 and PLD2, and it plays pivotal roles as signaling mediators in various cellular functions, such as cell survival, differentiation, and migration. These isoforms are predominantly expressed in diverse cell types, including many immune cells, such as monocytes and macrophages, as well as non-immune cells, such as epithelial and endothelial cells. Several previous studies have revealed that the stimulation of these cells leads to an increase in PLD expression and its enzymatic products, potentially influencing the pathological responses in a wide spectrum of diseases. Metabolic diseases, exemplified by conditions, such as diabetes, obesity, hypertension, and atherosclerosis, pose significant global health challenges. Abnormal activation or dysfunction of PLD emerges as a potential contributing factor to the pathogenesis and progression of these metabolic disorders. Therefore, it is crucial to thoroughly investigate and understand the intricate relationship between PLD and metabolic diseases. In this review, we provide an in-depth overview of the functional roles and molecular mechanisms of PLD involved in metabolic diseases. By delving into the intricate interplay between PLD and metabolic disorders, this review aims to offer insights into the potential therapeutic interventions.

The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

A Brief Overview of the Toxic Sphingomyelinase Ds of Brown Recluse Spider Venom and Other Organisms and Simple Methods To Detect Production of Its Signature Cyclic Ceramide Phosphate.

A special category of phospholipase D (PLD) in the venom of the brown recluse spider (Loxosceles reclusa) and several other sicariid spiders accounts for the dermonecrosis and many of the other clinical symptoms of envenomation. Related proteins are produced by other organisms, including fungi and bacteria. These PLDs are often referred to as sphingomyelinase Ds (SMase Ds) because they cleave sphingomyelin (SM) to choline and "ceramide phosphate." The lipid product has actually been found to be a novel sphingolipid: ceramide 1,3-cyclic phosphate (Cer1,3P). Since there are no effective treatments for the injury induced by the bites of these spiders, SMase D/PLDs are attractive targets for therapeutic intervention, and some of their features will be described in this minireview. In addition, two simple methods are described for detecting the characteristic SMase D activity using a fluorescent SM analog, (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-SM (C12-NBD-SM), that is cleaved to C12-NBD-Cer1,3P, which is easily separated from other potential metabolites by thin-layer chromatography and visualized under UV light. Besides confirming that C12-NBD-Cer1,3P is the only product detected upon incubation of C12-NBD-SM with brown recluse spider venom, the method was also able to detect for the first time very low levels of activity in venom from another spider, Kukulcania hibernalis The simplicity of the methods makes it relatively easy to determine this signature activity of SMase D/PLD. SIGNIFICANCE STATEMENT: The sphingomyelinase D/phospholipase D that are present in the venom of the brown recluse spider and other sources cause considerable human injury, but detection of the novel sphingolipid product, ceramide 1,3-cyclic phosphate, is not easy by previously published methods. This minireview describes simple methods for detection of this activity that will be useful for studies of its occurrence in spider venoms and other biological samples, perhaps including lesions from suspected spider bites and infections.

Phospholipase D Immobilization on Lignin Nanoparticles for Enzymatic Transformation of Phospholipids.

Lignin nanoparticles (LNPs) are promising components for various materials, given their controllable particle size and spherical shape. However, their origin from supramolecular aggregation has limited the applicability of LNPs as recoverable templates for immobilization of enzymes. In this study, we show that stabilized LNPs are highly promising for the immobilization of phospholipase D (PLD), the enzyme involved in the biocatalytic production of high-value polar head modified phospholipids of commercial interest, phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine. Starting from hydroxymethylated lignin, LNPs were prepared and successively hydrothermally treated to obtain c-HLNPs with high resistance to organic solvents and a wide range of pH values, covering the conditions for enzymatic reactions and enzyme recovery. The immobilization of PLD on c-HLNPs (PLD-c-HLNPs) was achieved through direct adsorption. We then successfully exploited this new enzymatic preparation in the preparation of pure polar head modified phospholipids with high yields (60-90 %). Furthermore, the high stability of PLD-c-HLNPs allows recycling for a number of reactions with appreciable maintenance of its catalytic activity. Thus, PLD-c-HLNPs can be regarded as a new, chemically stable, recyclable and user-friendly biocatalyst, based on a biobased inexpensive scaffold, to be employed in sustainable chemical processes for synthesis of value-added phospholipids.

The wide world of non-mammalian phospholipase D enzymes.

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Phospholipase D1 activity is crucial for cytosolic phospholipase A2 -dependent prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells.

In bone, prostaglandin E2 (PGE2) is highly osteogenic and formed by osteoblasts, a key modulatory event in the regulation of bone cell activity. MC3T3-E1 cells are widely used as an in vitro model of osteoblast function. It is still not clear which pathways contribute to the release of AA in these cells. In this study we have focussed on the contribution of phospholipase D (PLD) enzymes to osteoblastic PGE2 formation after stimulation with endothelin-1 (ET-1). Using specific inhibitors of PLD1 and PLD2 we could show that PGE2 formation was strictly dependent on PLD1 but not PLD2 activity and cytosolic phospholipase A2 (cPLA2) was activated by triggering through PLD1. We have identified diacyl glycerol (DAG) as a possible effector molecule which may serve as a triggering signal for PKC activation and subsequent cPLA2 phosphorylation.

A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation.

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

NME3 binds to phosphatidic acid and mediates PLD6-induced mitochondrial tethering.

Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.

Small Molecule Activation of NAPE-PLD Enhances Efferocytosis by Macrophages.

N-Acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N-acyl-phosphatidylethanolamines (NAPEs) to form N-acyl-ethanolamines (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld-/- BMDM or after Nape-pld inhibition. Together, these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.

Comparative Biochemical, Structural, and Functional Analysis of Recombinant Phospholipases D from Three Loxosceles Spider Venoms.

Spiders of Loxosceles genus are widely distributed and their venoms contain phospholipases D (PLDs), which degrade phospholipids and trigger inflammatory responses, dermonecrosis, hematological changes, and renal injuries. Biochemical, functional, and structural properties of three recombinant PLDs from L. intermedia, L. laeta, and L. gaucho, the principal species clinically relevant in South America, were analyzed. Sera against L. gaucho and L. laeta PLDs strongly cross-reacted with other PLDs, but sera against L. intermedia PLD mostly reacted with homologous molecules, suggesting underlying structural and functional differences. PLDs presented a similar secondary structure profile but distinct melting temperatures. Different methods demonstrated that all PLDs cleave sphingomyelin and lysophosphatidylcholine, but L. gaucho and L. laeta PLDs excelled. L. gaucho PLD showed greater "in vitro" hemolytic activity. L. gaucho and L. laeta PLDs were more lethal in assays with mice and crickets. Molecular dynamics simulations correlated their biochemical activities with differences in sequences and conformations of specific surface loops, which play roles in protein stability and in modulating interactions with the membrane. Despite the high similarity, PLDs from L. gaucho and L. laeta venoms are more active than L. intermedia PLD, requiring special attention from physicians when these two species prevail in endemic regions.

Identification of a common epitope in knottins and phospholipases D present in Loxosceles sp venom by a monoclonal antibody.

In the Americas and specially in Brazil, the Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta are the three most medically relevant brown spider species, and whose bites can lead to the condition known as loxoscelism. Here, we report the development of a tool capable of identifying a common epitope amongst Loxosceles sp. venom's toxins. A murine monoclonal antibody (LmAb12) and its recombinant fragments (scFv12P and diabody12P) have been produced and characterized. This antibody and its recombinant constructs were able to recognize proteins of Loxosceles spider venoms with specificity. The scFv12P variant was also able to detect low concentrations of Loxosceles venom in a competitive ELISA assay, displaying potential as a venom identification tool. The primary antigenic target of LmAb12 is a knottin, a venom neurotoxin, that has a shared identity of 100 % between the L. intermedia and L. gaucho species and high similarity to L. laeta. Furthermore, we observed LmAb12 was able to partially inhibit in vitro hemolysis, a cellular event typically induced by the Loxosceles sp. venoms. Such behavior might be due to LmAb12 cross-reactivity between the antigenic target of LmAb12 and the venom's dermonecrotic toxins, the PLDs, or even the existence of synergism between these two toxins.