Serine pseudoproteases in physiology and disease.

Serine proteases (SPs) constitute a very important family of enzymes, both physiologically and pathologically. The effects produced by these proteins have been explained by their proteolytic activity. However, the discovery of pharmacologically active SP molecules that show no enzymatic activity, as the so-called pseudo SPs or SP homologs (SPHs), has exposed a profoundly neglected possibility of nonenzymatic functions of these SP molecules. In this review, the most thoroughly described SPHs are presented. The main physiological domains in which SPHs operate appear to be in reproduction, embryonic development, immune response, host defense, and hemostasis. Hitherto unexplained actions of SPs should therefore be considered also as the result of the ligand-like attributes of SPs. The gain of a novel function by an SPH is a consequence of specific amino acid replacements that have resulted in a novel interaction interface or a 'catalytic trap'. Unraveling the SP/SPH interactome will provide a description of previously unknown physiological functions of SPs/SPHs, aiding the creation of innovative medical approaches.

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, ß-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom ß-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

The Phospholipase Activity of Ammodytoxin, a Prototype Snake Venom ß-Neurotoxin, Is Not Obligatory for Cell Internalisation and Translocation to Mitochondria.

ß-Neurotoxins are secreted phospholipase A2 molecules that inhibit transmission in neuromuscular synapses by poisoning the motor neurons. These toxins specifically and rapidly internalise into the nerve endings of motor neurons. Ammodytoxin (Atx) is a prototype ß-neurotoxin from the venom of the nose-horned viper (Vipera ammodytes ammodytes). Here, we studied the relevance of the enzymatic activity of Atx in cell internalisation and subsequent intracellular movement using transmission electron microscopy (TEM). We prepared a recombinant, enzymatically inactive mutant of Atx, Atx(D49S), labelled with gold nanoparticles (GNP), and incubated this with PC12 cells, to analyse its localisation by TEM. Atx(D49S)-GNP internalised into the cells. Inside the cells, Atx(D49S)-GNP was detected in different vesicle-like structures, cytosol, endoplasmic reticulum and mitochondria, where it was spotted in the intermembrane space and matrix. Co-localization of fluorescently labelled Atx(D49S) with mitochondria in PC12 cells by confocal fluorescence microscopy confirmed the reliability of results generated using Atx(D49S)-GNP and TEM and allowed us to conclude that the phospholipase activity of Atx is not obligatory for its cell internalisation and translocation into the mitochondrial intermembrane space and matrix.

Secreted Phospholipases A2 - not just Enzymes: Revisited.

Secreted phospholipases A2 (sPLA2s) participate in a very broad spectrum of biological processes through their enzymatic activity and as ligands for membrane and soluble receptors. The physiological roles of sPLA2s as enzymes have been very well described, while their functions as ligands are still poorly known. Since the last overview of sPLA2-binding proteins (sPLA2-BPs) 10 years ago, several important discoveries have occurred in this area. New and more sensitive analytical tools have enabled the discovery of additional sPLA2-BPs, which are presented and critically discussed here. The structural diversity of sPLA2-BPs reveals sPLA2s as very promiscuous proteins, and we offer some structural explanations for this nature that makes these proteins evolutionarily highly advantageous. Three areas of physiological engagement of sPLA2-BPs have appeared most clearly: cellular transport and signalling, and regulation of the enzymatic activity of sPLA2s. Due to the multifunctionality of sPLA2s, they appear to be exceptional pharmacological targets. We reveal the potential to exploit interactions of sPLA2s with other proteins in medical terms, for the development of original diagnostic and therapeutic procedures. We conclude this survey by suggesting the priority questions that need to be answered.

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration.

BACKGROUND: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. METHODS: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. RESULTS: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. CONCLUSION: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration.

The neurotoxic secreted phospholipase A2 from the Vipera a. ammodytes venom targets cytochrome c oxidase in neuronal mitochondria.

The ß-neurotoxic secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals. Damage inflicted by such sPLA2s (ß-ntx) on neuronal mitochondria is characteristic, very similar to that induced by structurally homologous endogenous group IIA sPLA2 when its activity is elevated, as, for example, in the early phase of Alzheimer's disease. Using ammodytoxin (Atx), the ß-ntx from the venom of the nose-horned viper (Vipera a. ammodytes), the sPLA2 receptor R25 has been detected in neuronal mitochondria. This receptor has been purified from porcine cerebral cortex mitochondria by a new Atx-affinity-based chromatographic procedure. Mass spectrometry analysis revealed R25 to be the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. CCOX was confirmed as being the first intracellular membrane receptor for sPLA2 by alternative Atx-affinity-labellings of purified CCOX, supported also by the encounter of Atx and CCOX in PC12 cells. This discovery suggests the explanation of the mechanism by which ß-ntx hinders production of ATP in poisoned nerve endings. It also provides a new insight into the potential function and dysfunction of endogenous GIIA sPLA2 in mitochondria.

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy.

Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157-loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Glioblastoma-specific anti-TUFM nanobody for in-vitro immunoimaging and cancer stem cell targeting.

Glioblastoma multiforme (GBM) is the most common and lethal form of brain tumor. The prognosis for patients remains poor, despite the combination of new preoperative and intraoperative neuroimaging, radical surgery, and recent advances in radiotherapy and chemotherapy. To improve GBM therapy and patient outcome, sustained drug delivery to glioma cells is needed, while minimizing toxicity to adjacent neurons and glia cells. This might be achieved through an anti-proteomic approach based on nanobodies, the single-domain antigen-binding fragments of heavy-chain antibodies of the camelid adaptive immune system. We report here on the validation and quantification of a nanobody raised against mitochondrial translation elongation factor (TUFM). Differential expression of TUFM was examined in different GBM cell lines and GBM tissue at the protein and mRNA levels, as compared to their expression in neural stem cells and normal brain tissue. We further used in-silico modelling and immunocytochemistry to define the specificity of anti-TUFM nanobody (Nb206) towards GBM stem cells, as compared to GBM cell lines (U251MG and U87MG cells). Due to its specificity and pronounced inhibitory effect on GBM stem cell growth, we propose the use of this anti-TUFM nanobody for GBM in vitro immunoimaging and potentially also cancer stem cell targeting.

Time-dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes.

Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa488 ) and fluorescent dextran (Dextran546 ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa488 from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa488 . Initially, S100B-Alexa488 and Dextran546 internalized with distinct rates into different endocytotic vesicles; S100B-Alexa488 internalized into smaller vesicles than Dextran546 . At a later stage, S100B-Alexa488 -positive vesicles substantially co-localized with Dextran546 -positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa488 , but not of Dextran546 . The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa488 -positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca2+ ]i ). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs in vivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications.

UNLABELLED: Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning - induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. BIOLOGICAL SIGNIFICANCE: Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.

On the role of protein disulfide isomerase in the retrograde cell transport of secreted phospholipases A2.

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A2 (sPLA2) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA2. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b'. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA2s have the same fold as Atx. The first three sPLA2s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA2 in the Atx-hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA2s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA2 and hPDI is however different from that of the other sPLA2s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA2s confirmed the model-based expectation that GV sPLA2 was a more effective inhibitor than GX sPLA2, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA2s by assisting the retrograde transport of these molecules from the cell surface. The sPLA2-hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA2s in relation to their action intracellularly.

Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A(2): an update.

ß-neurotoxins are enzymes, secreted phospholipases A2, that inhibit neurotransmission in neuromuscular synapses by poisoning the motoneuron. They were reviewed extensively several years ago (Pungercar and Krizaj, 2007). Here we present and critically discuss the most important experimental facts reported since then. Evidence has been presented for specific internalization of ß-neurotoxins into the nerve endings of motoneurons, their in vivo binding to some cytosolic proteins, direct action on mitochondria, disruption of Ca(2+) homoeostasis and inhibition of amphiphysin function. New insights have led to a more confident interpretation of the action of these toxins at the molecular level. The most important questions that remain to be answered are listed.

Secreted Phospholipases A2 - not just Enzymes.

Secreted phospholipases A2 (sPLA2s) constitute, physiologically and pathologically, a very important family of enzymes. Most actions of sPLA2s have been explained by their phosphatidylglycerol sn-2 hydrolytic activity. However, since pharmacologically active sPLA2 molecules without enzymatic activity have been discovered, first in snake venom and then also in human, it has become increasingly evident that, on many occasions, the action of these proteins has to be considered as arising from the interplay of their receptor-binding and enzymatic functions. The number of known sPLA2-interacting molecules is growing and, with the development of more sensitive biochemical techniques, further discoveries are expected. In this paper we are reviewing all the currently known sPLA2-binding proteins. The structural versatility of these molecules establishes sPLA2s as ligands with a broad interaction spectrum, in agreement with the already recognized multifunctional nature of these proteins. Mechanistic descriptions of the multitude of actions of sPLA2s is today one of the most exciting and promising research areas, and the description of the sPLA2-interactome is for sure one of its vital parts.

A new phospholipase A2 isolated from the sea anemone Urticina crassicornis - its primary structure and phylogenetic classification.

Disulfide pairings and active site residues are highly conserved in secretory phospholipases A(2) (PLA(2)s). However, secretory PLA(2)s of marine invertebrates display some distinctive structural features. In this study, we report the isolation and characterization of a PLA(2) from the northern Pacific sea anemone, Urticina crassicornis (UcPLA(2)), containing a C27N substitution and a truncated C-terminal sequence. This novel cnidarian PLA(2) shares about 60% identity and almost 70% homology with two putative PLA(2)s identified in the starlet sea anemone (Nematostella vectensis) genome project. UcPLA(2) lacks hemolytic and neurotoxic activities. A search of available sequences revealed that Asn27-'type' PLA(2)s are present in a few other marine animal species, including some vertebrates. The possibility that the C27N replacement represents a structural adaptation for PLA(2) digestion/activity in the marine environment was not supported by experiments testing the influence of ionic strength on UcPLA(2) enzymatic activity. Because of the highly divergent sequences among invertebrate group I PLA(2)s, it is currently not possible to identify orthologous relationships. As the Asn27-containing PLA(2)s are scattered among the other invertebrate group I PLA(2)s, they do not constitute a new, monophyletic PLA(2) clade.

The first phospholipase inhibitor from the serum of Vipera ammodytes ammodytes.

Ammodytoxins are neurotoxic secretory phospholipase A(2) molecules, some of the most toxic components of the long-nosed viper (Vipera ammodytes ammodytes) venom. Envenomation by this and by closely related vipers is quite frequent in southern parts of Europe and serotherapy is used in the most severe cases. Because of occasional complications, alternative medical treatment of envenomation is needed. In the present study, ammodytoxin inhibitor was purified from the serum of V. a. ammodytes using two affinity procedures and a gel exclusion chromatography step. The ammodytoxin inhibitor from V. a. ammodytes serum consists of 23- and 25-kDa glycoproteins that form an oligomer, probably a tetramer, of about 100 kDa. N-terminal sequencing and immunological analysis revealed that both types of subunit are very similar to gamma-type secretory phospholipase A(2) inhibitors. The ammodytoxin inhibitor from V. a. ammodytes serum is a potent inhibitor of phospholipase activity and hence probably also the neurotoxicity of ammodytoxins. Discovery of the novel natural inhibitor of these potent secretory phospholipase A(2) toxins opens up prospects for the development of new types of small peptide inhibitors for use in regulating the physiological and pathological activities of secretory phospholipases A(2).

A new photoprobe for studying biological activities of secreted phospholipases A2.

Ammodytoxin (Atx) is a snake venom phospholipase A2 (sPLA2s) with presynaptic toxicity, anticoagulant activity and the ability to influence cell cycle progression. These multiple physiological activities make this molecule a promising tool for studying processes influenced by the highly homologous mammalian sPLA2s-for example cell proliferation and apoptosis. Secreted PLA2s can act on cells as enzymes or as ligands for cellular receptors. To further characterize the sPLA2-binding molecules in cells we have developed a new method based on AtxC and a biotin-containing cross-linking reagent sulfo-SBED which possesses both an amine-reactive and a photo-reactive site, together with a biotin moiety that enables specific detection and affinity-based concentration. The biological activity of the AtxC derivatized by sulfo-SBED was demonstrated by biotin-tagging of calmodulin and R25, both known AtxC targets, but not of other proteins. In addition, using the new protocol we specifically labelled 14-3-3 proteins, protein disulfide isomerase and two unknown proteins of 45 and 46kDa in the mitochondrial-synaptosomal fraction of porcine cerebral cortex, none of which could be tagged by the previously used methods. The new methodology, which can be used for any sPLA2, constitutes a novel approach to discovering and purifying sPLA2-binding proteins, to studying the topology of their respective complexes and to following sPLA2s in different biological systems.

Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae.

Ammodytoxin (Atx), an sPLA2 (secretory phospholipase A2), binds to g and e isoforms of porcine 14-3-3 proteins in vitro. 14-3-3 proteins are evolutionarily conserved eukaryotic regulatory proteins involved in a variety of biological processes, including cell-cycle regulation. We have now shown that Atx binds to yeast 14-3-3 proteins with an affinity similar to that for the mammalian isoforms. Thus yeast Saccharomyces cerevisiae can be used as a model eukaryotic cell, which lacks endogenous phospholipases A2, to assess the in vivo relevance of this interaction. Atx was expressed in yeast cells and shown to be biologically active inside the cells. It inhibited G2 cell-cycle arrest in yeast, which is regulated by 14-3-3 proteins. Interference with the cell cycle indicates a possible mechanism by which sPLA2s are able to cause the opposing effects, proliferation and apoptosis, in mammalian cells.

Protein disulphide isomerase binds ammodytoxin strongly: possible implications for toxin trafficking.

Ammodytoxin, a group IIA secreted phospholipase A(2) from the venom of the long-nosed viper (Vipera ammodytes ammodytes), is a potent presynaptically acting neurotoxin. It blocks the secretion of neurotransmitter from the nerve cell, thus hindering the communication with the neighbouring neuron or muscle cell. To express the neurotoxicity, ammodytoxin should interact with specific receptors in the axon terminal and express phospholipase activity. Our previous results indicate that, following the association with a receptor on the external side of the presynaptic membrane, the toxin penetrates into the cytosol of the nerve cell. Here, we show that the toxin associates specifically with protein disulphide isomerase, a protein in the lumen of endoplasmic reticulum, which may be crucial for the retention and concentration of the toxin in this cellular compartment and for its subsequent transport across the membrane of endoplasmic reticulum into the cytosol of the nerve cell.

Ammodytoxin, a neurotoxic secreted phospholipase A(2), can act in the cytosol of the nerve cell.

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A(2) acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment.

R25 is an intracellular membrane receptor for a snake venom secretory phospholipase A(2).

Ammodytoxin is a presynaptically neurotoxic (beta-neurotoxic) snake venom secretory phospholipase A(2) (sPLA(2)). We detected a 25 kDa protein which binds the toxin with very high affinity (R25) in porcine cerebral cortex. Here we show that R25 is an integral membrane protein with intracellular localisation. It is the first sPLA(2) receptor known to date that localises to intracellular membranes. Centrifugation on sucrose gradients was used to fractionate porcine cerebral cortex. The subcellular composition of the fractions was determined by following the distribution of organelle-specific markers. The distribution of R25 in the fractions matched the distribution of the mitochondrial marker succinate dehydrogenase, but not the markers for plasma membrane, lysosomes, endoplasmic reticulum, synaptic and secretory vesicles. R25 most likely resides in mitochondria, which are known to be targets for sPLA(2) neurotoxins in the nerve ending and are potentially implicated in the process of beta-neurotoxicity.