A Grasso

National Research Council, Roma, Latium, Italy

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Publications (14)41.88 Total impact

  • M Pescatori · A Grasso
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    ABSTRACT: alpha-Latrotoxin, a protein toxin present in the venom of black widow spider, interacts with membrane receptors of neurons and other secretory cells to stimulate exocytosis. Two types of receptors have been identified and cloned. Our attention has been focused on the calcium independent receptor, a G-protein coupled receptor, named latrophilin to see whether alpha-latrotoxin interaction was capable to produce an ionotropic effect, in alternative to the metabotropic hypothesis. Expression of latrophilin receptor is sufficient for the alpha-latrotoxin effect to become manifest. By inducing the transient expression of latrophilin receptor in non-neuronal human embryonic cells, we made them susceptible to toxin action as demonstrated by the increase in 45Ca(2+) accumulation detected after toxin treatment. Since the presence of a monoclonal antibody against alpha-latrotoxin (4C4.1 mAb) was able to obliterate toxin-dependent effects, we further investigated the nature of toxin-antibody interaction by characterization of the binding epitope using phage display-peptide libraries. A conformational epitope was recognized and partially localized on a region of the peptide toxin whereby a tetrameric structure is formed and inserted into the membrane of target cells where it functions as a pore.
    No preview · Article · Sep 2000 · Biochimie
  • A Grasso

    No preview · Article · Jan 2000 · Toxicon
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    ABSTRACT: Electrophysiological recording demonstrates that alpha-latrotoxin, a 125,000 mol. wt component of black widow spider venom, promotes high frequency quantal discharges at larval neuromuscular junctions of Drosophila. Concomitantly, fluorescence imaging of presynaptic calcium ion activity reveals that this toxin qualitatively elevates cytosolic ionized calcium in this preparation. These activities of alpha-latrotoxin are selectively antagonized by a monoclonal antibody, 4C4.1, that was previously shown to inhibit the action of this toxin in PC-12 cells. However, 4C4.1 does not block the release-promoting activity of gel-filtered extracts of black widow spider venom. This indicates that black widow spider venom has multiple components that promote quantal transmitter secretion in invertebrates. This investigation demonstrates that alpha-latrotoxin is among the active principles in black widow spider venom that enhance transmitter release and raise cytosolic ionized calcium in Drosophila. These results suggest that Drosophila, because of the relative ease of genetic manipulation, may be useful to study the target protein(s) that mediate the binding and action of alpha-latrotoxin at nerve endings. Moreover, the procedure that we report for loading Drosophila nerve terminals with the calcium ion-sensing dye, Calcium Crimson, may have utility for studying calcium dynamics in mutant alleles with alterations in synapse development and function in this organism.
    No preview · Article · Jan 1999 · Neuroscience
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    Jochen Lang · Yuri Ushkaryov · Alfonso Grasso · Claes B. Wollheim
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    ABSTRACT: alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.
    Full-text · Article · Mar 1998 · The EMBO Journal
  • A Grasso · M Pescatori

    No preview · Article · Feb 1996 · Advances in Experimental Medicine and Biology
  • A. Grasso · M. Pescatori
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    ABSTRACT: Spider venoms are a rich source of potential probes for ion channels and receptors in nerve cells (Kawai and Nakajima, 1993). From a biochemical point of view, the study of the molecular composition of spider venoms, is not easy because a large number of venom glands are necessary from animals which are generally small, and frequently, difficult to collect in large number. To avoid these limitations, we have been recently induced to use a molecular biology approach, to study spider venoms. In fact, cloning and expression of cDNAs offers an alternative to secure sufficient material to the purpose of detecting molecules having pharmacological activities. In the venom of the Mediterranean black widow spider (Latrodectus tredecimguttatus) we developed the initial idea to sequence and clone the most interesting neurotoxin, named alpha-latrotoxin. The potentialities of this project were based on a limited amino acid sequence available for the toxin (Pescatori et al., 1995). In fact, alpha-latrotoxin was considered to be a single-polypeptide toxin that exerts its neurotoxic action, by dramatically affecting synaptic vesicles exocytosis at the nerve endings (Grasso, 1988). The finding that this toxin could stimulate neurosecretion has been of great interest in neurobiology and the possibility of developing its use, as a pharmacological tool for the study of neurotransmitter release at various nerve terminals, has been greatly developed, afterwards. At nerve endings, the toxin stimulates and supports secretion even in the absence of external calcium, thus offering a great variation, not even considered before, of experimental conditions to test. Furthermore, alpha-latrotoxin binds with a high-affinity to receptor which is localised exclusively at the presynaptic plasma membrane (Petrenko et al., 1990). The molecular organisation of presynaptic structures has been derived by the development of functional analysis on toxin-receptor interactions (Petrenko, 1993; O’Connor et al., 1993).
    No preview · Chapter · Jan 1996
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    ABSTRACT: A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
    No preview · Article · Jun 1995 · European Journal of Biochemistry
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    [Show abstract] [Hide abstract]
    ABSTRACT: A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from α-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified α-latrotoxin, suggesting that latrodectin is intimately associated with α-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
    Full-text · Article · Apr 1995
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    ABSTRACT: alpha-Latrotoxin causes massive release of norepinephrine from clonal rat pheochromocytoma PC12 cells, in the absence of external Ca2+, by an unknown mechanism. The effect almost disappeared in PC12 variant cells deficient in synaptotagmin I, a synaptic vesicle protein, and was rescued by transfecting the synaptotagmin I gene. These results indicate that synaptotagmin I is essential for the Ca(2+)-independent action of alpha-latrotoxin in PC12 cells.
    No preview · Article · Nov 1994 · FEBS Letters
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    ABSTRACT: α-Latrotoxin causes massive release of norepinephrine from clonal rat pheochromocytoma PC12 cells, in the absence of external Ca2+, by an unknown mechanism. The effect almost disappeared in PC12 variant cells deficient in synaptotagmin I, a synaptic vesicle protein, and was rescued by transfecting the synaptotagmin I gene. These results indicate that synaptotagmin I is essential for the Ca2+-independent action of α-latrotoxin in PC12 cells.
    Full-text · Article · Oct 1994 · FEBS Letters
  • M Pescatori · A Grasso

    No preview · Article · Apr 1994 · Annals of the New York Academy of Sciences
  • Mario Pescatori · Alfonso Grasso

    No preview · Article · Mar 1994 · Annals of the New York Academy of Sciences
  • A Grasso · A Mastrogiacomo
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    ABSTRACT: A toxin that causes a massive presynaptic activation of transmitter release from nerve terminals is alpha-latrotoxin, isolated from Latrodectus tredecimguttatus spider venom. This toxin has been highly purified, utilizing as a biological assay a toxin-dependent increase in 45Ca(2+)-accumulation by PC12 cells. The purification protocol includes an ion-exchange step and a gel-filtration column, by fast-flow liquid chromatography. The resulting toxin is a polypeptide of about 125 kDa in molecular mass. At nmol concentrations it specifically activates calcium influx and transmitter secretion after interacting with neuronal acceptors of the presynaptic membrane. The inhibitory effect of trivalent ions (which may develop as degradation product of 45Ca2+) on toxin-dependent calcium accumulation by PC12 cells is described. The results obtained suggest that calcium fluxes directly involved in the neurosecretory event, may occur through newly formed toxin-dependent channels.
    No preview · Article · Oct 1992 · FEMS microbiology immunology
  • Joy A. Umbach · Alfonso Grasso · Cameron B. Gundersen
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    ABSTRACT: When Xenopus oocytes are injected with rat brain mRNA, they acquire the ability to respond to bath applied alpha-latrotoxin. This spider venom toxin is normally highly selective for nerve endings, where its binding is associated with a high-frequency, quantal discharge of neurotransmitter. By 'transplanting' toxin acceptor sites to Xenopus oocytes, we have observed both a toxin-mediated rise in cellular ionized Ca along with the triggering of a calcium-dependent chloride channel in these cells. This approach may contribute both to a better understanding of the mechanism of action of this toxin and to efforts to clone the cDNA for this binding site.
    No preview · Article · Jul 1990 · Molecular Brain Research

Publication Stats

164 Citations
41.88 Total Impact Points

Institutions

  • 1994-2000
    • National Research Council
      • Institute of Cell Biology IBC
      Roma, Latium, Italy
    • INO - Istituto Nazionale di Ottica
      Florens, Tuscany, Italy
  • 1995
    • National Institute of Geophysics and Volcanology
      Roma, Latium, Italy