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Functional expression of α-latrotoxin binding sites in Xenopus oocytes
Report and Abstracts on Lutrodectus Neurotoxins
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binding sites in Xenopus oocytes. C. B. GUNDERSEN, J. A. UMBACHand A. GRA.~.YCJ (Department of Pharmacology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.; and Istituto di Biologia Cellulare, Rome, Italy) Functional expression of a-latrotoxin
ALPHAlatrotoxin (aLtx), a 130 kD protein component of black widow spider venom, binds with high affinity and great selectivity to the presynaptic membrane of a wide range of organisms. The binding of aLtx at the nerve ending normally triggers a high frequency, quanta1 discharge of neurotransmitter. Neither the basis for the selectivity of altx’s association with nerve terminal membranes nor the mechanism of its release promoting activity has been fully elucidated. One approach toward furthering our understanding of these problems would be to clone the cDNA for the nerve terminal protein that binds aLtx (and that has been tentatively purified and reconstituted by Scheer, Prestipino & Meldolesi). Our efforts in this direction have begun with the demonstration that binding/effecter sites for aLtx can be detected under appropriate circumstances in Xenopus oocytes that are injected with rat brain mRNA. Specifically, in contrast to control oocytes, those injected with rat brain mRNA exhibit a rise of intracellular Ca and the triggering of a Ca-dependent Cl conductance in response to 10-30 nM aLtx. These effects are generally observed only when bath Ca is raised to 5 mM. The responses are not reversed by washing. From these data, and based on precedents for other ligands acting on these oocytes, we conclude that aLtx indirectly elicits the activation of the native Ca-dependent Cl channel in the plasma membrane of these cells. Moreover, our data imply that the source of the Ca that triggers these responses involves both intracellular and extracellular pools. However, in no circumstances have we detected any currents that might arise directly from the channel forming activity of aLtx that has been reported for planar lipid bilayers and cultured cells. While this system may eventually prove useful for exploring further the actions of aLtx, the relatively small signals we detect in the mRNA injected oocytes are not conveniently suited to expression cloning techniques.
Structure of low molecular weight protein componentfrom
black widow spider venom. N. I. KIYATKIN(Shemyakin
Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, Moscow, Russia) THE BLACKwidow spider venom contains a family of high mol. wt neurotoxins selectively acting on presynaptic nerve endings of different animals. These neurotoxins can associate with low mol. wt proteins with unknown functions (TASHMLJKHAME~KJV et al., 1983). We have recently cloned and sequenced cDNA encoding the putative a-latrotoxin (a-LTX) precursor. According to structural analysis of the 5408 bp cDNA, it is the 156855 Da protein composed of 1401 amino acid residues. Two peptides from a tryptic digest of a-LTX were not found in the deduced amino acid sequence. To reveal whether a-LTX was co-purified with a low mol. wt component, these peptides were used to derive oligonucleotide hybridization probes. The cDNA library prepared from the venom glands of the black widow spider was screened with these probes. We isolated two groups of hybridization-positive clones, that contained cDNA inserts of about 0.5 and 1.2 kb, respectively. Both tryptic peptides were included in these sequences. Surprisingly, one group of the clones (0.5 kb) contained full-length cDNA corresponding to mRNA for a new low mol. wt protein. The deduced protein sequence (XTX) comprised a signal peptide, hydrophilic protein chain of 70 residues and 6 Cys residues. Analysis of cDNA from another group of clones (1.2 kb) showed that respective mRNA corresponded to mRNA from the first group of clones, but incorporated a novel sequence block (0.5 kb), which introduced an in-frame stop codon. Thus large mRNA cannot generate a new polypeptide chain differing from XTX, and translation XTX protein occurs after this mRNA processing at the level of splicing. Northern-blot analysis of mRNA from venom glands showed that XTX mRNA amounted to more than 90% of total pool mRNA. Specific interaction of XTX protein with a-LTX is under study. TASHMUKHAMEM)~, B. A. et al. (1983) Effects of different spider venoms on artificial and biological membranes. In: Toxins as Tools in Neurochemistry, pp. 31 l-323. Berlin: Walter de Gruyter. The effects of” presynaptic insectotoxin (a-latroinsectotoxin) isolated from black widow spider venom on neuromuscular preparations. L. G. MAGAZANIK(I. M. Sechenov Institute of Evolutionary Physiology and Bio-
chemistry, U.S.S.R. Academy of Sciences, St Petersburg, Russia) THE VENOM of the black widow spider Lutroa’ectus mactans tredecimguttatus (BWSV) contains a potent neurotoxin, a-latrotoxin (a-LT; mol. wt 130 kD), which affects transmitter release only from nerve endings of vertebrate animals, whereas the crude BWSV is also toxic for crustacea and insects (ROSEN~AL and MELDOLESI, 1989). As a result of subsequent procedures of chromatography separation a homogeneous protein of mol. wt 120 kD was isolated from black widow spider venom and referred to as a-latroinsectotoxin (a-LIT). It was highly potent (4 nM) in the induction of an increase of the frequency of miniature excitatory postsynaptic potentials in blowfly Calliphora vicina larva neuromuscular preparations (KOVALEVSKAYA et al., 1990; MAGAZANIKet al., 1991). In the frog nerve ending, however, even 50 nM a-LIT failed to affect transmitter release. Pretreatment of insect preparations with a-LT or frog preparations with a-LIT did not prevent the specific effect of consequent applications of a-LIT (insect) and a-LT (frog), respectively. The binding of labeled [“‘I]-a-latroinsectotoxin to
119
binding sites in Xenopus oocytes. C. B. GUNDERSEN, J. A. UMBACHand A. GRA.~.YCJ (Department of Pharmacology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.; and Istituto di Biologia Cellulare, Rome, Italy) Functional expression of a-latrotoxin
ALPHAlatrotoxin (aLtx), a 130 kD protein component of black widow spider venom, binds with high affinity and great selectivity to the presynaptic membrane of a wide range of organisms. The binding of aLtx at the nerve ending normally triggers a high frequency, quanta1 discharge of neurotransmitter. Neither the basis for the selectivity of altx’s association with nerve terminal membranes nor the mechanism of its release promoting activity has been fully elucidated. One approach toward furthering our understanding of these problems would be to clone the cDNA for the nerve terminal protein that binds aLtx (and that has been tentatively purified and reconstituted by Scheer, Prestipino & Meldolesi). Our efforts in this direction have begun with the demonstration that binding/effecter sites for aLtx can be detected under appropriate circumstances in Xenopus oocytes that are injected with rat brain mRNA. Specifically, in contrast to control oocytes, those injected with rat brain mRNA exhibit a rise of intracellular Ca and the triggering of a Ca-dependent Cl conductance in response to 10-30 nM aLtx. These effects are generally observed only when bath Ca is raised to 5 mM. The responses are not reversed by washing. From these data, and based on precedents for other ligands acting on these oocytes, we conclude that aLtx indirectly elicits the activation of the native Ca-dependent Cl channel in the plasma membrane of these cells. Moreover, our data imply that the source of the Ca that triggers these responses involves both intracellular and extracellular pools. However, in no circumstances have we detected any currents that might arise directly from the channel forming activity of aLtx that has been reported for planar lipid bilayers and cultured cells. While this system may eventually prove useful for exploring further the actions of aLtx, the relatively small signals we detect in the mRNA injected oocytes are not conveniently suited to expression cloning techniques.
Structure of low molecular weight protein componentfrom
black widow spider venom. N. I. KIYATKIN(Shemyakin
Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, Moscow, Russia) THE BLACKwidow spider venom contains a family of high mol. wt neurotoxins selectively acting on presynaptic nerve endings of different animals. These neurotoxins can associate with low mol. wt proteins with unknown functions (TASHMLJKHAME~KJV et al., 1983). We have recently cloned and sequenced cDNA encoding the putative a-latrotoxin (a-LTX) precursor. According to structural analysis of the 5408 bp cDNA, it is the 156855 Da protein composed of 1401 amino acid residues. Two peptides from a tryptic digest of a-LTX were not found in the deduced amino acid sequence. To reveal whether a-LTX was co-purified with a low mol. wt component, these peptides were used to derive oligonucleotide hybridization probes. The cDNA library prepared from the venom glands of the black widow spider was screened with these probes. We isolated two groups of hybridization-positive clones, that contained cDNA inserts of about 0.5 and 1.2 kb, respectively. Both tryptic peptides were included in these sequences. Surprisingly, one group of the clones (0.5 kb) contained full-length cDNA corresponding to mRNA for a new low mol. wt protein. The deduced protein sequence (XTX) comprised a signal peptide, hydrophilic protein chain of 70 residues and 6 Cys residues. Analysis of cDNA from another group of clones (1.2 kb) showed that respective mRNA corresponded to mRNA from the first group of clones, but incorporated a novel sequence block (0.5 kb), which introduced an in-frame stop codon. Thus large mRNA cannot generate a new polypeptide chain differing from XTX, and translation XTX protein occurs after this mRNA processing at the level of splicing. Northern-blot analysis of mRNA from venom glands showed that XTX mRNA amounted to more than 90% of total pool mRNA. Specific interaction of XTX protein with a-LTX is under study. TASHMUKHAMEM)~, B. A. et al. (1983) Effects of different spider venoms on artificial and biological membranes. In: Toxins as Tools in Neurochemistry, pp. 31 l-323. Berlin: Walter de Gruyter. The effects of” presynaptic insectotoxin (a-latroinsectotoxin) isolated from black widow spider venom on neuromuscular preparations. L. G. MAGAZANIK(I. M. Sechenov Institute of Evolutionary Physiology and Bio-
chemistry, U.S.S.R. Academy of Sciences, St Petersburg, Russia) THE VENOM of the black widow spider Lutroa’ectus mactans tredecimguttatus (BWSV) contains a potent neurotoxin, a-latrotoxin (a-LT; mol. wt 130 kD), which affects transmitter release only from nerve endings of vertebrate animals, whereas the crude BWSV is also toxic for crustacea and insects (ROSEN~AL and MELDOLESI, 1989). As a result of subsequent procedures of chromatography separation a homogeneous protein of mol. wt 120 kD was isolated from black widow spider venom and referred to as a-latroinsectotoxin (a-LIT). It was highly potent (4 nM) in the induction of an increase of the frequency of miniature excitatory postsynaptic potentials in blowfly Calliphora vicina larva neuromuscular preparations (KOVALEVSKAYA et al., 1990; MAGAZANIKet al., 1991). In the frog nerve ending, however, even 50 nM a-LIT failed to affect transmitter release. Pretreatment of insect preparations with a-LT or frog preparations with a-LIT did not prevent the specific effect of consequent applications of a-LIT (insect) and a-LT (frog), respectively. The binding of labeled [“‘I]-a-latroinsectotoxin to