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Structure of a protein copurified with α-latrotoxin, specifically expressed in the venom gland of the spider Latrodectus tredecimguttatus
Tenth European Symposium
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mouth, and erection of tail . The heart cafe, pulse and blood propuce wero railed following injection, sad continued to rise, followrod by a fall a~ the death of the animal. The lungs showed congestion, oedema and peteacheal haemorrhages . The heart showed myocardial degeneration and cellular in5ltration. The liver ceW showed a vacuolated appearance with complete depletion of glycogen grsaules; some liver ceW showed degenerative changes. The kidney showed collapse of glomerular capillaries, increased eellularity in glomeru6 aced glomerular ~. The adrenal medulla showed depletion of elxtron~ense vesicles, aced some medulLuy odb ahawed complete disruption and disorganiation . The observatiom are compatible with `autonomic storm' weds. B~w~s, H. 3. and Bawwsua, P. H. (1991) J. WiJdernesr Mad. 2, 164-175.
Sodihen cGmusr! sito-directed orrtibodka distirtgwiclY betweet tGe b sétta of two nwt~aHy dlsplaoaa6k inmt nleetire tsesrotoxlnc. D. Goanox, H. Massowmz and E. Zc.ors~r (Institute of Life Sciences, The Hebcew University, Jerusalem, Israel). Sere-nntECt~ antibodies corresponding to conserved putative extracllular segments of sodium channels, coupled with binding studies of radiolabelled insect-selective scorpion neurotoxins were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LghIT~ was shown to possess two non-interacting binding :ites in locust neuronal membranes: a high affinity (1C, - 0.9 f0.6 nI~ and low capacity (Bin.,, = 0.1 f 0.07 p~mole/mg) binding site a: well as a low a®nity (Km ~ 185 t 13 nI~ a~ high capacity (B~ = 10.0 t0.6 pmok/mg) binding site. (2) The high a®nity site serves es a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LghITz was sigaiScantiy inhibited in a dosadependent manner by each of four sito-directod snti'bodies. The binding inhibition resulted from the reduction in the number of the binding sites. (4) The antibody-mediated inhibition of the ['~I]AaIT binding differs from that of LghITr three out of the four antibodies which inhibited the LghIT= binding only Partially affected the AaIT binding. Two antibodies, one wrmaponding to extracelular and one to intracellular segments of the channel, did not affect the binding of either toxins . These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium c snn~1 ; (b) are fonmed by segments of external loops in domains I, III and IV of the sodium channel; e~ (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.
Strwctrve of o protein rnpwifud wüh a-latrotoxln, specifically expressed is tht venons glmrd of the apidrr Latrodedus trededmguttatus. M. P~roaa,' A. Baeusuax' and A. Gruvo' ('Istituto di Biologie Cdluhue, C.N .R. V.k Marx 43, Roma ; and =SISSA, via Beirut 2~, Trieste, Italy) . T~ seQVexce of a tryptic fragment of the a-latrotoxin purißed from the venom of black widow spider (Lotrodccdu tredecWguttahar) as prsvioualy described (Gs~sso e~ M~tneoaucwso, 1992) was found to be: I3PADIGJtTDISQADFDEDN. Considering the absence of the above-mentioned sequence in the published sequence of the putative a-Istrotoxin precursor (ICrxe~r et aL, 1990) wro believed it important to clone it, on the suspicion that it belonged to a protein strongly associated with the toxin. Two overlapping oligonudeotides [Q-E and BamHI-Q-N] were designed to clone the 3' end of the toxin by the RACE method (FaotnuN, et al., 1988). cDNA was synthesimd es descdbed using poly A* mRNA isolated from the glands of the black widow spider. The primer used for cDNA synthesis wu the sine as that (dT),r adaptor, used by Fhasttux et a!. (1988). PCR using this primer and Q-E prodnoed a weak band of 350 bp. This was gel purißed and subjected to 30 finther cycles of PCR using HsmIß-Q-N sad the adaptor primer described by FsasnuN et d. (1988). The stron®er band produced was gel purißed, digested with HamHI sad CIaI, and cloned into Hh>csaipt K3*. On the basis of the sequence obtained, two ~nested oligon»deotides weinedesigned to amplify sad done the S' end of the mRNA. DNA sequencing of the two dosed fragments allowed the deduction of the following amino adds sequence: MSKLFFWFLCLIL4VFA] [ISPADIGCTDISQADFDERNNNCIKCGBDGFGEEMVNRCRDKCPTDNFYQSCVDLLNKVYEEICDTPPVQE. According to the hydropathy pro81e the ßrst 18 amino acids, mainly hydrophobic, were considered to correspond to a signal peptide. The resulting protein was found to be highly hydrophilic with a calailated pl 6.8 and mol. wt 7945 . Northern blot analysis of RNA from spider venom gLnds and cephelothoaoes showed that mRNA of the dosed molecule was speciflcally and highly expressed in the glands, thus suggesting a functional role for the protein in venom action . The interaction of this protein with alatrotoxin is under study. Ftiwsnux, M. et al. (1988) PNAS USA ~ 8998-+9002 . Ga~o, A. aced M oos~, A. (1992) FSAIS AlMroblol. Inoraarol. (in press). Krxe~xnv N. et al. (1990) FEBS Lett. 270, 127-131.
52l
mouth, and erection of tail . The heart cafe, pulse and blood propuce wero railed following injection, sad continued to rise, followrod by a fall a~ the death of the animal. The lungs showed congestion, oedema and peteacheal haemorrhages . The heart showed myocardial degeneration and cellular in5ltration. The liver ceW showed a vacuolated appearance with complete depletion of glycogen grsaules; some liver ceW showed degenerative changes. The kidney showed collapse of glomerular capillaries, increased eellularity in glomeru6 aced glomerular ~. The adrenal medulla showed depletion of elxtron~ense vesicles, aced some medulLuy odb ahawed complete disruption and disorganiation . The observatiom are compatible with `autonomic storm' weds. B~w~s, H. 3. and Bawwsua, P. H. (1991) J. WiJdernesr Mad. 2, 164-175.
Sodihen cGmusr! sito-directed orrtibodka distirtgwiclY betweet tGe b sétta of two nwt~aHy dlsplaoaa6k inmt nleetire tsesrotoxlnc. D. Goanox, H. Massowmz and E. Zc.ors~r (Institute of Life Sciences, The Hebcew University, Jerusalem, Israel). Sere-nntECt~ antibodies corresponding to conserved putative extracllular segments of sodium channels, coupled with binding studies of radiolabelled insect-selective scorpion neurotoxins were employed to clarify the relationship between the toxins' receptor sites and the insect sodium channel. (1) The depressant insect toxin LghIT~ was shown to possess two non-interacting binding :ites in locust neuronal membranes: a high affinity (1C, - 0.9 f0.6 nI~ and low capacity (Bin.,, = 0.1 f 0.07 p~mole/mg) binding site a: well as a low a®nity (Km ~ 185 t 13 nI~ a~ high capacity (B~ = 10.0 t0.6 pmok/mg) binding site. (2) The high a®nity site serves es a target for binding competition by the excitatory insect toxin AaIT. (3) The binding of LghITz was sigaiScantiy inhibited in a dosadependent manner by each of four sito-directod snti'bodies. The binding inhibition resulted from the reduction in the number of the binding sites. (4) The antibody-mediated inhibition of the ['~I]AaIT binding differs from that of LghITr three out of the four antibodies which inhibited the LghIT= binding only Partially affected the AaIT binding. Two antibodies, one wrmaponding to extracelular and one to intracellular segments of the channel, did not affect the binding of either toxins . These data suggest that the receptors to the depressant and excitatory insect toxins (a) comprise an integral part of the insect sodium c snn~1 ; (b) are fonmed by segments of external loops in domains I, III and IV of the sodium channel; e~ (c) are localized in close proximity but are not identical in spite of the competitive interaction between these toxins.
Strwctrve of o protein rnpwifud wüh a-latrotoxln, specifically expressed is tht venons glmrd of the apidrr Latrodedus trededmguttatus. M. P~roaa,' A. Baeusuax' and A. Gruvo' ('Istituto di Biologie Cdluhue, C.N .R. V.k Marx 43, Roma ; and =SISSA, via Beirut 2~, Trieste, Italy) . T~ seQVexce of a tryptic fragment of the a-latrotoxin purißed from the venom of black widow spider (Lotrodccdu tredecWguttahar) as prsvioualy described (Gs~sso e~ M~tneoaucwso, 1992) was found to be: I3PADIGJtTDISQADFDEDN. Considering the absence of the above-mentioned sequence in the published sequence of the putative a-Istrotoxin precursor (ICrxe~r et aL, 1990) wro believed it important to clone it, on the suspicion that it belonged to a protein strongly associated with the toxin. Two overlapping oligonudeotides [Q-E and BamHI-Q-N] were designed to clone the 3' end of the toxin by the RACE method (FaotnuN, et al., 1988). cDNA was synthesimd es descdbed using poly A* mRNA isolated from the glands of the black widow spider. The primer used for cDNA synthesis wu the sine as that (dT),r adaptor, used by Fhasttux et a!. (1988). PCR using this primer and Q-E prodnoed a weak band of 350 bp. This was gel purißed and subjected to 30 finther cycles of PCR using HsmIß-Q-N sad the adaptor primer described by FsasnuN et d. (1988). The stron®er band produced was gel purißed, digested with HamHI sad CIaI, and cloned into Hh>csaipt K3*. On the basis of the sequence obtained, two ~nested oligon»deotides weinedesigned to amplify sad done the S' end of the mRNA. DNA sequencing of the two dosed fragments allowed the deduction of the following amino adds sequence: MSKLFFWFLCLIL4VFA] [ISPADIGCTDISQADFDERNNNCIKCGBDGFGEEMVNRCRDKCPTDNFYQSCVDLLNKVYEEICDTPPVQE. According to the hydropathy pro81e the ßrst 18 amino acids, mainly hydrophobic, were considered to correspond to a signal peptide. The resulting protein was found to be highly hydrophilic with a calailated pl 6.8 and mol. wt 7945 . Northern blot analysis of RNA from spider venom gLnds and cephelothoaoes showed that mRNA of the dosed molecule was speciflcally and highly expressed in the glands, thus suggesting a functional role for the protein in venom action . The interaction of this protein with alatrotoxin is under study. Ftiwsnux, M. et al. (1988) PNAS USA ~ 8998-+9002 . Ga~o, A. aced M oos~, A. (1992) FSAIS AlMroblol. Inoraarol. (in press). Krxe~xnv N. et al. (1990) FEBS Lett. 270, 127-131.