Functional Characterization Of Novel Conotoxins And Preparation Of ?-conotoxin GI Antiserum

Objective: A series of the novel A-,M-superfamily conotoxins with new framework,which were cloned from the South China Sea,were synthesized and functionally characterized using nicotinic acetylcholine receptors(n ACh Rs)expressed on Xenopus oocytes.In addition,the antitoxin of ?-conotoxin GI with the high toxicity was prepared and its neutralization activity was tested.All the efforts will make the foundation for finding new analgesic molecules or neuropharmacological research tools as well as the development of therapeutical treatments of conotoxin poisonings.Methods: the linear peptide toxins were synthesized using Fmoc-solid phase synthsis method.After the cleavage of peptide-resins and the following air oxidation of linear peptides,the aim conotoxins with two or three disulfide bridges were prepared.The effects of conotoxins on neuronal and muscular n ACh Rs were determined using two electrode voltage clamp method.The ?-GI-BSA hapten was prepared with the BSA coupled by the reaction product of ?-GI and glutaraldehyde,the other GI haptens were prepared by the Fmoc-protected GI coupled with multiple antigen peptide resins or pentaethylenehexmine.After the immunization of the GI-BSA hapten in mice,the antiserum was collected and its neutralization activity was determined.Results:(1)Ten A-superfamily conotoxins with four single cysteines and two M-superfamily conotoxins Mr6 and Eb5 with a new framework were prepared.(2)The effects of Bt14.12,Bt14.5,Bt22.1,Eb5 and Mr6 on the ?9?10 n ACh Rs were determined.The results showed that Bt 22.1 potently inhibited the ?9?10 n ACh Rs with the IC50 of 124.6 n M,while Bt14.12?Bt14.5?Eb5?Mr6 did not exhibit apparent inhibitory activity(IC50 > 10 ?M).Further investigation showed that Bt 22.1 had not significant inhibitory activity to the ?2?4,?3?2 and ?4?4(IC50>10 ?M).(3)Eb5 selectively inhibited the human muscular n ACh Rs with the IC50 of 124.6 n M,10 ?M of Mr6 also inhibited the human muscular n ACh Rs by 95%.(5)The SDS-PAGE protein electrophoresis showed that the coupling reaction of GI with BSA was successful.Each mouse was immunized four times with 99 ?g every two weeks.After the fourth immunization,the serum neutralization titer was more than 1:64000.After the intraperitoneal injection of the mixture of 100 ?l and 200 ?l of the antiserum and1×LD50 or higher dose of GI LD50 with,75% of mice survived in the group of 100 ?l of the antiserum and 1× LD50 GI(12.5 ?g/kg),75% of mice also survived in the group of 200 ?l of serum and 25.8 ?g/kg of GI.(5)The synthesis of the ?-GI-branched peptide and the coupling reaction of ?-GI with pentaethylenehexmine were also investigated.The results showed that the couping reaction products were complex,and the reaction conditions need to be improved.Conclusion: Bt22.1 specifically inhibited the neuronal ?9?10 n ACh Rs,Eb5 and Mr6 specifically inhibited muscular n ACh Rs.The two conotoxins contain novel Cys framework,our discovery on their targets will lay the foundation for the further studies of the function,modification and application.In addition,the antiserum produced by immunizing mice with GI-BSA hapten exhibits significant detoxication activity to conotoxin GI.