Cloning And Phylogenetic Analysis Of Novel Conotoxins From The South China Sea

Conotoxins are neurophysiologically active peptides isolated from venomousmarine cone snails. Most conotoxins contain12-46amino acids with rich disulfidebonds.In comparison with other animal toxins, conotoxins have smaller molecularweight, better structural stability, high activity and high selectivity, and could beeasily synthesized. They can specifically act on calcium, sodium, potassium and otherion channels as well as a variety of neurotransmitter receptor subtypes (nicotinicacetylcholine receptors and NMDA receptors, etc). Conotoxins have become apowerful tool of pharmacology and neuroscience and treasury of drug development.Object：Though cone snails are rich in our country, a great quantity of conotoxinshave not been cloned and elucidated. This study aims to isolate and clone newconotoxins with novel amino acid alignments, disulfide bridge arrangements andpost-translational modifications. This will be very important for the discovery of newfunctional conotoxins and related drug development.Method：Using highly conserved signal sequences of conotoxins, the sequences ofnovel conotoxins were cloned by3’-rapid amplification of cDNA ends (RACE) andNest PCR method from seven Conus species from the South China Sea. Then weanalyzed the sequence and phylogenetic tree of these conotoxins. The completecDNA sequences of the conotoxins with a novel cysteine framework were cloned by5’end amplification.Results:1. We obtained60novel conotoxins and29known conotoxins from more than800positive clones. These novel conotoxins covered A, M, O and J superfamilyconotoxins including2of A superfamily conotoxins,11of J superfamilyconotoxins,10of M-superfamily conotoxins,21of O-superfamily conotoxins.10novel superfamily conotoxins were also cloned, such as S, I, conomarphin andcontryphan conotoxins.In addition, two new cysteine frameworks (XXIV and XXV) were found.2. A conotoxin with20cysteines was found by3’ and5’ RACE methed. This is thefirst report on the conotoxin with20cyeteines.3. Some novel conotoxins are significantly different from the reported conotoxins. Forexample, two α4/7type A-superfamily conotoxins contain the V and I amino acidsin the second and third cystine, some M-superfamily conotoxins with less or nocysteine framework were found.19novel O-superfamily conotoxins are dividedinto three families, and the cysteine framework is the CC-CC-CC; In the11novelJ-superfamily conotoxins, a novel cysteine framework “CX3CX9CXC” was foundbesides the framework “CX3CX8CXC” and “CX3CX10CXC”.4. The analysis of M, O and J-superfamily conotoxin phylogenetic tree showed thatthese novel toxins located in the different branch. The new M-superfamilyconotoxins are low in homology and scattered in different branches of thephylogenetic tree for the different or same species. In addition, O3familyconotoxins are in a small branch, the rest of the new toxins are low homology andlocated in different branches of the phylogenetic tree. Novel J-superfamilyconotoxins exhibit less homology to the reported except Di14.1and Mr14.2, andlocated in branch II in the evolutionary tree.Conclusion：1. We have got60novel conotoxins and29known conotoxins including A, M, O, Jsuperfamily conotoxins and so on.2. Two novel frameworks of conotoxins were found and were named as frameworkXXIV and framework XXV.3. Conotoxins with20cysteines were firstly found.