Precursor Conotoxin Sequences From Conus Achatinus And Conus Monile
Dewan, Kalyan Kumar
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The numerous toxic peptides, called conotoxins (Olivera et al 1990, 1991), that marine cone snails produce and use for capturing prey, deterring predators and presumably for other biotic interactions, are known to target several classes of mammalian voltage and ligand gated receptors with excellent specificity and good receptor-subtype discrimination (Terlau and Olivera 2004). This fine specificity has placed upon the conotoxins a great deal of scientific and medical interest, and cone snail venom is currently considered a vast natural resource of peptides that have the potential of eventually benefiting the prognosis of many human afflictions, either directly as therapeutics or indirectly as research tools. In this regard, the characterization of conotoxins and the identification of the specific receptors they target remains an actively pursued area of study in several countries. There has been an active effort to characterize peptides from Indian populations of cone snails that has resulted in several reports describing novel peptides from them. This thesis is part of these ongoing efforts and largely relates to the isolation and identification of cDNA sequences of precursor conotoxins from cone snails prospected in India. One final section of the thesis is concerned with the assessment of secondary structure predictions of conotoxin genes and discusses how such formations may determine the regions where variations and conservations are taking place among the conotoxins. Structure and Outline of the thesis Chapter 1 is an overview of the cone snails and the conotoxins that they produce. There is some emphasis on the variability and diversity that are found among conotoxins since these are some of the aspects that are discussed in subsequent chapters. Chapter 2 describes the identification of new cDNA sequences isolated from the relatively rare Indian population of the piscivorous cone snail, Conus achatinus. This species was chosen for the study since it is one of the few piscivorous cone species that have been reported from the coastlines of India. However, the limited availability of specimens belonging to this species precluded a direct study of individual venom peptides from it. To overcome this bottleneck of specimen availability, a molecular biological route to obtain cDNA sequences of superfamily specific conotoxins was considered. The steps of PCR mediated amplification and cloning that are incorporated within the procedures of preparing cDNA for sequencing, to some extent overcome the limitations of large sample requirements and in this respect have been used to investigate conotoxin sequences from this species. Using the cDNA route and comparative sequence analysis, it has been possible to identify 5 novel O-superfamily conotoxin sequences from C.achatinus. The precursor sequences have been classified as delta, omega and omega-like conotoxins that potentially target voltage gated sodium and calcium channels. A parallel study trying to detect the specific cDNA related conotoxins using mass spectrometry (MALDI-MS) as a screening tool is also discussed. In this search it has been possible to detect 3 of the 5 cDNA related peptides using small amounts of unpurified venom. The cDNA and mass spectrometric results show that precursor sequences of conotoxins from a relatively rare population of Conus species can be successfully identified and the existence of the peptides that they specify verified through the combined approach of obtaining cDNA sequences and MALDI-MS screening of total unpurified venom. Chapter 3 similarly relates to the isolation of cDNA sequences from a single specimen of the vermivorous cone snail, Conus monile. Three precursor sequences, Mo3.1, Mo15.1 and Mo16.1, of M-superfamily conotoxins have been isolated from this species out of which two precursors (Mo15.1 and Mo16.1) show unexpected cysteine distribution patterns within their putative mature toxin regions. In addition, several other features of these precursors do not fit into the description of known M-superfamily conotoxins. The sequence analyses and the deviations that have been noted among these sequences are described. Chapter 4 describes the efforts that have gone in towards detecting one of these deviant conotoxins (Mo16.1) in the venom of Conus monile using MALD-MS as a means for screening the venom. A peptide having the Mo16.1 predicted mass (1512 Da) has been detected as a minor component of the venom. The chapter describes additional mass-spectrometric experiments that strongly support the assignment of this detected peak being the specific Mo16.1 peptide. A speculative discussion on the role of minor peptide deviants such as the Mo16.1 peptide concludes the chapter. Chapter 5 is concerned with the assessment of secondary structure predictions of conotoxin genes and discusses how such formations may determine the regions where variations and conservations are taking place among the conotoxins. The comparative analysis of DNA sequences corresponding to the variable mature conotoxins reveal that it is possible to differentiate mature conotoxin sequences into variable and conserved regions. Using the prediction program mFold (Markham and Zuker 2005) it has been noted that regions of the DNA encompassing the conserved codons (including the highly conserved cysteine codons) correspond to predicted secondary structures of higher stabilities. In contrast the regions of the conotoxin that have a higher degree of variation correlate to regions of lower stability. These correlations have been observed quite consistently across several classes of conotoxins that show different patterns of variability and conservation in their sequence and representing different categories of conotoxins i.e intra species, inter species, and hyperconserved conotoxins. The observation on these co-relations allows for a simple model of inaccessibility of a mutator to these relatively structured regions of the conotoxin gene (including the cysteine codons) allowing them a relative degree of resistance towards change. Chapter 6 summarizes the findings of the thesis, briefly recapitulating the discussion of the individual chapters from a broader perspective.
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