Junliang Liu

Diversity and evolution of conotoxins based on gene expression profiling of Conus litteratus

Cone snails are attracting increasing scientific attention due to their unprecedented diversity of invaluable channel-targeted peptides. As arguably the largest and most successful evolutionary genus of invertebrates, Conus also may become the model system to study the evolution of multigene families and biodiversity. Here, a set of 897 expressed sequence tags (ESTs) derived from a Conus litteratus venom duct was analyzed to illuminate the diversity and evolution mechanism of conotoxins. Nearly half of these ESTs represent the coding sequences of conotoxins, which were grouped into 42 novel conotoxin cDNA sequences (seven superfamilies), with T-superfamily conotoxins being the dominant component. The gene expression profile of conotoxin revealed that transcripts are expressed with order-of-magnitude differences, sequence divergence within a superfamily increases from the N to the C terminus of the open reading frame, and even multiple scaffold-different mature peptides exist in a conotoxin gene superfamily. Most excitingly, we identified a novel conotoxin superfamily and three novel cysteine scaffolds. These results give an initial insight into the C. litteratus transcriptome that will contribute to a better understanding of conotoxin evolution and the study of the cone snail genome in the near future.

Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern

Conus litteratus is a worm-hunting cone snail with a highly sophisticated neuropharmacological defense strategy using small peptides in its venom. By analyzing different clones in the cDNA library of venom ducts from C. litteratus, we identified the peptide lt14a which displays a characteristic signal peptide sequence in its precursor and a unique arrangement of Cys residues (-C-C-C-C-) in its mature peptide region. RT-PCR analysis suggested that lt14a is abundantly expressed throughout the whole venom duct. An intensive analysis in sequence suggested that lt14a is similar to alpha-conotoxin qc1.1 cloned from Conus quercinus. We conducted the chemical synthesis of lt14a. The synthetic lt14a has a remarkable biological activity to suppress pain and inhibits the neuronal-type nicotinic acetylcholine receptors.

Identification of a novel M-superfamily conotoxin with the ability to enhance tetrodotoxin sensitive sodium currents

In this work, a novel M-superfamily conotoxin, designated lt3a, was purified from the crude venom of Conus litteratus. Combined with peptide sequencing, MALDI-TOF mass spectrometry and cDNA cloning techniques, the amino acid sequence of lt3a was supposed to be DγCCγ OQWCDGACDCCS, where O is hydroxyproline and γ is carboxyglutamate. The Cys framework of lt3a (–CC–C–C–CC–) is similar to that of ψ-, μ-, κM-conotoxins, which are representatives of M-conotoxins. Peptide lt3a is categorized into M1 branch based on the number of residues in the last Cys loop. Whole cell patch-clamp study on adult rat dorsal root ganglion neurons indicated that lt3a could enhance tetrodotoxin-sensitive sodium currents. This is a previously unknown function of M-superfamily conotoxins.

Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels.

A T-1-conotoxin, lt5d, was purified and characterized from the venom of vermivorous hunting cone snails Conus litteratus. The complete amino acid sequence of lt5d (DCCPAKLLCCNP) has been determined by Edman degradation. With two disulfide bonds, the calculated average mass is 1274.57 Da, which is confirmed by MALDI-TOF mass spectrometry (average mass 1274.8778). Under whole cell patch-clamp mode, lt5d inhibits tetrodotoxin-sensitive sodium currents on adult rat dorsal root ganglion neurons, but has no effects on tetrodotoxin-resistant sodium currents. The inhibition of TTX-sensitive sodium currents by lt5d was found to be concentration-dependent with the IC(50) value of 156.16 nM. Thus, this is the first T-superfamily conotoxin identified to block TTX-sensitive sodium channels.

Identification and characterization of a novel O‐superfamily conotoxin from Conus litteratus

A novel conotoxin named lt6c, an O‐superfamily conotoxin, was identified from the cDNA library of venom duct of Conus litteratus. The full‐length cDNA contains an open reading frame encoding a predicted 22‐residue signal peptide, a 22‐residue proregion and a mature peptide of 28 amino acids. The signal peptide sequence of lt6c is highly conserved in O‐superfamily conotoxins and the mature peptide consists of six cysteines arranged in the pattern of CCCCCC that is defined the O‐superfamily of conotoxins. The mature peptide fused with thioredoxin, 6‐His tag, and a Factor Xa cleavage site was successfully expressed in Escherichia coli. About 12 mg lt6c was purified from 1L culture. Under whole‐cell patch‐clamp mode, lt6c inhibited sodium currents on adult rat dorsal root ganglion neurons. Therefore, lt6c is a novel O‐superfamily conotoxin that is able to block sodium channels. Copyright

Soluble expression and sodium channel activity of lt16a, a novel framework XVI conotoxin from the M-superfamily.

A peptide toxin, lt16a, from the venom of the worm-hunting Conus litteratus, shares the typical signal peptide sequences of M-superfamily conotoxins, which usually contain six cysteine residues that are arranged in a CC-C-C-CC pattern. Interestingly, lt16a comprises 21 amino acid residues in its mature region and has a cysteine framework XVI, which is arranged in a C-C-CC pattern. The coding region of lt16a was cloned into the pTRX vector and the fusion protein was overexpressed in Escherichia coli. After cleaving the fusion protein and purifying the protein lt16a using chromatography, the mass of lt16a was found by mass spectrometry to be consistent with the expected mass of 2357.7 Da. Whole-cell patch clamp experiments demonstrated that lt16a could inhibit both the TTX-sensitive and TTX-resistant sodium currents in adult rat dorsal root ganglion neurons. The inhibition of lt16a on TTX-resistant sodium currents was stronger than on TTX-sensitive sodium currents. To our knowledge, this is the first report of a framework XVI conotoxin that can inhibit voltage-gated sodium channel currents in mammalian sensory neurons. This report helps facilitates an understanding of the sequence diversity of conotoxins.

Discovery of two P-superfamily conotoxins, lt9a and lt9b, with different modifications on voltage-sensitive sodium channels

ABSTRACT In this work, two P‐superfamily conotoxins, lt9a and lt9b, were purified and characterized from the crude venom of Conus litteratus. The amino acid sequences of lt9a and lt9b were determined by the Edman degradation method. It has been suggested that both lt9a and lt9b are produced from the precursor encoded by the gene Lt9.1. During the conotoxin maturation process, different post‐translational modifications occurred between lt9a and lt9b. Conotoxin lt9b was predicted to have two prolines that underwent hydroxylation and one glutamate that underwent carboxylation, while lt9a had no hydroxyproline and carboxyglutamate residue. The calculated mass weights of two P‐superfamily conotoxins with three proposed disulfide bonds were confirmed by matrix‐assisted laser desorption/ionization–time‐of‐flight (MALDI‐TOF) mass spectrometry after considering corresponding post‐translational modifications. These two conotoxins showed different effects on tetrodotoxin‐sensitive sodium currents. Conotoxin lt9a (300 nM) resulted in marked slowing of the tetrodotoxin‐sensitive sodium current decay, a notable increase in the peak current, and an alteration in reversal potential. However, lt9b inhibits tetrodotoxin‐sensitive sodium currents, and the inhibition showed a concentration‐dependent with the half maximal inhibitory concentration (IC50) value of 504.04 nM, but there were no change in the activation and inactivation kinetics of currents. To the best of our knowledge, this is the first investigation of two P‐superfamily conotoxins identified to act on voltage‐sensitive sodium channels with different modifications. HIGHLIGHTSConotoxins lt9a and lt9b were identified from the venom of Conus litteratus.Lt9b is the natural mutant of lt9a with posttranslational modifications.Lt9a and lt9b show different modifications on TTX‐S sodium currents.