Chemical synthesis and functional characterization of LaIT3, an insecticidal two-domain peptide in Liocheles australasiae venom.

LaIT3, belonging to the ß-KTx family, is an insecticidal peptide in the venom of the Liocheles australasiae scorpion. Peptides in the family consist of two structural domains: an N-terminal domain with an α-helical structure common to antimicrobial peptides and a C-terminal domain with a structure stabilized by three disulfide bonds common to ion-channel blocking peptides. However, the contribution of each domain of LaIT3 to its activity remained unknown. In addition, some peptidic components are known to be enzymatically cleaved in the venom, which generates partial peptides. In our study, we searched for partial peptides of LaIT3 using LC/MS analysis and found peptides generated by cleavage at the central region of LaIT3. We subsequently synthesized full-length LaIT3 and its partial peptides to evaluate their insecticidal activity. The results, showing that only full-length LaIT3 is active, indicate that the insecticidal activity of LaIT3 depends on the presence of both N-terminal and C-terminal domains. Furthermore, LaIT3 did not exhibit the cytolytic activity against insect cells and showed only weak antibacterial activity. These findings suggest that its action is not due to a simple membrane disruption effect but instead due to actions on specific target molecules, including ion channels.

Isolation and characterization of the insecticidal, two-domain toxin LaIT3 from the Liocheles australasiae scorpion venom.

A novel insecticidal peptide (LaIT3) was isolated from the Liocheles australasiae venom. The primary structure of LaIT3 was determined by a combination of Edman degradation and MS/MS de novo sequencing analysis. Discrimination between Leu and Ile in MS/MS analysis was achieved based on the difference in side chain fragmentation assisted by chemical derivatization. LaIT3 was determined to be an 84-residue peptide with three intrachain disulfide bonds. The sequence similarity search revealed that LaIT3 belongs to the scorpine-like peptides consisting of two structural domains: an N-terminal α-helical domain and a C-terminal cystine-stabilized domain. As observed for most of the scorpine-like peptides, LaIT3 showed significant antibacterial activity against Escherichia coli, which is likely to be caused by its membrane-disrupting property.

Chemical synthesis of a two-domain scorpion toxin LaIT2 and its single-domain analogs to elucidate structural factors important for insecticidal and antimicrobial activities.

Scorpion venom contains various bioactive peptides. Among them, peptides having two different structural domains constitute a toxin family known as ß-KTx or scorpine-like peptides. These peptides consist of an α-helical structure in the N-terminal region and a cysteine-stabilized structure in the C-terminal region. This unique structure of ß-KTx peptides contributes to their diverse biological functions, but the importance of each domain for their activities is not fully understood. LaIT2 is a ß-KTx peptide isolated from the venom of the scorpion Liocheles australasiae, which shows both insecticidal and antimicrobial activities. In this study, we chemically synthesized full-length LaIT2 using a native chemical ligation technique as well as its N-terminally or C-terminally truncated single-domain analogs to evaluate structural factors important for the activities. Biological evaluation of these peptides revealed that the N-terminal α-helical domain of LaIT2 is essential for the expression of both insecticidal and antibacterial activities. This suggests that the disruption of membrane structures largely accounts for the biological activities of LaIT2.

A Facile Method for Preferential Modification of the N-Terminal Amino Group of Peptides Using Triazine-Based Coupling Reagents.

It has been shown that chemical modification of the peptide N-terminus with a charged tag greatly affects the fragmentation process caused by collision-induced dissociation to obtain more interpretable product ion spectra. In this study, we examined the selective introduction of a charged tag, 4-(guanidinomethyl)benzoic acid (Gmb), into the peptide N-terminus. After optimization of the reaction conditions, we found that the most effective conversion in terms of the reaction rate and selectivity was achieved by reacting the peptide with the active ester of Gmb, prepared using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) at pH 7. This method is applicable to the introduction of various carboxylic acid-containing compounds into the N-terminus of peptides, which will be useful not only for improvement of MS/MS fragmentation but also for various biochemical studies of peptides and proteins.

Isolation, structural identification and biological characterization of two conopeptides from the Conus pennaceus venom.

A novel anti-mollusk conopeptide pn4c was isolated from the Conus pennaceus venom by repeated HPLC fractionation based on the activity against freshwater snails. The primary structure of pn4c was determined by the mass spectrometric de novo sequencing analysis. In addition, pn3a was isolated from the same fraction containing pn4c, as a peptide with unknown functions.

Characterization of the venom of the vermivorous cone snail Conus fulgetrum.

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.