Cheng-Wu Chi

Characterization of novel M-superfamily conotoxins with new disulfide linkage

The M‐superfamily with the typical Cys framework (–CC–C–C–CC–) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M‐superfamily conotoxins can be provisionally categorized into four branches (M‐1, M‐2, M‐3, M‐4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176–8186]. Here we report the purification of seven M‐superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M‐1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M‐superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M‐1 and M‐2 and only one to M‐3. The disulfide analyses of two M‐1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1–C5, C2–C4, C3–C6) which is different from those of the M‐4 branch (C1–C4, C2–C5, C3–C6) and M‐2 branch (C1–C6, C2–C4, C3–C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

BmTx3, a scorpion toxin with two putative functional faces separately active on A-type K+ and HERG currents

A novel HERG channel blocker was isolated from the venom of the scorpion Buthus martensi Karsch, sequenced and characterized at the pharmacological level after chemical synthesis. According to the determined amino acid sequence, the cDNA and genomic genes were then cloned. The genomic gene consists of two exons interrupted by an intron of 65 bp at position -6 upstream from the mature toxin. The protein sequence of this toxin was completely identical with that of a known A-type K+ current blocker BmTx3, belonging to scorpion alpha-KTx subfamily 15. Thus BmTx3 is the first reported alpha-KTx peptide also showing HERG-blocking activity, like gamma-KTx peptides. Moreover, different from classical alpha-KTx peptides, such as charybdotoxin, BmTx3 cannot block Shaker -type K+ channels. Phylogenetic tree analysis reveals that this toxin takes an intermediate position between classical alpha-KTx and gamma-KTx toxins. From a structural point of view, we propose that two separate functional faces might exist on the BmTx3 molecule, responsible for the two different K+-current-blocking functions. Face A, composed of Arg18 and Lys19 in the alpha-helix side, might correspond to HERG blocking activity, whereas Face B, containing a putative functional dyad (Lys27 and Tyr36) in the beta-sheet side, might correspond to A-type blocking activity. A specific deletion mutant with the disrupted Face B, BmTx3-Y36P37del, loses the A-type current-blocking activity, but keeps a similar HERG-blocking activity, as seen with the wild-type toxin.

Purification and structural characterization of a d‐amino acid‐containing conopeptide, conomarphin, from Conus marmoreus

Cone snails, a group of gastropod animals that inhabit tropical seas, are capable of producing a mixture of peptide neurotoxins, namely conotoxins, for defense and predation. Conotoxins are mainly disulfide‐rich short peptides that act on different ion channels, neurotransmitter receptors, or transporters in the nervous system. They exhibit highly diverse compositions, structures, and biological functions. In this work, a novel Cys‐free 15‐residue conopeptide from Conus marmoreus was purified and designated as conomarphin. Conomarphin is unique because of its d‐configuration Phe at the third residue from the C‐terminus, which was identified using HPLC by comparing native conomarphin fragments and the corresponding synthetic peptides cleaved by different proteases. Surprisingly, the cDNA‐encoded precursor of conomarphin was found to share the conserved signal peptide with other M‐superfamily conotoxins, clearly indicating that conomarphin should belong to the M‐superfamily, although conomarphin shares no homology with other six‐Cys‐containing M‐superfamily conotoxins. Furthermore, NMR spectroscopy experiments established that conomarphin adopts a well‐defined structure in solution, with a tight loop in the middle of the peptide and a short 310‐helix at the C‐terminus. By contrast, no loop in l‐Phe13‐conomarphin was found, which suggests that d‐Phe13 is essential for the structure of conomarphin. In conclusion, conomarphin may represent a new conotoxin family, whose biological activity remains to be identified.

Critical effect of peptide cyclization on the potency of peptide inhibitors against Dengue virus NS2B-NS3 protease.

Dengue virus (DENV) infection is a serious public health threat worldwide that demands effective treatment. In the search for potent virus protease inhibitors, several cone snail venoms were screened against serotype 2 DENV NS2B-NS3 protease, and one conotoxin, MrIA, was identified to have inhibitory activity. The inhibitory activity was attributed to a disulfide bond-mediated loop, from which rational optimization was made to improve the potency and stability. An eight-residue cyclic peptide inhibitor was finally obtained with high potency (inhibitory constant 2.2 μM), stability, and cell permeability. This inhibitor can thus serve as a good lead for DENV drug development. In addition, this work highlights the critical effect of peptide cyclization on the potency of oligopeptide inhibitors against DENV protease, which may advance the design of peptide inhibitors for homologous virus proteases.

A Helical Conotoxin from Conus imperialis Has a Novel Cysteine Framework and Defines a New Superfamily

Background: Cone snail venoms are a rich source of novel peptide toxins. Results: Conus imperialis contains two new toxins that define a new cysteine framework and adopt an uncommon all-helical structure. Conclusion: im23a is a novel conotoxin with respect to amino acid sequence, cysteine framework, and three-dimensional structure. Significance: These new toxins extend the repertoire of novel peptides derived from cone snails. Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.

Solution Structure of BmBKTx1, a New BKCa1 Channel Blocker from the Chinese Scorpion Buthus martensi Karsch†,‡

BmBKTx1 is a 31-amino acid peptide identified from the venom of the Chinese scorpion Buthus martensi Karsch, blocking high-conductance calcium-activated potassium channels. Sequence homology analysis indicates that BmBKTx1 is a new subfamily of short-chain alpha-KTx toxins of the potassium channel, which we term alpha-KTx19. Synthetic BmBKTx1 was prepared by using solid-phase peptide synthesis. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmBKTx1. The results show that the BmBKTx1 forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins. The structure of BmBKTx1 consists of a two-stranded antiparallel beta-sheet (residues 20-29) and an alpha-helix (residues 5-15). The three-dimensional structure of BmBKTx1 was also compared with those of two function-related scorpion toxins, charybdotoxin (ChTx) and BmTx1, and their structural and functional implications are discussed.

Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels

The γ‐KTx–type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while α‐KTx3.2 Agitoxin‐2 binds to the pore region of the Shaker K+ channel, and α‐KTx5.3 BmP05 binds to the intermediate region of the small‐conductance calcium‐activated K‐channel (SKCa). In order to explore the critical residues for γ‐KTx binding, we determined the NMR structure of native γ‐KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruroïdes noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H‐NMR three‐dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3JNH‐Hα and 10 hydrogen bonds. The structure is characterized by 2 segments of α‐helices and a triple‐stranded antiparallel β‐sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed. Proteins 2004.

α4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors

Cone snails comprise approximately 700 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. alpha-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An alpha4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, alpha-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat alpha3beta2 and alpha6alpha3beta2 nAChRs. Comparing the distinct primary structure with other functionally related alpha-conotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.

Interaction of Mint3 with Furin regulates the localization of Furin in the trans-Golgi network

Furin is a proprotein convertase that cycles between the plasma membrane, endosomes and the trans-Golgi network (TGN), maintaining a predominant distribution in the latter. Mint3, a member of the Mint protein family, is involved in the signaling and trafficking of membrane proteins. Until now, little has been known about the roles of Mint3 in the localization or trafficking of Furin. Here, using co-immunoprecipitation and immunofluorescence assays, we show that Mint3 interacts with Furin in the Golgi compartment of HeLa cells. Knockdown of endogenous Mint3 expression by RNA interference disrupts the TGN-specific localization of Furin and increases its distribution in endosomes. We further demonstrate that the phosphotyrosine-binding (PTB) domain of Mint3 is essential for the binding of Furin and that this binding affects the TGN-specific localization of Furin. Moreover, mutation studies of Furin indicate that Mint3 regulates Furin distribution mainly through interaction with the acidic peptide signal of Furin. Collectively, these data suggest that the interaction between the PTB domain of Mint3 and the acidic peptide signal of Furin regulates the specific localization of Furin in the TGN.

Solution structure of an M-1 conotoxin with a novel disulfide linkage.

The M‐superfamily of conotoxins has a typical Cys framework (‐CC‐C‐C‐CC‐), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M‐superfamily family can be divided into four branches, namely M‐1, ‐2, ‐3 and ‐4. Recently, two M‐1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M‐2 and M‐4 branches. Here we report the 3D structure of mr3e determined by 2D 1H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six ϕ dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 ± 0.19 Å. Although mr3e has the same Cys arrangement as M‐2 and M‐4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a ‘flying bird’. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.