Conantokins derived from the Asprella clade impart conRl-B, an N-methyl d-aspartate receptor antagonist with a unique selectivity profile for NR2B subunits

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ω-conotoxin GVIA and its selenocysteine analogs

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.     

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels

Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.     

Characterization of (Boc‐Cys/Sec‐NHMe)2 and (Boc‐Cys/Sec‐OMe)2: Evidence of local conformational difference between disulfide and diselenide

Conformations of disulfide and diselenide were compared in (Boc‐Cys/Sec‐NHMe)₂ and (Boc‐Cys/Sec‐OMe)₂ using X‐ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, density functional theory (DFT), and circular dichroism (CD) spectroscopy. Conformations of disulfide/diselenide in polypeptides are defined based on the sign of side chain torsion angle χ₃ (–CH₂–S/Se–S/Se–CH₂–); negative indicates left‐handed and positive indicates right‐handed orientation. In the crystals of (Boc‐Cys‐OMe)₂ and (Boc‐Sec‐OMe)₂, the disulfide exhibits a left‐handed and the diselenide a right‐handed orientation. Characterization of cystine and selenocystine derivatives in solution using ¹H‐NMR, natural abundant ⁷⁷Se NMR, 2D‐ROESY, and chemical shift analysis coupled to DMSO titration has indicated the symmetrical nature and antiparallel orientation of Cys/Sec residues about the disulfide/diselenide bridges. Structural calculations of cystine and selenocystine derivatives using DFT further support the antiparallel orientation of Cys/Sec residues about disulfide/diselenide. The far‐ultraviolet (UV) region CD spectra of cystine and selenocystine derivatives have exhibited the negative Cotton effect (CE) for disulfide and positive for diselenide confirming the difference in the conformational preference of disulfide and diselenide. In the previously reported polymorphic structure of (Boc‐Sec‐OMe)₂, the diselenide has right‐handed orientation. In the X‐ray structures of disulfide and diselenide analogues of Escherichia coli protein encoded by curli specific gene C (CgsC) retrieved from Protein Databank (PDB), disulfide has left‐handed and the diselenide right‐handed orientation. The current report provides the evidence for the local conformational difference between a disulfide and a diselenide group under unconstrained conditions, which may be useful for the rational replacement of disulfide by diselenide in polypeptide chains.     

Characterization of conantokin Rl‐A: molecular phylogeny as structure/function study

A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl‐A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl‐A is a 24‐residue peptide containing three γ‐carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl‐A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl‐A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca⁺⁺. The order of potency for blocking the four NMDA receptor subtypes by ConRl‐A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl‐A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Dietary fibers as emerging nutritional factors against diabetes: focus on the involvement of gut microbiota

Diabetes mellitus (DM) increases the risk of cardiovascular diseases and other secondary complications, such as nephropathy, neuropathy, retinopathy, etc. The important risk factors for the pathogenesis of DM are aging, family history, sedentary lifestyle, unhealthy dietary habits, and obesity. Evidence from epidemiological studies also indicates that DM is characterized by specific alterations in the human gut microbiota (GM). GM transplantation in rodents and humans revealed that a specific GM constituent can be the cause and not just the consequence of the DM condition and complications. These findings suggest a potential role of GM in human health, disease prevention, and treatment. Dietary intervention studies using dietary fibers (DFs) suggested that modulation of the GM can suppress the metabolic risk markers in humans. However, a causal role of GM in such studies remains unexplored. Long-term follow-up studies disclosed that the diet rich in insoluble and non-viscous fibers are responsible for DF-mediated antidiabetic activities, while soluble and viscous fibers have little influence on DM despite having a profound impact on glycemia. However, general conclusions cannot be drawn simply based on these findings. Long-term follow-up studies are urgently required in this area to explore the therapeutic potential of different DFs in treating DM and to delineate the exact role of GM involvement. Here we review and discuss the signature of GM during DM, antidiabetic activity of metformin via GM modulation, DFs from different sources and their antidiabetic activity, and the possible role of GM involvement.