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αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABA receptors (GABAR) inhibiting HVA N-type calcium (Cav2.2) channels and reducing membrane excitability in mouse DRG neurons. The inhibition of HVA Ba currents and neuroexcitability by GeXIVA[1,3] was partially reversed by the selective GABAR antagonist CGP 55845. In transfected HEK293T cells co-expressing human GABAR1 and R2 subunits and Cav2.2 channels, both GeXIVA[1,3] and GeXIVA[1,4] inhibited depolarization-activated Ba currents mediated by Cav2.2 channels, whereas GeXIVA[1,2] had no effect. The effects of three cyclized GeXIVA[1,4] ribbon isomers were also tested, with cGeXIVA GAG being the most potent at human GABAR-coupled Cav2.2 channels. Interestingly, globular GeXIVA[1,3] also reversibly potentiated inwardly-rectifying K currents mediated by human GIRK1/2 channels co-expressed with GABAR in HEK293T cells. This study highlights GABAR as a potentially important receptor target for the activity of αO-conotoxin GeXIVA to mediate analgesia. (Figure presented.).

Voltage-activated potassium (Kv) channels open in response to membrane depolarization and subsequently inactivate through distinct mechanisms. For the model Shaker Kv channel from Drosophila, fast N-type inactivation is thought to occur by a mechanism involving blockade of the internal pore by the N-terminus, whereas slow C-type inactivation results from conformational changes in the ion selectivity filter in the external pore. Kv channel inactivation plays critical roles in shaping the action potential and regulating firing frequency, and has been implicated in a range of diseases including episodic ataxia and arrhythmias. Although structures of the closely related Shaker and Kv1.2 channels containing mutations that promote slow inactivation both support a mechanism involving dilation of the outer selectivity filter, mutations in the outer pores of these two Kv channels have been reported to have markedly distinct effects on slow inactivation, raising questions about the extent to which slow inactivation is related in both channels. In this study, we characterized the influence of a series of mutations within the external pore of Shaker and Kv1.2 channels and observed many distinct mutant phenotypes. We find that mutations at four positions near the selectivity filter promote inactivation less dramatically in Kv1.2 when compared to Shaker, and they identify one key variable position (T449 in Shaker and V381 in Kv1.2) underlying the different phenotypes in the two channels. Collectively, our results suggest that Kv1.2 is less prone to inactivate compared to Shaker, yet support a common mechanism of inactivation in the two channels.

αO-Conotoxin GeXIVA is a 28 amino acid peptide derived from the venom of the marine snail Conus generalis. The presence of four cysteine residues in the structure of GeXIVA allows it to have three different disulfide isomers, that is, the globular, ribbon or bead isomer. All three isomers are active at α9α10 nicotinic acetylcholine receptors, with the bead isomer, GeXIVA[1,2], being the most potent and exhibiting analgesic activity in animal models of neuropathic pain. The original report of GeXIVA activity failed to observe any effect of the isomers on high voltage-activated (HVA) calcium channel currents in rat dorsal root ganglion (DRG) neurons. In this study, we report, for the first time, the activity of globular GeXIVA[1,3] at G protein-coupled GABA

The ribbon isomer of α-conotoxin AuIB has 10-fold greater potency than the wild-type globular isomer at inhibiting nicotinic acetylcholine receptors (nAChRs) in rat parasympathetic neurons, and unlike its globular isoform, ribbon AuIB only targets a specific stoichiometry of the α3β4 nAChR subtype. Previous electrophysiological recordings of AuIB indicated that ribbon AuIB binds to the α3(+)α3(−) interface within the nAChR extracellular domain, which is displayed by the (α3)3(β4)2 stoichiometry but not by (α3)2(β4)3. This specificity for a particular stoichiometry is remarkable and suggests that ribbon isoforms of α-conotoxins might have great potential in drug design. In this study, we investigated the binding mode and structure-activity relationships of ribbon AuIB using a combination of molecular modeling and electrophysiology recording to determine the features that underpin its selectivity. An alanine scan showed that positions 4 and 9 of ribbon AuIB are the main determinants of the interaction with (α3)3(β4)2 nAChR. Our computational models indicate that the first loop of ribbon AuIB binds in the “aromatic box” of the acetylcholine orthosteric binding site, similar to that of globular AuIB. In contrast, the second loop and the termini of the ribbon isomer have different orientations and interactions in the binding sites to those of the globular isomer. The structure-activity relationships reported herein should be useful to design peptides displaying a ribbon α-conotoxin scaffold for inhibition of nAChR subtypes that have hitherto been difficult to selectively target.

This PhD project focused on investigating the structure-activity relationships (SARs) of the ribbon isomer of a-conotoxins, providing the basis for using a-conotoxin ribbon isomers as scaffolds in drug design to target nicotinic acetylcholine receptors (nAChRs). Chaptern1 provides an introduction to the structure and function of nAChRs and to a-conotoxins, as well as introducing the concept of disulfide isomers of a-conotoxins. Chapters 2 to 5 describe the engineering of ribbon isomers of a-conotoxins for drug design application, as summarised below.Chapter 2 describes a strategy to improve the yield of synthesis of a ribbon conotoxin, enabling the creation of mutants for SAR studies. The three-disulfide isomers of aO-conotoxin GeXIVA, i.e. the globular, ribbon and beads isomers, have similar potency at the a9a10 nAChR, which is considered to be a pain target. Owing to its potent activity, GeXIVA prompts further studies to investigate its analgesic potential. Unfortunately, the directed folding using orthogonal protection of cysteine residues is difficult for GeXIVA because it has a long sequence and has poor assembly yields arising from numerous Arg residues. Chapter 2 of this thesis shows that the folding of the ribbon GeXIVA could be achieved directly using rationally designed backbone cyclising linkers instead of using an orthogonal protection strategy. Additionally, backbone cyclisation of the ribbon GeXIVA was shown to improve peptide stability in human serum and have comparable activity as the parent peptide.Chapter 3 focuses on the determination of the binding mode of an a-conotoxin, AuIB, which is an essential step for structure-based design of ribbon a-conotoxins. The ribbon isomer of a-conotoxin AuIB has 10-fold higher potency than the wild-type globular isomer at inhibiting nAChRs in rat parasympathetic neurons, and unlike its globular isoform, ribbon AuIB targets a specific stoichiometry of the a3b4 nAChR subtype. This specificity for a particular stoichiometry is remarkable and suggests that the ribbon isoform of a-conotoxins has potential applications in drug design. In Chapter 3, the binding mode and SAR of ribbon AuIB were investigated to determine the features that underpin its specific activity using a combination of molecular modelling and electrophysiology recording. An alanine scan showed that positions 4 and 9 of ribbon AuIB are the main determinants of the interactions with rat (a3)3(b4)2 nAChR. Computational models indicated that the first loop of ribbon AuIB bound in the laromatic boxr of the acetylcholine orthosteric binding site. In contrast, the second loop and the termini of the ribbon isomer had different orientations and interactions in the binding sites than those of the globular isomer.In Chapter 4, several computation methods for predicting mutational energies of a-conotoxins were compared, and the optimal method could be used for rational computational design. Binding free energy predictions are potentially an important tool for designing selective inhibitors based on globular and ribbon a-conotoxins but these computational methods need to be benchmarked to assess their accuracy in the context of nAChR/conotoxin systems. Computational free energy predictions are especially sensitive to the accuracy of the structural models; ultimately they should be used with high-resolution experimental structures. Unfortunately, there is so far no crystal structure of nAChR in complex with a-conotoxin. By contrast, the acetylcholine binding proteins (AChBPs), which are structurally homologous to the extracellular domains (ECDs) of the nAChRs, can be relatively easily crystallised and studied using X-ray crystallography. The crystal structures of AChBP/a-conotoxin complexes provide important information on the binding mode of the a-conotoxin. In Chapter 4, we evaluated four mutational energy prediction methods (BeAtMuSic, Foldx, MMPBSA/MMGBSA, and coarse-grained umbrella sampling) using the crystal structure of the complex between AChBP and a-conotoxin LsIA and between AChBP and LvIA, and associated experimental affinity change of these complexes after mutations. Foldx was identified as the most reliable method for mutational energy prediction. Notably, this method was successful at predicting variations with increased or decreased affinities.Chapter 5 combined results from the two previous chapters to the study of the binding mode of the a-conotoxin GID with the a4b2 nAChR. The a4b2 nAChR is linked to a range of diseases and disorders including nicotine addiction, epilepsy, and Parkinsonrs and Alzheimerrs diseases. Designing a4b2 nAChR selective inhibitors could help refine the role of a4b2 nAChR in disease states. In Chapter 5, we aimed to modify globular and ribbon a-conotoxin GID to selectively target the a4b2 nAChR through competitive inhibition of the a4(+)b2(-) or a4(+)a4(-) interfaces. The binding modes of the globular a-conotoxin [Gla4E]GID with rat a3b2, a4b2 and a7 nAChRs were built using computational methods, and they were validated using published experimental data. The binding mode of globular [Gla4E]GID at a4b2 nAChR can rationally explain the experimental mutagenesis data, suggesting that it could be used to design GID variants rationally. The predicted mutational energy results showed that globular [Gla4E]GID seemed to be already optimal for binding to a4b2 nAChR and its activity cannot further be improved through amino-acid substitutions. The cryo-electron microscopy structure of (a4)3(b2)2 nAChR has been recently released, providing an optimal template to build the binding mode of the ribbon GID with (a4)3(b2)2 nAChR using information on the binding mode of ribbon AuIB studied in Chapter 3. The Foldx predicted the mutational energies of ribbon [Gla4E]GID at a4(+)a4(-) interface, and a number of ribbon [Gla4E]GID mutants were suggested to have desirable properties to inhibit (a4)3(b2)2 nAChR.Chapter 6 provides an overview of my findings and highlights the major conclusions of the thesis, as well as suggesting ideas for future studies in this field. In summary, my PhD thesis has shown that ribbon a-conotoxins can be used in the design of specific nAChR inhibitors.

Conotoxins are disulfide-rich peptides found in the venoms of marine snails of the genus Conus. They have attracted great attention from the pharmaceutical industry because of their potential uses as drug leads, but like most peptides, conotoxins are susceptible to proteolysis and typically are not orally bioavailable. Here we discuss approaches that have been used to stabilise conotoxins to improve their potential pharmaceutical use. Specifically, we focus on the use of backbone cyclisation to improve their stability in biological fluids. The Microreview provides an introduction to the various classes of conotoxins, including their frameworks (cysteine patterns) and a background on the receptors that they interact with, as well as an analysis of the binding interactions between conotoxins and their receptors.

Summary Compositions and antioxidant properties of protein hydrolysates prepared from four carp skins: black carp, grass carp, silver carp and bighead carp, using pepsin, with a degree of hydrolysis (DH) of 6–15%, were investigated. The yield of freeze-dried hydrolysates was in the range of 54–62 g/100 g (dry skin). The content of protein and ash in four freeze-dried hydrolysates was 72–81% and 8–17%, respectively. All hydrolysates contained high amount of hydrophobic amino acid residues (389–480 residues/1000 residues). Meanwhile, their antioxidant properties were evaluated by in vitro assays. The results revealed that all hydrolysates possessed potent antioxidant activities and showed dose dependency as the activity increased with sample concentration, capable of scavenging 72–88% of DPPH and 61–69% of hydroxyl radicals, respectively, at the highest tested concentration. The hydrolysates exhibited high reducing power and β-carotene–linoleic acid oxidation inhibition. Among the four hydrolysates, the hydrolysate derived from bighead carp skin was superior to others in terms of yield, DH and antioxidant activities.

BACKGROUND Collagen has a wide range of applications in food, biomedical and pharmaceutical products. RESULTS The collagens in grass carp (Ctenopharyngodon idella) skin and swim bladder were extracted using acetic acid and pepsin. Higher yield of pepsin-soluble collagen (PSC) was obtained from skin (178 g kg−1) than from swim bladder (114 g kg−1). Not surprisingly, yields of PSC from skin and swim bladder were also higher than those of acid-soluble collagen (ASC) from the same organs (89 and 51 g kg−1). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that ASC and PSC were type I collagens, with PSC containing a higher proportion of α components than ASC. Fourier transform infrared spectra revealed that ASC and PSC were very similar in their protein secondary structures. Scanning electron micrographs showed that the collagens had a spongy structure, with more pores being obtained in swim bladder than in skin collagens. The collagens showed high solubility in the acidic pH range. However, their solubility decreased in the presence of NaCl at concentrations above 20 g kg−1. CONCLUSION Collagens were successfully extracted from the skin and swim bladder of grass carp. These fish by-products could serve as an alternative source of collagens for a wide variety of applications in the food and nutraceutical industries. © 2015 Society of Chemical Industry

Antimicrobial activity of essential oils is widely known, but their application to fish preservation is already limited. The objective was to evaluate the microbiological characteristics and quality changes in turbot (Scophthalmus maximus) fillets with essential oil fumigation treatments over 20 days stored at 2 ± 1 °C. The turbot fillets were fumigated with a series of concentrations (1, 4, and 8 μL/L) of essential oils of clove, cumin, and spearmint. Changes in the color, texture profile analysis (TPA), peroxide value (PV), 2-thiobarbituric acid (TBA), total volatile basic nitrogen (TVB-N), trimethylamine nitrogen (TMA-N), microbial characteristics (total viable count, psychrotrophic count, pseudomonads, Shewanella putrefaciens, Enterobacteriaceae, and lactic acid bacteria count) were measured. The results showed the turbot fillets from the control group were the first (day 10) to indicate signs of degradation reaching rejection threshold values for all evaluated parameters. All essential oils could inhibit the oxidation of turbot fillets, and the most effective essential oil was spearmint oil. Fumigated with 4 μL/L spearmint oil maintained the color and texture, retarded the lipid and protein oxidation, and reduced the microorganism counts. Therefore, post-mortem essential oil fumigation treatment has positive effects on improving the quality of refrigerated turbot fillets.

In order to establish an effective freezing method for quality control, the present research evaluated the effects of the different freezing treatments on the quality of Japanese sea bass ( Lateolabrax japonicas ) over a period of 20 days storage at 0 ± 1 °C. Fish pH value, total volatile basic nitrogen (TVB-N), K-value, trimethylamine nitrogen (TMA-N), drip loss, hardness, color, biogenic amines, microbiological characteristics were measured. Sea bass fillets were stored at −18 °C (T1), −55 °C for 24 h and then −18 °C (T2), −55 °C (T3) for 3 months prior to refrigerated storage. T2 showed lower TVB-N, pH value, biogenic amines and drip loss than T3 and T1 did. Significant lower value of bacterial loads, b* value and hardness were observed in T1, T2 and T3 than those of control group. No significant differences were observed among T1, T2 and T3 for TMA-N and a* value. The study demonstrated that Japanese sea bass fillets treated with −55 °C for 24 h and then −18 °C up to 3 months maintain better quality during refrigerated storage.

Cone snails express a sophisticated arsenal of small bioactive peptides known as conopeptides or conotoxins (CTxs). Through evolutionary selection, these peptides have gained the ability to interact with a range of ion channels and receptors, such as nicotinic acetylcholine receptors (nAChRs). Here, we used reversed-phase high performance liquid chromatography (RP-HPLC) and electrospray ionization-mass spectrometry (ESI-MS) to explore the venom peptide diversity of Conus textile, a species of cone snail native to Hainan, China. One fraction of C. textile crude venom potently blocked α3β2 nAChRs. Subsequent purification, synthesis, and tandem mass spectrometric analysis demonstrated that the most active compound in this fraction was identical to α-CTx TxIA, an antagonist of α3β2 nAChRs. Then three disulfide isoforms of α-CTx TxIA were synthesized and their activities were investigated systematically for the first time. As we observed, disulfide isomerisation was particularly important for α-CTx TxIA potency. Although both globular and ribbon isomers showed similar retention times in RP-HPLC, globular TxIA potently inhibited α3β2 nAChRs with an IC50 of 5.4 nM, while ribbon TxIA had an IC50 of 430 nM. In contrast, beads isomer had little activity towards α3β2 nAChRs. Two-step oxidation synthesis produced the highest yield of α-CTx TxIA native globular isomer, while a one-step production process based on random oxidation folding was not suitable. In summary, this study demonstrated the relationship between conotoxin activity and disulfide connectivity on α-CTx TxIA.

Conotoxin GeXIVA inhibits the α9α10 nicotinic acetylcholine receptor (nAChR) and is analgesic in animal models of pain. α-Conotoxins have four cysteines that can have three possible disulfide connectivities: globular (CysI–CysIII and CysII–CysIV), ribbon (CysI–CysIV and CysII–CysIII), or bead (CysI–CysII and CysIII–CysIV). Native α-conotoxins preferably adopt the globular connectivity, and previous studies of α-conotoxins have focused on the globular isomers as the ribbon and bead isomers typically have lower potency at nAChRs than the globular form. A recent report showed that the bead and ribbon isomers of GeXIVA are more potent than the globular isomer, with low nanomolar half-maximal inhibitory concentrations (IC50). Despite this high potency, the therapeutic potential of GeXIVA is limited, because like most peptides, it is susceptible to proteolytic degradation and is challenging to synthesize in high yield. Here we used backbone cyclization as a strategy to improve the folding yield as well as increase the serum stability of ribbon GeXIVA while preserving activity at the α9α10 nAChR. Specifically, cyclization of ribbon GeXIVA with a two-residue linker maintained the biological activity at the human α9α10 nAChR and improved stability in human serum. Short linkers led to selective formation of the ribbon disulfide isomer without requiring orthogonal protection. Overall, this study highlights the value of backbone cyclization in directing folding, improving yields, and stabilizing conotoxins with therapeutic potential.

The effect of chitosan–ergothioneine coating (CHER) on the post-mortem quality and shelf life of Japanese sea bass (Lateolabrax japonicas) stored at 4 ± 1 °C for 16 days was investigated. Four different treatments were used: control without coating, CH with chitosan coating, ER with 0.3 % ergothioneine immersion, and CHER with chitosan containing 0.3 % ergothioneine coating. Sea bass pH value, total volatile basic nitrogen, peroxide value, thiobarbituric acid-reactive substances, biogenic amines, color, texture profile, and microbiological and sensory qualities were measured. The results indicate that treatment with CHER coating inhibited increase of total volatile basic nitrogen, peroxide value, and TBA value; maintained tissue hardness; and reduced microorganism counts, such as Pseudomonas, compared with control treatment. The efficiency was better than that of ER or CH treatment. Furthermore, sea bass treated with chitosan–ergothioneine coating also exhibited a positive effect, causing low biogenic amine content, especially putrescine, cadaverine, and histamine. Sensory evaluation proved the efficacy of chitosan–ergothioneine coating by maintaining the overall quality of sea bass during the storage period. Our study suggests that chitosan–ergothioneine coating might be a promising candidate for maintaining sea bass quality and extending their shelf life.

μO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.

Collagen has a wide range of applications in food, biomedical and pharmaceutical products.The collagens in grass carp (Ctenopharyngodon idella) skin and swim bladder were extracted using acetic acid and pepsin. Higher yield of pepsin-soluble collagen (PSC) was obtained from skin (178 g kg(-1)) than from swim bladder (114 g kg(-1)). Not surprisingly, yields of PSC from skin and swim bladder were also higher than those of acid-soluble collagen (ASC) from the same organs (89 and 51 g kg(-1)). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles showed that ASC and PSC were type I collagens, with PSC containing a higher proportion of α components than ASC. Fourier transform infrared spectra revealed that ASC and PSC were very similar in their protein secondary structures. Scanning electron micrographs showed that the collagens had a spongy structure, with more pores being obtained in swim bladder than in skin collagens. The collagens showed high solubility in the acidic pH range. However, their solubility decreased in the presence of NaCl at concentrations above 20 g kg(-1).Collagens were successfully extracted from the skin and swim bladder of grass carp. These fish by-products could serve as an alternative source of collagens for a wide variety of applications in the food and nutraceutical industries.

To observe the neuroprotective and antioxidant activities of the grass carp protein hydrolysates (GPH) obtained from grass carp (Ctenopharyngodon idella) skin by enzymatic hydrolysis. GPH prepared using Protamex, at different (5, 10, 15, 20 and 30 %) degrees of hydrolysis (DH) were investigated. The DPPH radial scavenging, reducing power and inhibition of linoleic acid oxidation activities of GPH were significantly improved by a low DH (5 %) compared with those of GPH with a higher DH (p