Edman Degradation Research Articles

Characterization of a Novel Mannose-Binding Lectin with Antiviral Activities from Red Alga, Grateloupia chiangii.

Lectins have the ability to bind specific carbohydrates and they have potential applications as medical and pharmacological agents. The unique structure and usefulness of red algal lectin have been reported, but these lectins are limited to a few marine algal groups. In this study, a novel mannose-binding lectin from Grateloupia chiangii (G. chiangii lectin, GCL) was purified using antiviral screens and affinity chromatography. We characterized the molecular weight, agglutination activity, hemagglutination activity, and heat stability of GCL. To determine the carbohydrate specificity, a glycan microarray was performed. GCL showed strong binding affinity for Maltohexaose-β-Sp1 and Maltoheptaose-β-Sp1 with weak affinity for other monosaccharides and preferred binding to high-mannan structures. The N-terminal sequence and peptide sequence of GCL were determined using an Edman degradation method and LC-MS/MS, and the cDNA and peptide sequences were deduced. GCL was shown to consist of 231 amino acids (24.9 kDa) and the N-terminus methionine was eliminated after translation. GCL possessed a tandem repeat structure of six domains, similar to the other red algal lectins. The mannose binding properties and tandem repeat structure of GCL may confer it the potential to act as an antiviral agent for protection against viral infection.

Myticusin-beta, antimicrobial peptide from the marine bivalve, Mytilus coruscus

We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-performance liquid chromatography, and displayed antibacterial activity. Total molecular mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M. coruscus. Complete sequence of the target peptide was determined through cDNA cloning and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta, which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-parasitic activity at various concentrations. A short AMP analog was designed and synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial activity. Expression of myticusin-beta was detected in the mantle at the highest level, followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an immune-related AMP of M. coruscus and an effective alternative to antibiotics.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/β, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell–derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

The structure of MP-4 from Mucuna pruriens at 2.22 Å resolution.

The structure of the MP-4 protein was previously determined at a resolution of 2.8 Å. Owing to the unavailability of gene-sequence information at the time, the side-chain assignment was carried out on the basis of a partial sequence available through Edman degradation, sequence homology to orthologs and electron density. The structure of MP-4 has now been determined at a higher resolution (2.22 Å) in another space group and all of the structural inferences that were presented in the previous report of the structure were validated. In addition, the present data allowed an improved assignment of side chains and enabled further analysis of the MP-4 structure, and the accuracy of the assignment was confirmed by the recently available gene sequence. The study reinforces the traditional concept that conservative interpretations of relatively low-resolution structures remain correct even with the availability of high-resolution data.

Reciprocal surface expression of arylsulfatase A and ubiquitin in normal and defective mammalian spermatozoa.

Defective mammalian spermatozoa are marked on their surface by proteolytic chaperone ubiquitin. To identify potential ubiquitinated substrates in the defective spermatozoa, we resolved bull sperm protein extracts on a two-dimensional gel and isolated a 64-65-kDa spot (p64) corresponding to one of the major ubiquitin-immunoreactive bands observed in the one-dimensional Western blots. Immune serum raised against this protein recognized a prominent, possibly glycosylated band/spot in the range of 55-68kDa, consistent with the original spot used for immunization. Internal sequences obtained by Edman degradation of this spot matched the sequence of arylsulfatase A (ARSA), the sperm acrosomal enzyme thought to be important for fertility. By immunofluorescence, a prominent signal was detected on the acrosomal surface (boar and bull) and on the sperm tail principal piece (bull). A second immune serum raised against a synthetic peptide corresponding to an immunogenic internal sequence (GTGKSPRRTL) of the porcine ARSA also labeled sperm acrosome and principal piece. Both sera showed diminished immunoreactivity in the defective bull spermatozoa co-labeled with ananti-ubiquitin antibody. Western blotting and image-based flow cytometry (IBFC) confirmed a reduced ARSA immunoreactivity in the immotile sperm fraction rich in ubiquitinated spermatozoa. Larger than expected ARSA-immunoreactive bands were found in sperm protein extracts immunoprecipitated with anti-ubiquitin antibodies and affinity purified with matrix-bound, recombinant ubiquitin-binding UBA domain. These bands did not show the typical pattern of ARSA glycosylation but overlapped with bands preferentially binding the Lens culinaris agglutinin (LCA) lectin. By both epifluorescence microscopy and IBFC, the LCA binding was increased in the ubiquitinated spermatozoa with diminished ARSA immunoreactivity. ARSA was also found in the epididymal fluid suggesting that in addition to intrinsic ARSA expression in the testis, epididymal spermatozoa take up ARSA on their surface during the epididymal passage. We conclude that sperm surface ARSA is one of the ubiquitinated sperm surface glycoproteins in defective bull spermatozoa. Defective sperm surface thus differs from normal sperm surface by increased ubiquitination, reduced ARSA binding, and altered glycosylation.

Isolation and structural identification of a new T1-conotoxin with unique disulfide connectivities derived from Conus bandanus.

Background:Conopeptides are neuropharmacological peptides derived from the venomous salivary glands of cone snails. Among 29 superfamilies based on conserved signal sequences, T-superfamily conotoxins, which belong to the smallest group, include four different frameworks that contain four cysteines denominated I, V, X and XVI. In this work, the primary structure and the cysteine connectivity of novel conotoxin of Conus bandanus were determined by tandem mass spectrometry using collision-induced dissociation.Methods:The venom glands of C. bandanus snails were dissected, pooled, and extracted with 0.1% trifluoroacetic acid in three steps and lyophilized. The venom was fractionated and purified in an HPLC system with an analytical reversed-phase C18 column. The primary peptide structure was analyzed by MALDI TOF MS/MS using collision-induced dissociation and confirmed by Edman's degradation. The peptide’s cysteine connectivity was determined by rapid partial reduction-alkylation technique.Results:The novel conotoxin, NGC1C2(I/L)VREC3C4, was firstly derived from de novo sequencing by MS/MS. The presence of isoleucine residues in this conotoxin was confirmed by the Edman degradation method. The conotoxin, denominated Bn5a, belongs to the T1-subfamily of conotoxins. However, the disulfide bonds (C1-C4/C2-C3) of Bn5a were not the same as found in other T1-subfamily conopeptides but shared common connectivities with T2-subfamily conotoxins. The T1-conotoxin of C. bandanus proved the complexity of the disulfide bond pattern of conopeptides. The homological analysis revealed that the novel conotoxin could serve as a valuable probe compound for the human-nervous-system norepinephrine transporter.Conclusion:We identified the first T1-conotoxin, denominated Bn5a, isolated from C. bandanus venom. However, Bn5a conotoxin exhibited unique C1-C4/C2-C3 disulfide connectivity, unlike other T1-conotoxins (C1-C3/C2-C4). The structural and homological analyses herein have evidenced novel conotoxin Bn5a that may require further investigation.

Jingzhaotoxin-X, a gating modifier of Kv4.2 and Kv4.3 potassium channels purified from the venom of the Chinese tarantula Chilobrachys jingzhao.

Background:The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels.Methods:JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice.Results:JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test.Conclusions:JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Purification and Biochemical Characterization of Selected F-Type Lectins.

The purification of fucose-binding lectins from the liver of striped bass (Morone saxatilis), a teleost fish, and the identification of a novel lectin sequence motif led to the identification of a new family of lectins, the F-type lectins (FTLs) (see overview of the FTL family in Chapter 23 ). Isolation and purification of these proteins from liver extracts of striped bass was accomplished by affinity chromatography and size exclusion, and their identification as FTLs, by direct Edman sequencing, and protein, transcript, and gene sequence analysis. The development of specific antibodies against the M. saxatilis FTL provided an additional tool for the identification of FTLs. These methods have been successfully used for the purification of the FTL family members from tissues and body fluids of various animal species. Production and characterization of FTLs has been facilitated by the expression of the recombinant proteins. In this chapter, the biochemical characterization of FTLs is focused on the analysis of their carbohydrate specificity.

Structural and antioxidant analysis of Tartary buckwheat (Fagopyrum tartaricum Gaertn.) 13S globulin.

The main component of buckwheat seed storage proteins is 13S globulin. In this study, Tartary buckwheat 13S globulin was separated and its structural features were investigated using Edman sequencing and matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS). The protective effect of its enzymatic hydrolysates against oxidative stress induced by H2 O2 was also evaluated to elucidate the antioxidant mechanism. Results showed that the isolated Tartary buckwheat 13S globulin contained one acidic and one basic subunit, which were linked by a disulfide bond. Six Tartary buckwheat active peptides were obtained from the enzymatic hydrolysates of Tartary buckwheat 13S globulin acidic subunit with a molecular weight of 38 kDa, namely Pep-1, Pep-2, Pep-3, Pep-4, Pep-5, and Pep-6. Pre-treatment of cells with Tartary buckwheat active peptides maintained the redox state balance of HepG2 cells and protected the activity of antioxidant enzymes in HepG2 cells. The Tartary buckwheat active peptides improved oxidative stress in HepG2 cells via the PPAR-α/HO-1 pathway. These results provide an insight into the antioxidant mechanism of Tartary buckwheat 13S globulin and suggest that Tartary buckwheat active peptides can be used as a functional ingredient in the food industry. © 2019 Society of Chemical Industry.

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.

Functional Characterization of a Rice Thioredoxin Protein OsTrxm and Its Cysteine Mutant Variant with Antifungal Activity.

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening Oryza sativa leaves to isolate novel AFPs. We identified a thioredoxin protein with antioxidant properties. Being ubiquitous, thioredoxins (Trxs) function in the redox balance of all living organisms. Sequencing by Edman degradation method revealed the AFP to be O. sativa Thioredoxin m-type isoform (OsTrxm). We purified the recombinant OsTrxm and its cysteine mutant proteins (OsTrxm C/S) in Escherichia coli. The recombinant OsTrxm proteins inhibited the growth of various pathogenic fungal cells. Interestingly, OsTrxm C/S mutant showed higher antifungal activity than OsTrxm. A growth inhibitory assay against various fungal pathogens and yeasts confirmed the pertinent role of cysteine residues. The OsTrxm protein variants penetrated the fungal cell wall and membrane, accumulated in the cells and generated reactive oxygen species. Although the role of OsTrxm in chloroplast development is known, its biochemical and molecular functions have not been elucidated. These findings suggest that in addition to redox regulation, OsTrxm also functions as an antimicrobial agent.

Isolation, Biochemical Characterization and Antiparasitic Activity of BmatTX-IV, A Basic Lys49-Phospholipase A2 from the Venom of Bothrops mattogrossensis from Paraguay.

Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential. A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated. Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 μg/mL against Leishmania infantum promastigotes and of 13.8 μg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 μg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 μg/mL. Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.

Identification and Sequencing of N-Terminal Peptides in Proteins by LC-Fluorescence-MS/MS: An Approach to Replacement of the Edman Degradation.

Automated Edman degradation for sequencing is the classic tool for determining the N-terminal sequences of proteins. Although it has been complemented or largely replaced by a variety of mass spectrometric methods in recent years, there is no simple mass spectrometric method for identification and de novo determination of the N-terminal sequence of a protein. Here, we present a simple and reliable approach which identifies N-terminal peptides of proteins by selectively labeling the N-terminal α-amino groups with a fluorescent reagent followed by enzyme digestion and tandem mass spectrometric analysis using an LC-MS/MS system coupled with a fluorescence detector. This approach also includes a differential peptide mapping step for identification of pyroglutamate-blocked N-terminal peptides. In addition, the strategy can be applied to proteins in gel. With this approach, complete N-terminal sequences of proteins can be obtained precisely and efficiently.

Identification and Functional Characterization of a Novel Insecticidal Decapeptide from the Myrmicine Ant Manica rubida.

Ant venoms contain many small, linear peptides, an untapped source of bioactive peptide toxins. The control of agricultural insect pests currently depends primarily on chemical insecticides, but their intensive use damages the environment and human health, and encourages the emergence of resistant pest populations. This has promoted interest in animal venoms as a source of alternative, environmentally-friendly bio-insecticides. We tested the crude venom of the predatory ant, Manica rubida, and observed severe fitness costs in the parthenogenetic pea aphid (Acyrthosiphon pisum), a common agricultural pest. Therefore, we explored the M. rubida venom peptidome and identified a novel decapeptide U-MYRTX-MANr1 (NH2-IDPKVLESLV-CONH2) using a combination of Edman degradation and de novo peptide sequencing. Although this myrmicitoxin was inactive against bacteria and fungi, it reduced aphid survival and reproduction. Furthermore, both crude venom and U-MYRTX-MANr1 reversibly paralyzed injected aphids and induced a loss of body fluids. Components of M. rubida venom may act on various biological targets including ion channels and hemolymph coagulation proteins, as previously shown for other ant venom toxins. The remarkable insecticidal activity of M. rubida venom suggests it may be a promising source of additional bio-insecticide leads.

A novel antimicrobial protein of the endophytic Bacillus amyloliquefaciens and its control effect against Fusarium chlamydosporum

Fusarium chlamydosporum is the causal agent of stem rot in Jacaranda acutifolia. Bacillus amyloliquefaciens is an endophytic bacterium that shows promising biological control activity against F. chlamydosporum. In this study, B. amyloliquefaciens was cultured in a fermentation broth, and the main antimicrobial substance in the fermentation broth was isolated using the ammonium sulfate precipitation method and purified by DEAE sepharose fast flow weak anion exchange chromatography and sephadex G-50 molecular sieve chromatography. Polyacrylamide gel electrophoresis revealed a single antimicrobial protein with a molecular weight of approximately 29.0 kDa. The antimicrobial protein is a strong antagonist against F. chlamydosporum, leading to mycelial deformity, unclear mycelial septa, and isolated and granulated intracellular protoplasm. The N-terminal eight-amino acid sequence, NH2-Gly-Arg-Pro-Leu-Pro-Leu-Ala-Ala, was determined using the automatic Edman degradation method. BLAST analysis indicated that the amino acid residue sequence at positions 154–161 shows 100% homology to the hypothetical protein of Chromobacterium sp. LK1. However, no known homologous antimicrobial protein was identified, indicating that we have isolated a novel antimicrobial protein.

Preparation and purification of Cry1Ah protein candidate reference material

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.

Linear Peptides Containing d-Leucine with Neuroprotective Activities from the Leech Whitmania pigra Whitman.

Three new linear peptides containing d-leucine, named whitmantides A-C (1-3), were isolated from the dried whole bodies of Whitmania pigra Whitman. Their structures with absolute configurations were elucidated by Edman degradation, mass spectrometry, Marfey's analysis, and solid-phase synthesis. It is the first time that peptides containing d-amino acid in leeches were discovered. Compounds 1-3 displayed neuroprotective activities against oxygen-glucose deprivation/reperfusion injury on Neuro-2a cells. In addition, ex vivo serum stability tests showed that 1-3 were resistant to protease degradation.

αD-Conotoxins in Species of the Eastern Pacific: The Case of Conus princeps from Mexico.

Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 μM, and it does not affect the hα3β2 subtype at 6.5 μM.

Different mechanisms of serum complement activation in the plasma of common (Chelydra serpentina) and alligator (Macrochelys temminckii) snapping turtles.

Reptiles are declining worldwide yet our understanding of their immune function lags far behind other taxa. The innate immune system is the primary mode of defense in reptiles, and the serum complement cascade is its major component. We assessed serum complement activity of plasma in two closely related aquatic turtle species, the common snapping turtle (CST; Chelydra serpentina) and alligator snapping turtle (AST; Macrochelys temminckii). We used a sheep red blood cell (SRBC) hemolysis assay to assess serum complement activity. Although the antibacterial activities of the plasma of these turtle species are similar, the hemolytic activity was much stronger in CST than AST. Treatment with inhibitors of the serum complement cascade indicated differences in the mechanisms of complement activation between the turtle species. We subjected plasma from both turtle species to mannan affinity chromatography and analyzed the eluate with SDS-PAGE, which revealed that plasma from the CSTs contained only small amounts of one C-type lectin protein while the AST plasma contained high concentrations of two C-type lectins (31.0 and 35.9 kDa). Edman degradation analyses confirmed that the two AST proteins contained identical N-terminal sequences. Thus, the CST appears to rely more heavily on the alternative mechanism of serum complement activation, while the AST appears to rely more on the lectin-mediated pathway, which is a pattern recognition response to prokaryotes not activated by the SRBCs. These results are unique in that the use of serum complement pathways are generally assumed to be conserved within clades.