Enzymatic sulfation of coelenterazine by human cytosolic aryl sulfotransferase SULT1A1: identification of coelenterazine C2-benzyl monosulfate by LC/ESI-TOF-MS

Coelenterazine (CTZ) is known as a light-emitting source for the bioluminescence reaction in marine organisms. CTZ has two phenolic hydroxy groups at the C2-benzyl and C6-phenyl positions, and a keto-enol type hydroxy group at the C3-position in the core structure of imidazopyrazinone (= 3,7-dihydroimidazopyrazin-3-one). These hydroxy groups in CTZ could be sulfated by sulfotransferase(s), and the sulfates of Watasenia luciferin (CTZ disulfate at the C2- and C6-positions) and Renilla pre-luciferin (CTZ 3-enol sulfate) have been identified in marine organisms. To characterize the sulfation process of CTZ, human cytosolic aryl sulfotransferase SULT1A1 (SUTase) was used as a model enzyme. The sulfated products catalyzed by SUTase with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) were analyzed by LC/ESI-TOF-MS. The product was the monosulfate of CTZ and identified as the C2-benzyl sulfate of CTZ (CTZ C2-benzyl monosulfate), but CTZ disulfate, CTZ 3-enol sulfate, and CTZ C6-phenyl monosulfate were not detected. The non-enzymatic oxidation products of dehydrocoelenterazine (dCTZ, dehydrogenated derivative of CTZ), coelenteramide (CTMD), and coelenteramine (CTM) from CTZ were also identified as their monosulfates.

Biochemical and Biophysical Research Communications


Formation of Coelenteramine from 2-Peroxycoelenterazine in the Ca2+-Binding Photoprotein Aequorin

Aequorin consists of apoprotein (apoAequorin) and (S)-2-peroxycoelenterazine (CTZ-OOH) and is considered to be a transient-state complex of an enzyme (apoAequorin) and a substrate (coelenterazine and molecular oxygen) in the enzymatic reaction. The degradation process of CTZ-OOH in aequorin was characterized under various conditions of protein denaturation. By acid treatment, the major product from CTZ-OOH was coelenteramine (CTM), but not coelenteramide (CTMD), and no significant luminescence was observed. The counterparts of CTM from CTZ-OOH were identified as 4-hydroxyphenylpyruvic acid (4HPPA) and 4-hydroxyphenylacetic acid (4HPAA) by liquid chromatography/electrospray ionization-time-of-flight mass spectrometry (LC/ESI-TOF-MS). In the luminescence reaction of aequorin with Ca , similar amounts of 4HPPA and 4HPAA were detected, indicating that CTM is formed by two pathways from CTZ-OOH through dioxetanone anion and not by hydrolysis from CTMD.


Enzymatic conversion of dehydrocoelenterazine to coelenterazine using FMN-bound flavin reductase of NAD(P)H:FMN oxidoreductase

Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.


Identification of a novel oxidation product from yellow fluorophore in the complex of apoPholasin and dehydrocoelenterazine

The complex of the recombinant fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) with non-fluorescent dehydrocoelenterazine (dCTZ) (GST-apoPholasin/dCTZ complex) shows yellow fluorescence at 539 nm by excitation at 430 nm. The GST-apoPholasin/dCTZ complex with a fluorophore (dCTZ*) has considerably weak luminescence activity, converting slowly to a blue fluorescence protein with the emission peak at 430 nm. The main oxidation products from dCTZ* for blue fluorescence were identified as coelenteramine (CTM) and an unreported pyrazine derivative, 3-benzyl-5-(4-hydroxyphenyl)pyrazin-2(1H)-one (CTO) that was confirmed by chemical synthesis.

Bioorganic & Medicinal Chemistry Letters


Efficient conversion to Cypridina luciferin from Cypridina luciferyl sulfate, coupled with enzymatic sulfation of acetic acid.

In Cypridina (Vargula) hilgendorfii, Cypridina luciferin is converted from Cypridina luciferyl sulfate by a sulfotransferase with adenosine 3', 5'-diphosphate (PAP), and is used for the luminescence reaction of Cypridina luciferase. We found that the luminescence activity of crude extracts of C. hilgendorfii was significantly stimulated by the addition of acetic acid. This stimulation may be explained by an efficient supply of PAP from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) catalyzed by a sulfotransferase. Thus, acetic acid acts as a sulfate acceptor from PAPS, followed by forming acetyl sulfate and PAP. The structure of acetyl sulfate was identified using mass spectrometry and it spontaneously decomposed to acetic acid and free sulfate ion in aqueous solutions. This enzymatic conversion from Cypridina luciferyl sulfate to Cypridina luciferin could be coupled with acetic acid and PAPS by a sulfotransferase.


A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λ = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-HO system, but not in sodium hypochlorite.


Expression, purification, and characterization of recombinant apoPholasin

Pholasin is a reactive oxygen-sensitive photoprotein that consists of an apoprotein (apoPholasin) and an unknown chromophore. The preferred human codon-optimized apoPholasin gene was transiently expressed in mammalian cells and apoPholasin was detected using an anti-recombinant apoPholasin antibody. For the first time, we found that apoPholasin secreted into the culture medium could catalyze the oxidation of coelenterazine (CTZ, a luciferin) to produce continuous luminescence. The fusion protein of apoPholasin and glutathione S-transferase (GST-apoPholasin) was successfully expressed as a soluble form in bacterial cells using the cold induction system. The purified GST-apoPholasin also had luminescence activity with CTZ, showing the bioluminescence emission peak at 461 nm, and the resultant product showed purple blue fluorescence under 365 nm light. Unexpectedly, the main oxidation product of CTZ was identified as coelenteramine (CTM), not coelenteramide (CTMD).


Enzymatic conversion of Cypridina luciferyl sulfate to Cypridina luciferin with coenzyme A as a sulfate acceptor in Cypridina (Vargula) hilgendorfii

In the luminous ostracod Cypridina (presently Vargula) hilgendorfii, Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) is converted to Cypridina luciferin by a sulfotransferase with 3'-phosphoadenosine-5'-phosphate (PAP) as a sulfate acceptor. The resultant Cypridina luciferin is used for the luciferase-luciferin reaction of Cypridina to emit blue light. The luminescence stimulation with major organic cofactors was examined using the crude extracts of Cypridina specimens, and we found that the addition of coenzyme A (CoA) to the crude extracts significantly stimulated luminescence intensity. Further, the light-emitting source in the crude extracts stimulated with CoA was identified as Cypridina luciferyl sulfate, and we demonstrated that CoA could act as a sulfate acceptor from Cypridina luciferyl sulfate. In addition, the sulfate group of Cypridina luciferyl sulfate was also transferred to adenosine 5'-monophosphate (5'-AMP) and adenosine 3'-monophosphate (3'-AMP) by a sulfotransferase. The sulfated products corresponding to CoA, 5'-AMP, and 3'-AMP were identified using mass spectrometry. This is the first report that CoA can act as a sulfate acceptor in a sulfotransferase reaction. This article is protected by copyright. All rights reserved.


Design and synthesis of a potent inhibitor of class 1 DYRK kinases as a suppressor of adipogenesis.

Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer's disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARγ and C/EBPα. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome.


Identification of 3-enol sulfate of Cypridina luciferin, Cypridina luciferyl sulfate, in the sea-firefly Cypridina (Vargula) hilgendorfii


Development of simple firefly luciferin analogs emitting blue, green, red, and near-infrared biological window light


Radical Scavenging Activity of Spring Mountain Herbs in the Shikoku Mountain Area and Identification of Antiradical Constituents by Simple HPLC Detection and LC-MS Methods

The functionality of spring mountain herbs, which were collected in the Kajigamori mountain area of Shikoku area in Japan, was investigated in the course of our studies for utilizing local plant resources. The radical scavenging activity of the extracts from seventeen herbs was measured. Among these herbs, two extracts from Polystichym ovato-paleaceum (Japanese name: Tsuyanashiinode) and Sambucus racemosa subsp. sieboldiana (Japanese name: Niwatoko) showed potent DPPH radical scavenging activity. The material evidence for the potent activity of the extracts was studied by a combination of our developed method for detecting antiradical compounds, LC-MS/MS, and enzymatic hydrolysis.

Bioscience, Biotechnology, and Biochemistry


Synthesis of manzacidin A and C: efficient construction of quarternary carbon stereocenters bearing nitrogen substituents

An efficient synthetic method for stereoselective construction of asymmetric quaternary carbon stereocenters, bearing nitrogen in the form of Boc-protected allyl amines, has been developed. This methodology is employed in the synthesis of marine alkaloids, manzacidin A and C.


Structure and function of the oxidation products of polyphenols and identification of potent lipoxygenase inhibitors from fe-catalyzed oxidation of resveratrol.

Polyphenols have recently attracted much attention as potent antioxidants and related bioactive substances. These potent antioxidative polyphenols are very oxidizable due to their chemical properties, and their oxidation products must accumulate in the oxidizing foods when they are contained as the active ingredients. In this investigation, 30 polyphenols and related phenolics were oxidized with oxygen in the presence of a catalytic amount of Fe ions. Piceatannol, catechin, epicatechin, hydroxytyrosol, carnosol, and carnosic acid were oxidized very quickly. Sinapic acid, caffeic acid, chlorogenic acid, rosmarinic acid, gallic acid, propyl gallate, α-tocopherol, quercetin, and nordihydroguaiaretic acid were moderately oxidized. Protocatechuic acid, syringic acid, taxifolin, resveratrol, gentisic acid, secoisolariciresinol, and ellagic acid were oxidized for 19-20 days; however, their oxidation was very slow and did not complete. The other phenolics were not oxidized. The obtained oxidation products were next subjected to a lipoxygenase inhibition assay and the results compared to those of the corresponding phenols. Very interestingly, the oxidation product from resveratrol showed a high inhibitory activity, whereas resveratrol itself had no activity and its oxidation efficiency was low. To clarify the inhibition principle of the oxidation product, an LC-MS analysis was carried out on the oxidation product. The analytical results showed that they are the oligomeric and degraded compounds of resveratrol. Among them, the structures of three dimeric compounds were successfully identified, and their activity data clarified that the closed ring dimers were potent lipoxygenase inhibitors, whereas the opened ring dimer was not. It should be noted that resveratrol had almost no lipoxygenase inhibitory activity, contrary to some researchers' findings.

Journal of Agricultural and Food Chemistry


Asymmetric synthesis of (+)-geranyllinaloisocyanide: assignment of absolute stereochemistry.

The first nonracemic synthesis of (+)-geranyllinaloisocyanide, starting with (-)-lactic acid methyl ester, has been accomplished by exploiting a [3.3] sigmatropic rearrangement of allyl cyanate. The synthesis enables assignment of the S configuration of the C(3) isocyano substituted, quaternary stereogenic center in natural geranyllinaloisocyanide.


A novel ring-expanded product with enhanced tyrosinase inhibitory activity from classical Fe-catalyzed oxidation of rosmarinic acid, a potent antioxidative Lamiaceae polyphenol

The iron-ion catalyzed oxidation of the ethanol solution of rosmarinic acid, a potent antioxidant polyphenol of Lamiaceae (Labiatae) plants, afforded a highly tyrosinase-inhibitory active product. The structure of the active product in the oxidation product mixture was determined using extensive NMR spectroscopy to have a novel oxygen-containing seven-membered ring system. The formation mechanism of the unique ring structure from the catechol part of the rosmarinic acid was proposed.

Bioorganic & Medicinal Chemistry Letters


Identification of cytotoxic dimers in oxidation product from sesamol, a potent antioxidant of sesame oil


DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation

Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.


Distinction of Sialyl Anomers on ESI- and FAB-MS/MS: Stereo-Specific Fragmentations

Journal of the American Society for Mass Spectrometry


A general mechanism for transcription regulation by Oct1 and Oct4 in response to genotoxic and oxidative stress

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure.


Development of a chemical screening system using aqueorin-fused protein

We developed a unique screening system that consists of combination of high photo-sensitivity of photoprotein aequorin (AQ) and our developed high-performance affinity purification system. In the present study, we demonstrated to detect the specific interaction between methotrexate (MTX) and its target dihydrofolate reductase (DHFR) fused with AQ. We succeeded to prepare highly purified AQ-fused DHFR, which showed high sensitive light emission. To test the screening system, we prepared the complex of MTX-immobilized magnetic nanobeads and AQ-fused DHFR. Bound AQ-fused DHFR with the beads was specifically released by addition of MTX. Thus, this methodology enables us to search a novel chemical that binds to target proteins without complicated processes. Furthermore, thank to the highly sensitive luminescence intensity of AQ, this methodology would be performed in very small scale with high responsibility, leading to development of high throughput screening systems.

Biochemical and Biophysical Research Communications


Stereoisomeric bio-inversion key to biosynthesis of firefly D-luciferin

The chirality of the luciferin substrate is critical to the luciferin-luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D-luciferin is still unclear, although it can be synthesized in vitro from D-cysteine. Here, we show that the firefly produces both D- and L-luciferin, and that the amount of active D-luciferin increases gradually with maturation stage. Studies of firefly body extracts indicate the possible conversion of L-cysteine via L-luciferin into D-luciferin, suggesting that the biosynthesis is enzymatically regulated by stereoisomeric bio-inversion of L-luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought.


Construction of a new firefly bioluminescence system using L-luciferin as substrate


Novel relationship beween the antifungal activity and cytotoxicity of marine-derived methabolite xestoquinone and its family

Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.

Bioscience, Biotechnology, and Biochemistry


Firefly luciferase exhibits bimodal action depending on the luciferin chirality

Firefly luciferase is able to convert L-luciferin into luciferyl-CoA even under ordinary aerobic luciferin-luciferase reaction conditions. The luciferase is able to recognize strictly the chirality of the luciferin structure, serving as the acyl-CoA synthetase for L-luciferin, whereas d-luciferin is used for the bioluminescence reaction. D-Luciferin inhibits the luciferyl-CoA synthetase activity of L-luciferin, whereas L-luciferin retards the bioluminescence reaction of D-luciferin, meaning that both enzyme activities are prevented by the enantiomer of its own substrate.


Bioluminescence activity of Latia luciferin analogues: Replacement of the 2,6,6-trimethylcyclohexene ring onto the methyl-substituted phenyl groups


Effects of modification at the fifth residue of mu-conotoxin GIIIA with bulky tags on the electrically stimulated contraction of the rat diaphragm

Mu-conotoxin GIIIA, a peptide toxin from the cone snail, blocks muscle-type sodium channels. Thr-5 of mu-conotoxin GIIIA, located on the opposite side of the active site in the globular molecule, was replaced by Cys to which the bulky tags were attached. The tagged mu-conotoxin GIIIA derivatives, except for the phospholipid-tagged one, exerted the biological activity with a potency slightly weaker than natural mu-conotoxin GIIIA. When the biotinylated tags of various lengths were added, the presence of avidin suppressed the action of the biotinylated toxins of <4 nm, but not with 5 nm. The bulky biotinylated tags are useful as a caliper to measure the depth of receptor sites in the channels.


Synthesis of Latia luciferin benzoate analogues and their bioluminescent activity


Synthesis of Biotinylated Xestoquinone That Retains Inhibitory Activity Against Ca2+ ATPase of Skeletal Muscle Myosin

Xestoquinone isolated from a marine sponge binds to skeletal muscle myosin and inhibits its Ca(2+) ATPase activity. In this study, we first examined xestoquinone and its analogues to assess the relationships between structure and myosin Ca(2+) ATPase inhibitory activity. On the basis of the resultant data, we then designed a biotinylated xestoquinone analogue. Xestoquinone and its analogues were derived from extracts of the marine sponge Xestospongia sapra. Four xestoquinone analogues with a quinone structure significantly inhibited Ca(2+) ATPase activity. In contrast, four xestoquinone analogues in which the quinone structure was converted to a quinol dimethyl ether did not inhibit Ca(2+) ATPase activity. This suggests that the quinone moiety is essential for inhibitory activity. Then, we synthesized a biotinylated xestoquinone in which a biotin tag was introduced to a site far from the quinone moiety, and this molecule exhibited stronger inhibitory activity than that of xestoquinone. This biotinylated xestoquinone could be useful as a probe in studies of the xestoquinone-myosin binding mode.


Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.


Identification of biotinylated lysine residues in the photoprotein aequorin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mapping after lysine-specific endopeptidase digestion

A method for identifying modified lysine residues in a protein, using lysine-specific endopeptidase treatment followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping, is described. As a model protein, the photoprotein aequorin was chosen and the N-hydroxysuccinimide ester of biotin was employed to chemically modify the lysine residues. After digestion with lysine-specific endopeptidase, the biotinylated residues of an amino terminus and five potential lysine residues were identified by MALDI-TOF-MS without any other separation procedure.

Analytical Biochemistry: Methods in the Biological Sciences


Thiolation of protein-bound carcinogenic aldehyde: An electrophilic acrolein-lysine adduct that covalently binds to thiols

Acrolein, a representative carcinogenic aldehyde that could be ubiquitously generated in biological systems under oxidative stress, shows facile reactivity with the epsilon-amino group of lysine to form N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product (Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) J. Biol. Chem. 273, 16058-16066). In the present study, we determined the electrophilic potential of FDP-lysine and established a novel mechanism of protein thiolation in which the FDP-lysine generated in the acrolein-modified protein reacts with sulfhydryl groups to form thioether adducts. When a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, was incubated with acrolein-modified bovine serum albumin in sodium phosphate buffer (pH 7.2) at 37 degrees C, a significant loss of sulfhydryl groups, which was accompanied by the loss of enzyme activity and the formation of high molecular mass protein species (>200 kDa), was observed. The FDP-lysine adduct generated in the acrolein-modified protein was suggested to represent a thiol-reactive electrophile based on the following observations. (i) N(alpha)-acetyl-FDP-lysine, prepared from the reaction of N(alpha)-acetyl lysine with acrolein, was covalently bound to glyceraldehyde-3-phosphate dehydrogenase. (ii) The FDP-lysine derivative reacted with glutathione to form a GSH conjugate. (iii) The acrolein-modified bovine serum albumin significantly reacted with GSH to form a glutathiolated protein. Furthermore, the observation that the glutathiolated acrolein-modified protein showed decreased immunoreactivity with an anti-FDP-lysine monoclonal antibody suggested that the FDP-lysine residues in the acrolein-modified protein served as the binding site of GSH. These data suggest that thiolation of the protein-bound acrolein may be involved in redox alteration under oxidative stress, whereby oxidative stress generates the increased production of acrolein and its protein adducts that further potentiate oxidative stress via the depletion of GSH in the cells.


Generation of Polyclonal Antibody against mu-Conotoxin GIIIA Using an Immunogen of [Cys5]mu-Conotoxin GIIIA Site-Specifically Conjugated with Bovine Serum Albumin

mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.

Biochemical and Biophysical Research Communications


Modification of Arg-13 of mu-conotoxin GIIIA with piperidinyl-Arg analogs and the relation to the inhibition of sodium channels

mu-Conotoxin GIIIA, a peptide toxin isolated from the marine snail Conus geographus, preferentially blocks skeletal muscle sodium channels in vertebrates. In this study, analogs of mu-conotoxin GIIIA in which essential Arg-13 was replaced with arginine analogs consisting of a piperidyl framework to regulate length and direction of the side chain were synthesized. Synthesized analogs exhibited similar CD and NMR spectra to that of GIIIA, suggesting a three-dimensional structure identical to that of the native toxin. The biological activities of piperidyl analogs were decreased or lost despite the small change in the side chain of Arg-13. The investigated structure-activity relationships in inhibiting electrically stimulated muscle contraction suggest that the guanidinium group at amino acid position 13 interacts best when spaced with three to four carbons and placed in a vertical direction from the peptide loop. Thus, the position of the guanidinium group at Arg-13 of GIIIA must be located in a certain range for its strong interaction with the channel protein.


Synthesis of [Cys5]mu-conotoxin GIIIA and its derivatives as a probe of Na+ channel analysis

The residue of Thr-5 in mu-conotoxin GIIIA (GIIIA), a receptor site I sodium channel blocker, was replaced with Cys. The synthesized [Cys(5)]GIIIA had a similar 3D structure to the native GIIIA, revealed by CD and NMR. [Cys(5)]GIIIA and its tagged peptides inhibited the electrically stimulated contraction of the rat diaphragm with relatively comparable potency to that of GIIIA. Since the contractile response to electrical stimuli is caused by the activation of sodium channels, [Cys(5)]GIIIA could be a prototype for synthesizing useful tools for the analysis of sodium channels. Thus, [Cys(5)]GIIIA could be a prototype for synthesizing useful tools for the analysis of sodium channels.