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Glycosphingolipids (GSLs) are a group of molecules composed of a hydrophilic glycan part and a hydrophobic ceramide creating a diverse family. GSLs are de novo synthesised from ceramides at the endoplasmic reticulum and Golgi apparatus, and transported to the outer surface of the plasma membrane. It has been known that the glycan structures of GSLs change reflecting disease states. We envisioned that analysing the glycan pattern of GSLs enables distinguishing diseases. For this purpose, we utilised a fluorescently tagged compound, LacCerBODIPY (1). At first, compound 1 was taken up by cultured PC12D cells and transformed into various GSLs. As a result, changes in the GSL patterns of differentiation states of the cells were successfully observed by using an analysis platform, nano-liquid chromatography (LC)-fluorescence detection (FLD)-electrospray ionisation (ESI)-mass spectrometry (MS), which could quantify and provide molecular ions simultaneously. We found that compound 1 remained for about 10 min on the plasma membrane before it was converted into other GSLs. We therefore investigated a more rapid way to discriminate different cellular states by fluorescence recovery after photobleaching, which revealed that it is possible to distinguish the differentiation states as well.

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with N-acetyl-d-mannosamine (ManNAc) and its derivatives. The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-O-GlcNAcylation on the HCRT gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT were effective at inducing orexin neurons from hiPSCs. The prolonged exposure of orexin neurons to high glucose in culture caused irreversible silencing of the HCRT gene, which was characterized by H3/H4 hypoacetylation and hyper-O-GlcNAcylation. The DNA hypomethylation status, once established in orexin neurogenesis, was maintained in the HCRT-silenced orexin neurons, indicating that histone modifications, but not DNA methylation, were responsible for the HCRT silencing. Thus, the epigenetic status of the HCRT gene is unique to the hyperglycemia-induced silencing. Intriguingly, treatment of ManNAc and its derivatives reactivated HCRT gene expression, while inhibitors SIRT1 and the OGT did not. The present study revealed that the HCRT gene was silenced by the hyperglycemia condition, and ManNAc and its derivatives were useful for restoring the orexin neurons.

Attempts were made to synthesize seven analogs of µ-conotoxin GIIIA, a specific blocker of muscle sodium channels, by replacing the three Hyp residues (Hyp6, Hyp7, and Hyp17) with various amino acids. Replacement with Ala residue at these positions resulted in a very low isolation yield, suggesting that these three Hyp residues are essential for the folding of the molecule. CD spectra of the synthesized analogs suggest that, once synthesized, the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the hydroxyl group at side chains of Hyp residues are not essential for the activity.

Despite the increasing biological interests on glycoconjugates, the synthetic mechanism of oligosaccharides has not yet been revealed except for the enzymes involved. To clarify the synthetic events that occur inside cells, spatiotemporal analysis of fluorescently tagged glycosphingolipids was carried out. Transformation of the incorporated lactosylceramide analogue carrying 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl group (BODIPY fluorophore) was analyzed using nanoLC-fluorescence detection-nanoelectrospray ionization-mass spectrometry. A complex process of glycan synthesis and degradation was our primary observation.

Analysis of anomericity is one of the most important issues in the structure elucidation of carbohydrates. Mass spectrometry (MS)-based methods are of particular interest and important to address the issue related to resolving anomericity of monosaccharide units in a glycan. However, direct analysis of hemiacetals has not been possible by MS because of the nonavailability of information regarding the gas-phase behavior of such ion species. We addressed this issue by using stage-discriminated energy-resolved mass spectrometry (ERMS) at the stages of MSn and MSn+1 and showed that such analysis can be made. This was achieved by proving that individual anomers can be identified and that the equilibrium of sodium adducted ion species of α- and β-anomers can be negated in the gas phase under collision-induced dissociation (CID) conditions. On the basis of these results, we could 1) observe the mutarotation of lactose and 2) speculate the hydrolysis mechanism of endo-glycosylceramidase by using mass spectrometry.(Communicated by Takao SEKIYA, M.J.A.)

Collision-induced dissociation (CID) in mass spectrometry is a powerful technique with which to understand gas-phase chemical reactions. A mass spectrometer is used to carry out the reaction, isolation, and analysis. On the other hand, structural analysis of glycan structures is of extreme importance in the analysis of biomolecules, such as glycoproteins and glycolipids. In the analysis of glycan structures based on CID, certain ion species, including B-/Y-, C-/Z-, and A-/X-ions, are produced. Among these ions, we are interested in C-ion species that carry a glycosyl oxygen atom at the anomeric center and that possibly provide information regarding anomeric configuration. A method for generating C-ion species when necessary is thus considered to be important; however, none is currently available. In this study, synthetic glycosides carrying a series of aglycons were analyzed with the aim of identifying suitable glycosides with which to produce C-ions to be used in the structural determination of oligosaccharides. The results showed a 4-aminobutyl group was an excellent candidate. Furthermore, the use of C-ion species obtained in this manner in the structural characterization of a ganglioside, GM3, is described. The type of glycoside is believed to be valuable not only in structural analysis but also in biological investigation, because of the existing amino functionality that has been proven to be useful by enabling the generation of conjugates with other molecules and materials.

Lactosyl ceramide analogues carrying novel bifunctional BODIPY-based fluorescent tags were designed and synthesised for live cell imaging. Addition of azide functionality on the fluorophore facilitated isobaric tagging for quantitative multiplexed analysis of biomolecules based on tandem mass spectrometry.

Conotoxins are multiple disulfide-bonded peptides isolated from marine cone snail venom. These toxins have been classified into several families based on their disulfide pattern and biological properties. Here, we report a new family ofConus peptides, which have a novel cysteine motif. Three peptides of this family (CMrVIA, CMrVIB, and CMrX) have been purified from Conus marmoreus venom, and their structures have been determined. Their amino acid sequences are VCCGYK-LCHOC (CMrVIA), NGVCCGYKLCHOC (CMrVIB), and GICCGVSFCYOC (CMrX), where O represents 4-trans-hydroxyproline. Two of these peptides (CMrVIA and CMrX) have been chemically synthesized. Using a selective protection and deprotection strategy during disulfide bond formation, peptides with both feasible cysteine-pairing combinations were generated. The disulfide pattern (C1-C4, C2-C3) in native toxins was identified by their co-elution with the synthetic disulfide-isomeric peptides on reverse-phase high pressure liquid chromatography. Although cysteine residues were found in comparable positions with those of α-conotoxins, these toxins exhibited a distinctly different disulfide bonding pattern; we have named this new family “λ -conotoxins.” CMrVIA and CMrX induced different biological effects when injected intra-cerebroventricularly in mice; CMrVIA induces seizures, whereas CMrX induces flaccid paralysis. The synthetic peptide with λ-conotoxin folding is about 1150-fold more potent in inducing seizures than the mispaired isomer with α-conotoxin folding. Thus it appears that the unique disulfide pattern, and hence the “ribbon” conformation, in λ-conotoxins is important for their biological activity.

A series of synthetic lactosides with aglycones that differed in length and structure were used to determine the substrate specificity of endo-type glycosylceramidases. Endoglycoceramidases (EGCase) from bacteria preferred lactosides with an acylamide structure over simple n-alkyl lactosides. While ceramide glycanase (CGase) from leech did not show preference. N -Acylaminoethyl beta-lactosides and n -alkyl lactosides were substrates for both EGCase and CGase, but N-acylaminobutyl beta-lactosides, whose acylamide residue differs from that in ceramide, were not hydrolyzed by EGCases. Thus, EGCases, but not CGase, appear to require an N-acyl group at the same position as that of intact glycosphingolipid for substrate recognition. A p-nitrophenyl lactoside derivative possessing an N-acyl chain was degraded by both EGCases and CGase and this chromogenic substrate may be an alternative substrate for endo-type glycosylceramidase activity. Km of the chromogenic lactoside for CGase and Rhodococcus EGCase were 28 microM and 2.9 mM, respectively.

To study the structure-activity relationships of brain injury-derived neurotrophic peptide (BINP), 12 analogs were synthesized by replacing each amino acid residue with Gly. BINP showed CD spectra typical of an α-helical conformation in TFE solution which mimics the membrane environment. In the α-helical conformation, BINP showed an amphiphilic profile. Neurotrophic activities of BINP and its analogs were estimated from the effects on supporting septal cholinergic neurons and on rescuing hippocampal neurons from injury caused by glutamate. Both assays showed that the residues on the hydrophobic side of the amphiphilic helix were essential for the neurotrophic activity.

OUR aim was to visualize the dynamic features of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) activity. In order to do so, we synthesized a new reagent by conjugating a fluoroprobe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), to syntide 2, a specific peptide substrate for CaMKII. In cell-free conditions, the conjugate was found to be an effective indicator of calmodulin activation by Ca 2+ and the subsequent activation of CaMKII. The reagent is cell-permeable and can stain living cells when bath-applied. Using this technique we were able to obtain fluorescence images of stained cells and analyse the dynamic features of CaMKII inside the cells by means of image processing. Regional heterogeneity of CaMKII activation in cultured hippocampal neurones was seen following L-glutamate administration.

BIT (a brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a brain-specific membrane protein which has two cytoplasmic TAMs (tyrosine-based activation motifs). Using the Far Western blotting technique, we detected association of a 70-kDa protein with the tyrosine-phosphorylated TAMs of BIT. A mouse brain cDNA library in lambdagt11 was screened for this association, and two positive clones encoding tyrosine phosphatase SH-PTP2 were isolated. SH-PTP2 has two SH2 domains and is believed to function as a positive mediator in receptor tyrosine kinase signaling. SH-PTP2 and BIT were coimmunoprecipitated from phosphorylated rat brain lysate, and BIT was a major tyrosine-phosphorylated protein associated with SH-PTP2 in this lysate. This interaction was also observed in Jurkat T cells transfected with BIT cDNA depending on tyrosine phosphorylation of BIT. Bisphosphotyrosyl peptides corresponding to BIT-TAMs stimulated SH-PTP2 activity 33-35-fold in vitro, indicating that two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. Our findings suggest that the tyrosine phosphorylation of BIT results in stimulation of the signal transduction pathway promoted by SH-PTP2 and that BIT is probably a major receptor molecule in the brain located just upstream of SH-PTP2.

An analog of ω-conotoxin MVIIC ( Y13A-MVIIC ) was synthesized by replacing Tyr 13 with Ala to study the role of Tyr 13 residue conserved in many ω-conotoxins. Y13A-MVIIC has an overall conformation similar to that of the native toxin, but an enormously reduced ability to displace 125 I-ω-conotoxin MVIIC binding to rat cerebellar P 2 membranes. These results suggest that Tyr 13 is essential for the activity of ω-conotoxins at P/Q-type calcium channels.

A cytidine 5′-monophosphate (CMP)-sialic acid analogue carrying a fluorescent reporter group, an inhibitor of sialyltransferase, was synthesised in order to investigate glycan synthesis events in cells. The compound was found to be a substrate of a CMP-sialic acid transporter, and specific Golgi vesicles were visualised in the cells.

An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide. Owing to the zero dead volume after LC separation, an extremely high sensitivity was achieved for the quantitative analysis (260 amol). It was also shown that a simultaneous online structural analysis based on MS could be achieved for the same quantity of analyte. To further demonstrate its potential, an enzymatic reaction of fluorescently tagged lactosylceramide using sialyltransferase was carried out, and the conversion yield was obtained on the basis of fluorescence detection. In addition, the structural details of a product, sialyl lactosylceramide, were obtained by MS and MS/MS analyses.

Gangliosides, a family of glycosphingolipids (GSLs) that comprise sialic acid residue(s), are an important class of molecules that exist on the outer surface of the plasma membrane. To assess the functions of a particular series of gangliosides that play important roles in brain functions, their structures and localizations need to be investigated. We studied the structures of these gangliosides by collision-induced dissociation using quadrupole ion-trap mass spectrometry. The dissociation processes were investigated in detail based on energy-resolved mass spectrometry using sodiated molecules. The decision of utilization of the positive mode was based on the assumption that it was the generally applicable method for GSLs, including neutral ones. In this investigation, sialic acid residues were esterified to stabilize the linkages and to generate multiple fragment ions for successful structural investigations. A detailed analysis of a series of sodiated species of gangliosides based on energy-resolved mass spectrometry revealed that the GM1-equivelent fragments generated from the precursor ions under low energy CID conditions had the structural characteristics of their individual precursors. It was suggested that this information will be useful in determining the structures of their precursor gangliosides.

Mass spectrometry, especially tandem mass spectrometry, has been widely used in the field of analytical sciences for handling biological and chemical samples. The technique resolves molecular and fragment ions based on the mass to charge ratio. Energy-resolved mass spectrometry (ERMS) further provides an activation energy-related factor in the dissociation reaction. Therefore, it is a very powerful technique that can discriminate isomeric compounds. Despite the power of ERMS, useful information cannot be obtained when an analyte contains structural isomers. Carbohydrates carry multiple chiral centers, thus oligomers of monosaccharides can form a vast number of structural isomers. We decided to use such species in our endeavors to establish a method of identifying the 'purity' of an analyte solely based on mass spectrometry. In the present paper, we describe a stage-discriminated spectral correlation of ERMS, which not only enables identification of the presence of contaminants in an analyte, but also provides information regarding the 'purity' of fragment ions.

Replacement of the N-terminal half of omega-conotoxin MVIIC, a peptide blocker of P/Q-type calcium channels, with that of omega-conotoxin MVIIA significantly increased the affinity for N-type calcium channels. To identify the residues essential for subtype selectivity, we examined single reverse mutations from MVIIA-type to MVIIC-type in this chimeric analog. A reverse mutation from Lys(7) to Pro(7) decreased the affinity for both P/Q- and N-type channels, whereas that from Leu(11) to Thr(11) increased the affinity for P/Q-type channels and decreased the affinity for N-type channels. The roles of these two residues were confirmed by synthesizing two MVIIC analogs in which Pro(7) and Thr(11) were replaced with Lys(7) and Leu(11), respectively.

International audience; Despite their high sequence homology, the peptide neurotoxins ¢o-conotoxin MVIIA and MVIIC selectively block N-and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of ¢o-conotoxin MVIIA and MVIIC were synthesized by exchanging their N-and C-terminal halves. Binding assay for both N-and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole ¢o-conotoxin molecule.

In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108-15 cells, PKA activity was localized to the cytosol around the nucleus. In cultured hippocampal neurons, addition of l-glutamate caused PKA activation associated with increase of the cellular cAMP.

The circular dichroism (CD) spectrum of omega-conotoxin GVIA is quite different from those of omega-conotoxin MVIIA and MVIIC, despite their distinct similarity in three dimensional structures. In order to characterize the unique CD spectrum of omega-conotoxin GVIA, we focused our attention on the aromatic chromophore and analyzed the CD spectra of three synthetic analogs, in which Tyr13, Tyr22, and Tyr27 were individually replaced by alanine. Replacement of Tyr27 caused a significant change in both the near- and far-ultraviolet CD spectrum of omega-conotoxin GVIA and resulted in the omega-conotoxin MVIIA/MVIIC-like pattern, suggesting that Tyr27 has a dominant contribution to the unique CD profile of omega-conotoxin GVIA.

Two analogs of omega-conotoxin MVIIA, a 25mer peptide neurotoxin, were synthesized by replacing Lys2 or Tyr13 with Ala. The activities of synthetic analogs were estimated from the inhibitory action on 125I-omega-conotoxin GVIA binding to chick brain synaptic plasma membranes. As in the case of omega-conotoxin GVIA, replacement of Tyr13 resulted in an enormous reduction in activity. In contrast, substitution of Ala for Lys2 gave only a small effect. These results indicate that Tyr13 is a critical amino acid of omega-conotoxin MVIIA and GVIA for blocking N-type calcium channel function.

During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S-Sepharose and AF-Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS-PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, β-tubulin, MAP2 and α-casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be -SR-. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau-tubulin kinase is novel and distinct from TPKI, II and CKI, II.

omega-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P(2) membranes suggested that Thr(11), Tyr(13) and Lys(2) are essential for binding to both N- and P/Q-type channels, whereas Lys(4) and Arg(22) are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs.