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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been transmitted across all over the world, in contrast to the limited epidemic of genetically- and virologically-related SARS-CoV. However, the molecular basis explaining the difference in the virological characteristics among SARS-CoV-2 and SARS-CoV has been poorly defined. Here we identified that host sialoglycans play a significant role in the efficient spread of SARS-CoV-2 infection, while this was not the case with SARS-CoV. SARS-CoV-2 infection was significantly inhibited by α2-6-linked sialic acid-containing compounds, but not by α2–3 analog, in VeroE6/TMPRSS2 cells. The α2-6-linked compound bound to SARS-CoV-2 spike S1 subunit to competitively inhibit SARS-CoV-2 attachment to cells. Enzymatic removal of cell surface sialic acids impaired the interaction between SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2), and suppressed the efficient spread of SARS-CoV-2 infection over time, in contrast to its least effect on SARS-CoV spread. Our study provides a novel molecular basis of SARS-CoV-2 infection which illustrates the distinctive characteristics from SARS-CoV.

Glycolipid metabolism occurs in the Golgi apparatus, but the detailed mechanisms have not yet been elucidated. We used fluorescently labeled glycolipids to analyze glycolipid composition and localization changes and shed light on glycolipid metabolism. In a previous study, the fatty chain of lactosyl ceramide was fluorescently labeled with BODIPY (LacCer-BODIPY) before being introduced into cultured cells to analyze the cell membrane glycolipid recycling process. However, imaging analysis of glycolipid recycling is complicated because of limited spatial resolution. Therefore, we examined the microscopic conditions that allow the temporal analysis of LacCer-BODIPY trafficking and localization. We observed that the glycolipid fluorescent probe migrated from the cell membrane to intracellular organelles before returning to the cell membrane. We used confocal microscopy to observe co-localization of the glycolipid probe with endosomes and Golgi markers, demonstrating that it recycles mainly through the trans-Golgi network (TGN). Here, a glycolipid recycling pathway was observed that did not require the lipids to pass through the lysosome.

A series of compounds associated with naturally occurring and biologically relevant glycans consisting of α-mannosides were prepared and analyzed using collision-induced dissociation (CID), energy-resolved mass spectrometry (ERMS), and 1H nuclear magnetic resonance spectroscopy. The CID experiments of sodiated species of disaccharides and ERMS experiments revealed that the order of stability of mannosyl linkages was as follows: 6-linked > 4-linked ≧ 2-linked > 3-linked mannosyl residues. Analysis of linear trisaccharides revealed that the order observed in disaccharides could be applied to higher glycans. A branched trisaccharide showed a distinct dissociation pattern with two constituting disaccharide ions. The estimation of the content of this ion mixture was possible using the disaccharide spectra. The hydrolysis of mannose linkages at 3- and 6-positions in the branched trisaccharide revealed that the 3-linkage was cleaved twice as fast as the 6-linkage. It was observed that the solution-phase hydrolysis and gas-phase dissociation have similar energetics.

Recently, a number of chemoenzymatic strategies have been explored for achieving preparation of homogeneous glycopeptides and glycoproteins, especially by using endoglycanases and glycosyl oxazolines. However, concomitant occurrence of non-enzymatic reactions has been reported, but no further characterization of the byproducts was conducted. In this work, we made an attempt to identify the side product by using model substrates. Analysis of the product allowed us to propose that the oxazoline ring was attacked by the amino group of lysine, leading to the formation of disubstituted acetamidine.

Objectives The purpose of this study was to develop a new compound to overcome influenza epidemics and pandemics as well as drug resistance. Methods We synthesized a new compound carrying: (i) Neu5Acα2-6Galβ1-4GlcNAc (6SLN) for targeting immutable haemagglutinins (HAs) unless switched from human-type receptor preference; (ii) an acyl chain (lipo) for locking the compound with the viral HA via hydrophobic interactions; and (iii) a flexible poly-α-L-glutamic acid (PGA) for enhancing the compound solubility and for coating the viral surface, precluding accessibility of the PGA-coated virus to the negatively charged sialic acid on the host cell surface. Results 6SLN-lipo PGA appears to subvert binding of pandemic H1 and seasonal H3 HAs to receptors, as assessed by using guinea pig erythrocytes, which is critical for virus entry into host cells for multiplication. It shows high potency with IC50 values in the range of 300-500 nM against multiplication of both influenza pandemic H1N1/2009 and seasonal H3N2/2004 viruses in cell culture. It acts in synergism with either of the two FDA-approved neuraminidase inhibitor (NAI) clinical drugs, zanamivir (Relenza(®)) and oseltamivir carboxylate (active form of Tamiflu(®)), and it has the potential to aid NAI drugs to achieve complete clearance of the virus from the culture. Conclusions 6SLN-lipo PGA is a new potential candidate drug for influenza control and is an attractive candidate for use in combination with an NAI drug for minimized toxicity, delayed development of resistance, prevention and treatment with the potential for eradication of human influenza.

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.

Defining chiral centres is addressed by introducing a pair of chiral auxiliary groups. Ions of diastereomeric pairs of molecules could be distinguished utilising energy-resolved mass spectrometry, and the applicability of the method to a series of compounds carrying amine, carboxylic acid, alcohol, and all the amino acids was verified. The method was further strengthened by distinguishing diastereomeric ions that did not undergo fragmentation. Mass spectrometric evaluation of the dissociation process of adducted sodium cations from the diastereomeric precursors agreed with the theoretical calculations, indicating the potential usefulness of the method for the determination of absolute configurations.

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.)

Summary A NKT cell repertoire is characterized by the expression of the Vα19-Jα26 invariant TCR α chain (Vα19 NKT cell). This repertoire, as well as a well-established Vα14-Jα281 invariant TCR α + NKT cell subset (Vα14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Vα19 NKT cells. Vα19 as well as Vα14 NKT cells exhibited reactivity to α-galactosyl ceramide (α-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Vα19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Vα19 NKT cells by α-mannosyl ceramide.

The PLLA and PDLLA samples consisted of three types of carbon: C-C (C-H), C-O and 0=C-O, which corresponded to their theoretical composition. However, the XPS analysis revealed that the surfaces of the PLA film highly resistant to acid hydrolysis were overspread with unknown constituents consisting of nitrogen and unoxidized carbon, not being announced officially. The AFM analysis offered a clear distinction in the surface deterioration of the PLA samples; and the alkaline treatment excavated numerous holes on the PLA surfaces. These results indicated that the commercial plastics are chemically modified on their surfaces to improve the physical properties for practical use, resulting in being deprived of biodegradability and eco-compatibility. It was also suggested that the enlargement in the specific surface area, which may occur at the early stage on PLA degradation, possibly accelerated the PLA decomposition rate. A new conception in the material design for paper-based composites was proposed; a local alkaline circumstance expected by alkaline fillers, e.g., CaO available in papermaking processes would

Chemical and biological degradation behavior of commercial polylactide (PLA) film, poly (L-lactide) (PLLA) and poly (DL-lactide) (PDLLA) was investigated by gravimetric analysis, X-ray diffractometry, differential scanning calorimetry and Fourier transform Raman (FT-Raman) spectroscopy. The PLA film exhibited an extremely high resistance to acid hydrolysis by hydrochloric acid, although alkaline treatment with a dilute sodium hydroxide brought about the quick decomposition of PLAs, not being reflected in their crystallinity. The X-ray and FT-Raman results implied the immediate decomposition occurring only on the PLA surfaces. These phenomena were observed both for crystalline PLLA and amorphous PDLLA; however, at temperature above their glass transition points, the PDLLA was greater deteriorated than the PLLA under acidic conditions, whereas in the alkaline system the tendency was reversed. On the biological degradation, the PLA filrn remained almost unchanged even after buried in soil for six months, as supported by various analytical results. Possibly, the PLA=related products commercially available have lower biodegradability than expected, and thus it was indicated that a strategic material design, e.g., the utilization of paper-based ecomaterials whose practicality and biodegradability can be compatible with each other, is of significance for high performance composite-manufacturing wlth biodegradable plastics.

A series of iminocyclitols was prepared using a versatile synthetic strategy, and their inhibition of glycosidases was evaluated using capillary electrophoresis. The study has demonstrated that remarkable specificities in enzyme inhibition can be achieved with small modifications on the aglycon side chain and the ring nitrogen. Among the compounds synthesized, (2R,3R,4R,5R)-N-methyl-2-(acetamidomethyl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine was found to be very potent against β-N-acetylhexosaminidase P with the Ki value of 80 nM.

糖あるいは糖鎖は代謝、細胞間認識、感染といった様々な生体認識現象に一次的に関与しているばかりでなく、癌の転移に伴い発現する糖転位酵素もあり、タンパク質と糖の間の認識機構を調べ阻害剤等の研究を行うことは、将来的に新しい医薬へと繋がる可能性を秘めており重要である。ところが、現時点においては如何なる化合物がこうした生体反応を効率良く阻害するかを予測することは不可能に近い。このような理由から多岐に渡る化合物群の中から、目的に合うものを検索しようとするコンビナトリアルケミストリーの手法に基づく研究が有効である。また、糖が連続するキラルな炭素から成っていることを考えると、糖をキラルテンプレートに利用することも考えられる。本レビューは、盛んになりつつある糖関連ライブラリーに関する研究についての概説を試みるが、あくまでも“糖”が中心になっている研究に限定した。また、今後のライブラリー研究に展開が期待されるものについても割愛させていただいた。

Due to high demand to access biologically important oligosaccharide structures, efficient and stereoselective synthetic methods are required. This article addresses these two issues from our recent studies. The first part deals with β-mannosylation and α-sialylation known as the two most difficult glycosylation reactions which were now made possible by means of an “intramolecular aglycon delivery” system and control by “auxiliary”, respectively. In the second part, the remarkably efficient “orthogonal glycosylation strategy” which was developed based on tactical analysis of oligosaccharide synthesis is described.

Development of inhibitors specific for the glycosyltransferases involved in the biosynthesis of asparagine-linked sugar chains has been undertaken in the hopes that these compounds may serve as tools to elucidate the roles of complex carbohydrates in biological recognition events. We report here the first example of a glycosyltransferase acceptor analog in which strategic replacement of a nonreacting hydroxyl group with a larger substituent produces a molecule which is recognized by the enzyme but does not react because of a steric block to the glycosyl transfer reaction. N-Acetylglucosaminyltransferase V catalyzes the transfer of GlcNAc from the sugar nucleotide donor UDP-GlcNAc to the 6-OH group of mannose in the synthetic trisaccharide acceptor beta GlcNAc(1-->2)alpha Man(1-->6)beta Glc-O(CH2)7CH3 (Km = 23 +/- 2 microM; Vmax = 116 +/- 3 pmol/h) to form the tetrasaccharide beta GlcNAc(1-->2)(beta GlcNAc(1-->6))alpha Man(1-->6)beta Glc-O(CH2)7CH3. The acceptor analog produced by replacement of the adjacent nonreacting 4-OH group of the mannose residue with an O-methyl group was not a substrate for the enzyme but was found to be a good competitive inhibitor of GlcNAc transferase V with Ki = 14 +/- 2 microM. To test the theory that it was the presence of the large methyl group which prevented the glycosyl transfer reaction the 4'-deoxygenated analog was synthesized. It was found to be a good substrate with Km = 74 +/- 6 microM and an almost 5-fold higher kcat (Vmax = 535 +/- 13 pmol/h). NMR data show no evidence of important conformational differences between the trisaccharide analogs, and kinetic experiments detected no differences for the binding of UDP-GlcNAc in their presence. The conclusion was therefore reached that the large methyl group introduced on O-4' sterically prevented the formation of product even though both potential substrates were bound by the enzyme. This "steric exclusion" strategy offers potential for the design of inhibitors for that class of glycosyltransferases in which the reactive hydroxyl group is also an essential recognition element.

Background A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. Methodology/Principal Findings In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. Conclusions/Significance These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.

Stereocontrolled syntheses of model compounds related to a major antigenic epitope against antibupleurum 2IIc/PG-1-IgG from antiulcer pectic polysaccharide are described. A trisaccharide derivative (13) was prepared as a precursor and a novel and simple approach for the rational design of a glycocluster and glycodendrimer was developed, through the syntheses of the fluorescence-labeled glycocluster (2) and glycodendrimer (3).

A NKT cell repertoire is characterized by the expression of the Valpha19-Jalpha26 invariant TCR alpha chain (Valpha19 NKT cell). This repertoire, as well as a well-established Valpha14-Jalpha281 invariant TCR alpha(+) NKT cell subset (Valpha14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Valpha19 NKT cells. Valpha19 as well as Valpha14 NKT cells exhibited reactivity to alpha-galactosyl ceramide (alpha-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Valpha19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Valpha19 NKT cells by alpha-mannosyl ceramide.

The compound Neu5Ac3alphaF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3alphaF-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 microM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.

Background: Oligosaccharide processing enzymes are important classes of catalysts involved in synthesizing specific oligosaccharide structures on proteins and sphingolipids. Development of specific inhibitors of such enzymes is of current interest as these inhibitors may be used to control cellular functions. Five-membered iminocyclitols have been shown to be potent inhibitors of such enzymes. Since a rational design and synthesis of inhibitors is often extremely difficult due to the limited information regarding the structure of the active site, we carried out a combinatorial library approach. Results: To create diversity, we decided to use an aldehyde group of a protected iminocyclitol for reductive amination and the Strecker reaction. After transformation of the nitrile group introduced by the Strecker reaction into an amine and amide and complete deprotection, a small library of five-membered iminocyclitols consisting of 27 compounds was synthesized. A series of compounds obtained by reductive amination was first screened as potential inhibitors of glycosidases and glycosyltransferases. Among them, compounds carrying a C 10 -alkyl group showed marked enhancement of inhibitory activity against α-mannosidase at 10 μM concentration when compared with its parent compound and deoxymannojirimycin. Furthermore, compounds having the phenylethyl group showed an extremely strong inhibitory effect against α-galactosaminidase at a K i value of 29.4 nM. Compounds with an aminomethyl and amide group at the C -1′ position of these two molecules showed a decrease in inhibitory activities. Conclusions: A combinatorial approach based on five-membered iminocyclitols with a galacto -configuration was exploited. The potential usefulness of the library as a source of inhibitors of glycoenzymes is clearly shown in this study.

This paper describes the synthesis of 1,1-linked galactosyl mannosides as sialyl Lewis X mimetics that contain a spiro-ring to position the carboxylate group in a well-defined orientation. It was found that compound 4 is more active as a P-selectin inhibitor (IC50 = 19 microM) than the parent disaccharide 2, which contains a flexible carboxyl group (IC50 = 193 microM). This result is consistent with that observed in the previous NMR study of sialyl Lewis X bound to P-selectin. The chemistry described here should be useful for the development of selective inhibitors of E-, P-, and L-selectins.

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA–binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells., Author Summary Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. Accordingly, some glycans are generated in a stage- and tissue-specific manner. To address how such distinct glycosylation is regulated in different tissues, we performed a large-scale screen for genes involved in glycosylation of a neural-specific protein. We identified 109 genes, 95 of which are assigned for the first time as directly or indirectly involved in glycosylation. We further found that neural-specific glycosylation is regulated at the RNA level, which is a novel regulatory mechanism of tissue-specific glycosylation.

Glycans, a family of compounds often attached to proteins and ceramides, are diverse molecules involved in a wide range of biological functions. Their structural analysis is necessary and is often carried out at the microscale level. Methods based on mass spectrometry are therefore used, although they do not provide information regarding isomeric structures often found in glycan structures. If one finds "factors" characteristic of a certain isomer, this information can be used to elucidate an unknown oligosaccharide sequence. One potential technique is to use energy-resolved mass spectrometry (ERMS) that has been used to distinguish a pair of isomeric compounds. Thus, compounds in a combinatorial library might be effectively used for this purpose. We analyzed a set of 16 isomeric disaccharides, the structures of which consisted of all possible combinations of anomeric configurations and interglycosidic linkage positions. All of the compounds were distinguished based on ERMS where normal collision-induced dissociation could distinguish only seven compounds. Furthermore, it was shown that alpha-glycosidic linkages of fucose were more reactive than the beta-isomers and the secondary glycosides were more reactive than the primary glycosides.

Glycosylation of proteins can modulate their function in a striking variety of systems, including immune responses, neuronal activities and development. The Drosophila protein, Chaoptin (Chp), is essential for the development and maintenance of photoreceptor cells. This protein is heavily glycosylated, but the possible role of this glycosylation is not well-understood. Here we show that mutations introduced into about 1/3 of 16 potential N-linked glycosylation sites within Chp impaired its cell adhesive activities when expressed in Drosophila S2 cells. Mutation of 2/3 of the glycosylation sites resulted in a marked decrease in Chp protein abundance. These results suggest that N-linked glycosylation of Chp is essential for its stability and activity.

細胞表面に存在する糖タンパク、糖脂質や細胞間マトリックスであるプロテオグリカン等の複合糖質は、複数の糖加水分解酵素、糖転移酵素の連続的な作用により合成される。したがって、各々の糖鎖関連酵素の選択的な阻害物質は糖鎖の機能解析のために重要な役割を担うと考えられる。従来、天然物質として種々のアルカロイドやヌクレオチド類似物質が阻害能を有することが知られている。近年、糖鎖関連酵素の3次元構造の解析、特に糖加水分解酵素の結晶構造に基づき酵素阻害剤のデザイン合成が行われるようになってきた。また、コンビナトリアル合成の方法論を取り込んだ半デザイン合成によるアプローチも注目される。