Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress.

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

Synthesis of a fluorescent probe for measuring the activity of endo-ß-N-acetylglucosaminidases recognizing hybrid-type N-glycans.

A fluorescence-quenching-based assay system was constructed to determine the hydrolytic activity of endo-ß-N-acetylglucosaminidases (ENGases) interacting with hybrid-type N-glycans. This was achieved using a dual-labeled fluorescent probe with a nonasaccharide structure. We produced the nonasaccharide skeleton by the stepwise glycosylation of the galactose residue on a galactosyl chitobiose derivative. Next, we introduced azido and acetoxy groups into the nonasaccharide derivative in a stepwise manner, which led to stereochemistry inversion at both the C-4 and C-2 hydroxy groups on its galactose residue. The protecting groups of the resulting nonasaccharide derivative were removed, and the derivative was labeled with an N-methylanthraniloyl group to obtain a reporter dye and a 2,4-dinitrophenyl group as a quenching molecule to obtain target probe 1. The use of this probe along with a microplate reader enabled a facile evaluation of the hydrolytic activities of ENGases Endo-H, Endo-M, Endo-F3, Endo-S, and Endo-CC. Furthermore, this probe could also assist in the search for novel ENGases that are specific to hybrid-type N-glycans.

GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice.

Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis.

Protecting-group-free glycosylation of phosphatidic acid in aqueous media.

The glycosylation of unprotected carbohydrates has emerged as an area of significant interest because it obviates the need for long reaction sequences involving protecting-group manipulations. Herein, we report the one-pot synthesis of anomeric glycosyl phosphates through the condensation of unprotected carbohydrates with phospholipid derivatives while retaining high stereo- and regioselective control. The anomeric center was activated using 2-chloro-1,3-dimethylimidazolinium chloride to facilitate condensation with glycerol-3-phosphate derivatives in an aqueous solution. A water/propionitrile mixture provided superior stereoselectivity while maintaining good yields. Under these optimized conditions, the condensation of stable isotope-labeled glucose with phosphatidic acid provided efficient access to labeled glycophospholipids as an internal standard for mass spectrometry.

Occurrence of free N-glycans with a single GlcNAc at the reducing termini in animal sera.

Recent studies demonstrated the occurrence of sialyl free N-glycans (FNGs) in sera from a variety of animals. Unlike the intracellular FNGs that mainly carry a single N-acetylglucosamine at their reducing termini (Gn1-type), these extracellular FNGs have an N,N'-diacetylchitobiose at their reducing termini (Gn2-type). The detailed mechanism for how they are formed, however, remains unclarified. In this study, we report on an improved method for isolating FNGs from sera and found that, not only sialyl FNGs, but also neutral FNGs are present in animal sera. Most of the neutral oligomannose-type FNGs were found to be Gn1-type. We also found that a small portion of sialyl FNGs were Gn1-type. The ratio of Gn1-type sialyl FNGs varies between species, and appears to be partially correlated with the distribution of lysosomal chitobiase activity. We also identified small sialylated glycans similar to milk oligosaccharides, such as sialyl lactose or sialyl N-acetyllactosamine in sera. Our results indicate that there are varieties of free oligosaccharides in sera and the mechanism responsible for their formation is more complicated than currently envisaged.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

Synthesis of 3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside (ODSG) as a lipase-resistant SQAP derivative.

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).

Fluorogenic probe for measuring high-mannose type glycan-specific endo-ß-N-acetylglucosaminidase H activity.

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-ß-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the ß-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.

Specificity of Donor Structures for endo-ß-N-Acetylglucosaminidase-Catalyzed Transglycosylation Reactions.

To demonstrate the structural specificity of the glycosyl donor for the transglycosylation reaction by using endo-ß-N-acetylglucosaminidase from Mucor hiemalis (endo-M), a series of tetrasaccharide oxazoline derivatives was synthesized. These derivatives correspond to the core structure of an asparagine-linked glycoprotein glycan with a ß-mannose unit of a non-natural-type monosaccharide, including ß-glucose, ß-galactose, and ß-talose in place of the ß-mannose moiety. The transglycosylation activity of wildtype (WT) endo-M and two mutants, N175Q and N175A, was examined by using these tetrasaccharide donors with p-nitrophenyl N-acetylglucosaminide (GlcNAc-pNp). The essential configuration of the hydroxy group for the transglycosylation reaction was determined. On the basis of these results, the transglycosylation reaction was investigated by using chemically modified donors, and transglycosylated products were successfully obtained.

A New Fluorogenic Probe for the Detection of endo-ß-N-Acetylglucosaminidase.

We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-ß-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and with a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N-glycanase 1 (NGLY1) deficiency.

A novel glucuronoyl esterase from Aspergillus fumigatus-the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters.

Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40-50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.

Reactivity of anti-HNK-1 antibodies to branched O-mannose glycans associated with demyelination.

Human natural killer-1 (HNK-1) epitope, a highly-expressed glycan in the nervous system, is critical for normal synaptic plasticity and spatial learning. HNK-1 epitope modifies N-glycans on several neural glycoproteins, and also modifies O-mannosyl glycans. A branching enzyme for O-mannosyl glycans (GnT-IX, Core M2 synthase) exhibits brain-specific expression, and the product core M2 glycans are also limited to the brain. In a previous study, we showed that cuprizone-induced demyelination increased HNK-1-capped core M2 glycan expression, while GnT-IX deficiency ameliorated demyelination, suggesting that these glycans could be useful diagnostic markers for demyelination status and act as therapeutic targets. Nevertheless, a lack of appropriate detection tools hampered further analysis of HNK-1-capped O-mannosyl glycans. In the present study, we chemoenzymatically synthesized HNK-1-capped core M2 glycans for antibody production, and confirmed that the resulting immune sera reacted with HNK-1-capped core M2 glycans. We then examined several HNK-1-related antibodies, including the Cat-315 antibody, for reactions with HNK-1-capped core M2 glycans. Finally, we confirmed the increased HNK-1 epitope expression in demyelinated brains of cuprizone-fed mice.

Endo-α-Mannosidase-Catalyzed Transglycosylation.

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo-α-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α-glycosyl-fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1- 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc1 Man9 GlcNAc2 ).

Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe.

The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc2Man3-Dansyl was faster than that of Glc1Man3-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2) although the activity was low.

Scotopic vision in the monkey is modulated by the G protein-coupled receptor 55.

The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the monkey retina, suggesting its possible role in scotopic vision. To test this hypothesis, we recorded full-field electroretinograms (ERGs) after the intravitreal injection of the GPR55 agonist lysophosphatidylglucoside (LPG) or the selective GPR55 antagonist CID16020046 (CID), under light- and dark-adapted conditions. Thirteen vervet monkeys (Chlorocebus sabaeus) were used in this study: four controls (injected with the vehicle dimethyl sulfoxide, DMSO), four injected with LPG and five with CID. We analyzed amplitudes and latencies of the a-wave (photoreceptor responses) and the b-wave (rod and cone system responses) of the ERG. Our results showed that after injection of LPG, the amplitude of the scotopic b-wave was significantly higher, whereas after the injection of CID, it was significantly decreased, compared to the vehicle (DMSO). On the other hand, the a-wave amplitude, and the a-wave and b-wave latencies, of the scotopic ERG responses were not significantly affected by the injection of either compound. Furthermore, the photopic ERG waveforms were not affected by either drug. These results support the hypothesis that GPR55 plays an instrumental role in mediating scotopic vision.

Preferential binding of E. coli with type 1 fimbria to D-mannobiose with the Manα1 → 2Man structure.

Manα1 → 2Man, Manα1 → 3Man, Manα1 → 4Man, and Manα1 → 6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco™ beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1 → 2Man1 → beads, Manα1 → 3Man1 → beads, Manα1 → 4Man1 → beads, and Manα1 → 6Man1 → beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1 → 2Man disaccharide.

Loss of branched O-mannosyl glycans in astrocytes accelerates remyelination.

In demyelinating diseases such as multiple sclerosis, a critical problem is failure of remyelination, which is important for protecting axons against degeneration and restoring conduction deficits. However, the underlying mechanism of demyelination/remyelination remains unclear. N-acetylglucosaminyltransferase-IX (GnT-IX; also known as GnT-Vb) is a brain-specific glycosyltransferase that catalyzes the branched formation of O-mannosyl glycan structures. O-Mannosylation of α-dystroglycan is critical for its function as an extracellular matrix receptor, but the biological significance of its branched structures, which are exclusively found in the brain, is unclear. In this study, we found that GnT-IX formed branched O-mannosyl glycans on receptor protein tyrosine phosphatase ß (RPTPß) in vivo. Since RPTPß is thought to play a regulatory role in demyelinating diseases, GnT-IX-deficient mice were subjected to cuprizone-induced demyelination. Cuprizone feeding for 8 weeks gradually promoted demyelination in wild-type mice. In GnT-IX-deficient mice, the myelin content in the corpus callosum was reduced after 4 weeks of treatment, but markedly increased at 8 weeks, suggesting enhanced remyelination under GnT-IX deficiency. Furthermore, astrocyte activation in the corpus callosum of GnT-IX-deficient mice was significantly attenuated, and an oligodendrocyte cell lineage analysis indicated that more oligodendrocyte precursor cells differentiated into mature oligodendrocytes. Together, branched O-mannosyl glycans in the corpus callosum in the brain are a necessary component of remyelination inhibition in the cuprizone-induced demyelination model, suggesting that modulation of O-mannosyl glycans is a likely candidate for therapeutic strategies.

Identification of ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) as a regulator of N-acetylglucosaminyltransferase GnT-IX (GnT-Vb).

Our previous studies on a ß1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase.

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407-4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434-1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.

Measurement of endo-α-mannosidase activity using a fluorescently labeled oligosaccharide derivative.

Endo-α-mannosidase, a GH99-family glycoside hydrolase, cleaves α-mannoside linkages with glucose residues. This enzyme is proposed to play a critical role in N-glycan processing for deglucosylation. To measure endo-α-mannosidase activity, we synthesized a fluorescently labeled tetrasaccharide derivative (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) in a stereocontrolled manner. The tetrasaccharide skeleton was prepared by step-wise coupling using mannose donors 4 and 7. The 1,2-cis α-glycosidic linkage on the non-reducing end of the glucose residue was constructed by inversion of the stereochemistry of the C-2 hydroxyl group in the α-mannose residue. Finally, the dansyl group was introduced at the reducing end via an aminopropyl linker. This probe successfully measured endo-α-mannosidase activity.