Endo-, beta -N-acetylglucosaminidases (ENGases) are endoglycosidases that hydrolyze the glycosidic bond between two N-acetylglucosamine residues in asparagine-linked glycans. ENGases are crucial tools for the structural analysis and glycan remodeling of glycoproteins. However, current ENGase activity assays are often complex and unsuitable for high-throughput analysis. To address this, we developed Forster resonance energy transfer (FRET)-based glycan molecular probes for the real-time detection of ENGase activity. We synthesized di-, tri-, and pentasaccharide probes bearing a fluorophore at the non-reducing end and a quencher at the reducing end, and evaluated their quenching efficiencies for activity detection. The pentasaccharide probe, MM3D, was efficiently cleaved by EndoM, resulting in a significant increase in fluorescence. These results successfully demonstrate that our Furthermore, we constructed a library of probes with diverse glycan structures. Using this library, we evaluated the activities of six commercially available ENGases and observed their distinct substrate specificities. This FRET probe library represents a valuable tool for detecting ENGase activity and will significantly contribute to advances in glycobiology research.

Cytosolic peptide:N-glycanase (Ngly1 in mammals) and endo-beta-N-acetylglucosaminidase (ENGase) are deglycosylating enzymes. Detection methods for the Ngly1 and ENGase activities in the cytosol are necessary for understanding the deglycosylation of the N-glycoproteins during endoplasmic reticulum-associated degradation. We previously reported the development of a detection method for Ngly1 using an engineered split intein that self-catalyzes protein trans-splicing (PTS) and NanoLuc luciferase. Here, we report a detection method for the ENGase activity using a PTS-based approach. By employing bioluminescence resonance energy transfer (BRET), we successfully detected the endogenous levels of Ngly1 and ENGase. The PTS-based BRET assay can be used to characterize the enzyme functions in the cell cytosol.

High-affinity inhibitors of specific receptors are a valuable tool to elucidate cellular signaling, control biological systems and develop therapeutic drugs. Here, we report mixed squaramide thioesters as a novel electrophilic handle, demonstrating an optimal balance between nucleophile accessibility and hydrolytic stability in aqueous environments. Density function theory calculations show that the energy of the lowest unoccupied molecular orbital (LUMO) of mixed squaramide thioesters is lower compared to mixed squaramide esters, rendering them more reactive to nucleophilic attack. Synthetic access to various lysophosphatidylglucoside (LysoPtdGlc) analogues incorporating mixed squaramide thioesters as phosphate bioisosteres was readily established by condensation of mixed squaramide esters with the corresponding thiols. Next, we characterised the inhibitory activity of these analogues in biological assays of axon growth cone chemotropism in cultured primary nociceptive neurons. These synthetic analogues induce both acute and sustained (more than 12 h) inhibition of the GPR55/LysoPtdGlc signaling. This inhibition resulted in sustained antagonistic attenuation of GPR55-mediated axon chemotropism while preserving growth cone sensitivity to other GPR55-independent chemotropic signaling molecules. Our findings demonstrate the potential of thiosquaramide-based phosphate bioisosteres as highly specific inhibitors with well controlled reactivity, expanding the repertoire of modulators for lipid-sensing GPCRs.

The silicon nanowire (SiNW) biosensors were fabricated on an n-type SOI substrate with optimized nanowire width and doping concentration to enhance sensitivity. Using SiNW biosensors with similar structural and electrical characteristics, we developed a biosensor platform capable of detecting concanavalin A (Con A) through lectin-glycan interactions by precisely controlling the mannose density on the sensor surface. Mannose molecules were immobilized on the SiNW surface at controlled ratios of 0%-100%, enabling specific systematic evaluation of Con A binding. The resistance change ratio increased significantly with higher mannose ratio compared to the non-glycosylated compound, while sensors without mannose showed no measurable response. Furthermore, the negligible response to peanut agglutinin confirmed the specificity of mannose-Con A recognition. These results demonstrate that mannose-functionalized SiNW biosensors enable selective and real-time detection of lectin-glycan interactions. This approach may contribute to glycan-based disease diagnosis and biosensing applications.

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme acting on asparagine(N)-linked glycans on glycoproteins. It is well established that fish possesses two PNGases with distinct properties. One is a cytosolic PNGase (NGLY1 in humans), active at neutral pH and widely conserved among eukaryotes. The other is called acid PNGase and is found in fish embryos; it is active at acidic pH and is believed to be of lysosomal origin. The gene encoding the acid PNGase has not been identified in animals, and its evolutionary distribution has remained unknown. In this study, we identified the gene encoding the acid PNGase, which we named Ngly2, in zebrafish (Danio rerio). Interestingly, zebrafish Ngly2 was found to have structural similarity with bacterial PNGase (PNGase F) and indeed appeared to share common catalytic residues, despite the fact that these two enzymes exhibit quite distinct pH profiles. The structure of zebrafish Ngly2 was determined by cryo-EM, showing that it forms homodimers and that its substrate is accommodated in the cleft between the protease-associated domain and PNGase domain, where the catalytic residues are located. Tissue distribution analysis indicated that ngly2 was almost exclusively expressed in the ovary. A zebrafish ngly2-KO line was found to be fertile, survive well, and show no overt phenotypes, although it had significantly smaller fertilized eggs. It was also revealed that ngly2 KO resulted in a substantial reduction in the level of free oligosaccharides in fertilized eggs, implying that Ngly2, not Ngly1, is responsible for the formation of most, if not all, egg-free glycans.

LacdiNAc (GalNAcβ1-4GlcNAc) is a distinctive epitope found at the non-reducing termini of both N- and O-glycans. In recent years, the physiological functions of LacdiNAc have attracted increasing attention. Consequently, there is a significant demand for pure glycans for use in biochemical experiments. In this study, a concise and practical synthetic approach was developed for biantennary complex-type nonasaccharide 1, which contains LacdiNAc structures at the non-reducing end. Specifically, nonasaccharide 2 was initially constructed in a stereoselective manner via the condensation of trisaccharide donor 3 with trisaccharide acceptor 4, which bears hydroxy groups at the 3- and 6-positions of the β-mannose residue. Notably, this was achieved via the remote neighboring group participation of a pivaloyl group. Subsequent conversion of the N-phthalimido group into an acetamido group, followed by global deprotection, furnished the target compound, 1. The developed synthetic route represents a valuable tool for future investigations into LacdiNAc-modified N-glycans.

Vertebrate glycerophospholipids typically exhibit a glycerol-3-phosphate (G3P)-configured backbone corresponding to the R-configured stereoisomer at the sn-2 chiral center. We previously found that the lysoglycerophospholipid lyso-phosphatidyl-β-d-glucoside (LysoPtdGlc) with G3P configuration, R-LysoPtdGlc, is an endogenous ligand of the G protein-coupled receptor GPR55, acting as an axon guidance cue via GPR55-Gα13 signaling. However, LysoPtdGlc is hydrolytically derived from phosphatidyl-β-d-glucoside (PtdGlc), which exists in vivo in a 6:1 mixture of two stereoisomers, G3P-configured R-PtdGlc and glycerol-1-phosphate-configured S-PtdGlc. To test whether the stereoconfiguration of LysoPtdGlc influences its biological activity, we combined molecular dynamics simulations of GPR55 activation by R-LysoPtdGlc and S-LysoPtdGlc with in vitro and in vivo biological assays of GPR55-mediated functions in nervous system. Molecular dynamics simulations predicted that R-LysoPtdGlc, but not S-LysoPtdGlc, remained in the putative ligand-binding pocket of the GPR55-Gα13 complex. Utilizing our previously established synthetic access to R-LysoPtdGlc and S-LysoPtdGlc, we investigated in vitro axonal chemotropic responses to these two stereoisomers. We observed R-LysoPtdGlc-mediated chemorepulsion corroborating our previous studies, and unexpectedly, S-LysoPtdGlc-induced chemoattraction via GPR55-GαS. Since these phenomena were observed in nociceptive neurons, we tested whether intrathecal administration of R-LysoPtdGlc or S-LysoPtdGlc induced a nociceptive phenotype in adult mice and found that R-LysoPtdGlc but not S-LysoPtdGlc increased behavioral sensitivity to mechanical stimuli, and that this response was dependent on GPR55. These data indicate that the stereoconfiguration of LysoPtdGlc determines its biological activity and suggest, at least in vitro, that LysoPtdGlc stereoisomers exert distinct GPR55-mediated functions via different Gα subunits.

Endo-beta-N-acetylglucosaminidases (ENGases) cleave the N-glycan core from glycoproteins and are important tools for structural analysis of glycans. Currently, only a few ENGases can specifically cleave the complex-type N-glycans, highlighting the need for discovering novel ENGases for glycoengineering applications. Nevertheless, conventional techniques for ENGase analysis are limited by low throughput and a lack of real-time detection. Herein, we describe the development of a fluorescence quenching assay to measure the hydrolytic activity of ENGases against fucosylated and afucosylated biantennary complex-type N-glycans. Two chemically synthesized probes, MG2FD (1) and MG2D (2)-a decasaccharide and a nonasaccharide, respectively-were labeled with an N-methylanthraniloyl group (fluorophore) and a 2,4-dinitrophenyl group (quencher). These probes were used to evaluate commercially available ENGases: Endo-M, Endo-CC, Endo-F3, Endo-H, and Endo-S. Endo-M and Endo-CC selectively cleaved probe 2 but not probe 1; Endo-F3 cleaved only probe 1; however, Endo-H and Endo-S showed no detectable activity. These findings align with known substrate specificities, validating the assay as a rapid and reliable method for assessing ENGase activity, profiling substrate specificity, and identifying novel ENGases targeting complex-type glycans.

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-β-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

The cytosolic peptide:N-glycanase (PNGase) is involved in the quality control of N-glycoproteins via the endoplasmic reticulum-associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F-box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man(3)GlcNAc(2), which corresponds to the core pentasaccharide of N-glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar-binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency.

Chemo-enzymatic glycan engineering is considered to be one of the most promising strategies to enhance efficiency in pharmaceutical research. However, it is assumed that this technology has limited industrial application for the production of biological therapeutics because of the high cost of the process. In this study, we developed a scheme for rapidly preparing a glycan oxazoline and a homogeneously glycosylated antibody. The enzymeimmobilized monolith and the flow chemistry-based approach enabled a glycan oxazoline and a homogeneously glycosylated antibody to be obtained at the gram scale from starting materials (sialylglycopeptide and heterogeneously glycosylated protein) within 2.5 h. This cost-effective scheme for obtaining a large amount of glycan donors and homogeneously glycosylated proteins in a short time will be helpful to implement glycan engineering technology for industrial purposes such as pharmaceutical production.

Human orthogonal enzymes (HOEs) do not show the same activities as the endogenous enzymes of human cells and thus are useful as amplification enzymes to detect antigen proteins in biological samples. Here, we evaluate a new HOE from Escherichia coli, , alpha-sulfoquinovosidase (alpha-SQase). We confirmed that the activity of alpha-SQase did not exist in examined human cell lines, and thus it was applicable to live-cell enzyme-linked immunosorbent assay (ELISA) in which the antigen membrane protein on cells was detected without inactivating endogenous enzymes, a pretreatment required for cell ELISA using conventional amplification enzymes. Here, we also developed a fluorescent substrate for alpha-SQase whose active residue is located at the end of the narrow, deep pocket of the substrate recognition site. The designed methylumbelliferyl substrate with a hydroxyl benzyl alcohol linker showed a similar reactivity to the p- nitrophenol substrate, a good substrate for alpha-SQase.

A fluorescence-quenching-based assay system was constructed to determine the hydrolytic activity of endo-β-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.

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.

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-D-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.

Oligomannose-type glycans on glycoproteins are important signaling molecules in the glycoprotein quality control system in the endoplasmic reticulum. Recently, free oligomannose-type glycans generated by the hydrolysis of glycoproteins or dolichol pyrophosphate-linked oligosaccharides were recognized as important signals for immunogenicity. Hence, there is a high demand for pure oligomannose-type glycans for biochemical experiments; however, the chemical synthesis of glycans to achieve high-concentration products is laborious. In this study, we demonstrate a simple and efficient synthetic strategy for oligomannose-type glycans. Sequential regioselective α-mannosylation at the C-3 and C-6 positions of 2,3,4,6-unprotected galactose residues in galactosylchitobiose derivatives was demonstrated. Subsequently, the inversion of the configuration of the two hydroxy groups at the C-2 and C-4 positions of the galactose moiety was successfully carried out. This synthetic route reduces the number of the protection–deprotection reactions and is suitable for constructing different branching patterns of oligomannose-type glycans, such as M9, M5A, and M5B.

Atherosclerosis is a major cause of cerebral and cardiovascular diseases. Intravascular plaques, a well-known pathological finding of atherosclerosis, have a necrotic core composed of macrophages and dead cells. Intraplaque macrophages, which are classified into various subtypes, play key roles in maintenance of normal cellular microenvironment. Excessive uptake of oxidized low-density lipoprotein causes conversion of macrophages to foam cells, and consequent progression/exacerbation of atherosclerosis. G-protein-coupled receptor 55 (GPR55) signaling has been reported to associate with atherosclerosis progression. We demonstrated recently that lysophosphatidylglucoside (lysoPtdGlc) is a specific ligand of GPR55, although in general physiological ligands of GPR55 are poorly understood. Phosphatidylglucoside is expressed on human monocytes and can be converted to lysoPtdGlc. In the present study, we examined possible involvement of lysoPtdGlc/GPR55 signaling in foam cell formation. In monocyte-derived M2c macrophages, lysoPtdGlc/GPR55 signaling inhibited translocation of ATP binding cassette subfamily A member 1 to plasma membrane, and cholesterol efflux. Such inhibitory effect was reversed by GPR55 antagonist ML193. LysoPtdGlc/GPR55 signaling in M2c macrophages was involved in excessive lipid accumulation, thereby promoting foam cell formation. Our findings suggest that lysoPtdGlc/GPR55 signaling is a potential therapeutic target for inhibition of atherosclerosis progression.

1-stearoyl (18:0)-2-arachidoyl (20:0)-sn-glycero-3-phospho-ß-D-glucoside (Phosphatidylglucoside or PtdGlc) was synthesized by direct coupling of D-glucose with the phosphate group of phosphatidic acid (18:0, 20:0). Selective in situ activation of the anomeric center of D-glucose by 2-chloro-1,3-dimethylimidazolinium chloride (DMC) in aqueous media allows the omission of protecting groups while furnishing the required ß-phosphate linkage with high selectivity. The described method is suitable to access PtdGlc in mg scale utilizing a simple two step purification protocol.