Rat hepatocytes secrete free oligosaccharides.

We recently established a method for the isolation of serum-free oligosaccharides, and characterized various features of their structures. However, the precise mechanism for how these glycans are formed still remains unclarified. To further investigate the mechanism responsible for these serum glycans, here, we utilized rat primary hepatocytes to examine whether they are able to secrete free glycans. Our findings indicated that a diverse array of free oligosaccharides such as sialyl/neutral free N-glycans (FNGs), as well as sialyl lactose/LacNAc-type glycans, were secreted into the culture medium by primary hepatocytes. The structural features of these free glycans in the medium were similar to those isolated from the sera of the same rat. Further evidence suggested that an oligosaccharyltransferase is involved in the release of the serum-free N-glycans. Our results indicate that the liver is indeed secreting various types of free glycans directly into the serum.

Mitotic phosphorylation inhibits the Golgi mannosidase MAN1A1.

N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle.

A method for assaying peptide: N-glycanase/N-glycanase 1 activities in crude extracts using an N-glycosylated cyclopeptide.

Cytosolic peptide: N-glycanase (PNGase; NGLY1), an enzyme responsible for de-glycosylation of N-glycans on glycoproteins, is known to play pivotal roles in a variety of biological processes. In 2012, NGLY1 deficiency, a rare genetic disorder, was reported and since then, more than 100 patients have now been identified worldwide. Patients with this disease exhibit several common symptoms that are caused by the dysfunction of NGLY1. However, correlation between the severity of patient symptoms and the extent of the reduction in NGLY1 activity in these patients remains to be clarified, mainly due to the absence of a facile quantitative assay system for this enzyme, especially in a crude extract as an enzyme source. In this study, a quantitative, non-radioisotope (RI)-based assay method for measuring recombinant NGLY1 activity was established using a BODIPY-labeled asialoglycopeptide (BODIPY-ASGP) derived from hen eggs. With this assay, the activities of 27 recombinant NGLY1 mutants that are associated with the deficiency were examined. It was found that the activities of three (R469X, R458fs and H494fs) out of the 27 recombinant mutant proteins were 30-70% of the activities of wild-type NGLY1. We further developed a method for measuring endogenous NGLY1 activity in crude extracts derived from cultured cells, patients' fibroblasts, iPS cells or peripheral blood mononuclear cells (PBMCs), using a glycosylated cyclopeptide (GCP) that exhibited resistance to the endogenous proteases in the extract. Our methods will not only provide new insights into the molecular mechanism responsible for this disease but also promises to be applicable for its diagnosis.

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.

Siblings with MAN1B1-CDG Showing Novel Biochemical Profiles.

Congenital disorders of glycosylation (CDG), inherited metabolic diseases caused by defects in glycosylation, are characterized by a high frequency of intellectual disability (ID) and various clinical manifestations. Two siblings with ID, dysmorphic features, and epilepsy were examined using mass spectrometry of serum transferrin, which revealed a CDG type 2 pattern. Whole-exome sequencing showed that both patients were homozygous for a novel pathogenic variant of MAN1B1 (NM_016219.4:c.1837del) inherited from their healthy parents. We conducted a HPLC analysis of sialylated N-linked glycans released from total plasma proteins and characterized the α1,2-mannosidase I activity of the lymphocyte microsome fraction. The accumulation of monosialoglycans was observed in MAN1B1-deficient patients, indicating N-glycan-processing defects. The enzymatic activity of MAN1B1 was compromised in patient-derived lymphocytes. The present patients exhibited unique manifestations including early-onset epileptic encephalopathy and cerebral infarction. They also showed coagulation abnormalities and hypertransaminasemia. Neither sibling had truncal obesity, which is one of the characteristic features of MAN1B1-CDG.

Glycometabolic Regulation of the Biogenesis of Small Extracellular Vesicles.

The biogenesis of small extracellular vesicles (sEVs) is regulated by multiple molecular machineries generating considerably heterogeneous vesicle populations, including exosomes and non-exosomal vesicles, with distinct cargo molecules. However, the role of carbohydrate metabolism in generating such vesicle heterogeneity remains largely elusive. Here, we discover that 2-deoxyglucose (2-DG), a well-known glycolysis inhibitor, suppresses the secretion of non-exosomal vesicles by impairing asparagine-linked glycosylation (N-glycosylation) in mouse melanoma cells. Mechanistically, 2-DG is metabolically incorporated into N-glycan precursors, causing precursor degradation and partial hypoglycosylation. N-glycosylation blockade by Stt3a silencing is sufficient to inhibit non-exosomal vesicle secretion. In contrast, N-glycosylation blockade barely influences exosomal secretion of tetraspanin proteins. Functionally, N-glycosylation at specific sites of the hepatocyte growth factor receptor, a cargo protein of non-exosomal vesicles, facilitates its sorting into vesicles. These results uncover a link between N-glycosylation and unconventional vesicle secretion and suggest that N-glycosylation facilitates sEV biogenesis through cargo protein sorting.

Free glycans derived from O-mannosylated glycoproteins suggest the presence of an O-glycoprotein degradation pathway in yeast.

In eukaryotic cells, unconjugated oligosaccharides that are structurally related to N-glycans (i.e. free N-glycans) are generated either from misfolded N-glycoproteins destined for the endoplasmic reticulum-associated degradation or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of free N-glycans is now well-understood, but the issue of whether other types of free glycans are present remains unclear. Here, we report on the accumulation of free, O-mannosylated glycans in budding yeast that were cultured in medium containing mannose as the carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, which encodes for a general transcription repressor, resulted in the accumulation of excessive amounts of free O-glycans, concomitant with a severe growth defect, a reduction in the level of an O-mannosylated protein, and compromised cell wall integrity. Our findings provide evidence in support of a regulated pathway for the degradation of O-glycoproteins in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.

A bioactive mammalian disaccharide associated with autoimmunity activates STING-TBK1-dependent immune response.

Glycans from microbial pathogens are well known pathogen-associated molecular patterns that are recognized by the host immunity; however, little is known about whether and how mammalian self-glycans activate the host immune response, especially in the context of autoimmune disease. Using biochemical fractionation and two-dimensional HPLC, we identify an abundant and bioactive free glycan, the Manß1-4GlcNAc disaccharide in TREX1-associated autoimmune diseases. We report that both monosaccharide residues and the ß1-4 linkage are critical for bioactivity of this disaccharide. We also show that Manß1-4GlcNAc is produced by oligosaccharyltransferase hydrolysis of lipid-linked oligosaccharides in the ER lumen, followed by ENGase and mannosidase processing in the cytosol and lysosomes. Furthermore, synthetic Manß1-4GlcNAc disaccharide stimulates a broad immune response in vitro, which is in part dependent on the STING-TBK1 pathway, and enhances antibody response in vivo. Together, our data identify Manß1-4GlcNAc as a novel innate immune modulator associated with chronic autoimmune diseases.

Occurrence of free sialyl oligosaccharides related to N-glycans (sialyl free N-glycans) in animal sera.

Free oligosaccharides that are structurally related to N-glycans [free N-glycans (FNGs)] are widely distributed in the cytosol of animal cells. The diverse molecular mechanisms responsible for the formation of these FNGs have been well clarified. In this study we demonstrate the wide occurrence of sialylated FNGs in sera of various animals. The features of these extracellular FNGs are quite distinct from the cytosolic FNGs, as they are Gn2-type glycans, bearing an N,N'-diacetylchitobiose unit at their reducing termini, while the cytosolic FNGs are predominantly Gn1-type, with a single GlcNAc at their reducing termini. The major structures observed varied from species to species, and the structures of the FNGs appear to be correlated with the major sialyl N-glycans on serum glycoproteins, suggesting that the serum FNGs are produced by hepatocytes. Interestingly, glycan-profiles of the FNGs indicated that they are altered in a developmental stage-dependent manner. Sialyl FNGs in the sera may not only be of biological relevance, in that they might reflect the functionality of the liver, but also can be attractive sources for obtaining uniform sialyl FNGs in the chemoenzymatic synthesis of glycoproteins.

Co-Expression of NEU2 and GBA3 Causes a Drastic Reduction in Cytosolic Sialyl Free N-glycans in Human MKN45 Stomach Cancer Cells-Evidence for the Physical Interaction of NEU2 and GBA3.

It is well known that the "free" form of glycans that are structurally related to asparagine (N)-linked glycans ("free N-glycans") are found in a wide variety of organisms. The mechanisms responsible for the formation/degradation of high mannose-type free N-glycans have been extensively studied in mammalian cells. Recent evidence, however, also suggests that sialylated, complex-type free N-glycans are also present in the cytosol of various mammalian-derived cultured cells/tissues. We report herein on an investigation of the mechanism responsible for the degradation of such sialyl free N-glycans. The findings show that the amount of glycans is dramatically reduced upon the co-expression of cytosolic sialidase NEU2 with cytosolic ß-glycosidase GBA3 in human stomach cancer-derived MKN45 cells. The physical interaction between NEU2 and GBA3 was confirmed by co-precipitation analyses as well as gel filtration assays. The NEU2 protein was found to be stabilized in the presence of GBA3 both in cellulo and in vitro. Our results thus indicate that cytosolic GBA3 is likely involved in the catabolism of cytosolic sialyl free N-glycans, possibly by stabilizing the activity of the NEU2 protein.

Endo-ß-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells.

The cytoplasmic peptide:N-glycanase (PNGase; Ngly1 in mice) is a deglycosylating enzyme involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) process. The precise role of Ngly1 in the ERAD process, however, remains unclear in mammals. The findings reported herein, using mouse embryonic fibroblast (MEF) cells, that the ablation of Ngly1 causes dysregulation of the ERAD process. Interestingly, not only delayed degradation but also the deglycosylation of a misfolded glycoprotein was observed in Ngly1(-/-) MEF cells. The unconventional deglycosylation reaction was found to be catalyzed by the cytosolic endo-ß-N-acetylglucosaminidase (ENGase), generating aggregation-prone N-GlcNAc proteins. The ERAD dysregulation in cells lacking Ngly1 was restored by the additional knockout of ENGase gene. Thus, our study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly emerging genetic disorder caused by mutation of the human NGLY1 gene.

Autophagy regulates the stability of sialin, a lysosomal sialic acid transporter.

Macroautophagy plays a critical role in catabolizing cytosolic components via lysosomal degradation. Recent findings from our studies indicate that basal autophagy is required for the efficient lysosomal catabolism of sialyloligosaccharides, and that the downregulation of sialin, a lysosomal transporter of sialic acids can cause a significant delay in the cytosolic accumulation of such glycans. The findings reported herein show that the sialin protein level was increased when the autophagy process was inhibited. This effect appears to be specific to sialin, since the amount of LAMP1, another lysosomal membrane protein, remains constant under the same conditions. Our results suggest that autophagy may regulate the stability of sialin, and it could lead to the cytosolic accumulation of sialyloligosaccharides in autophagy-defective cells.

Glucoamylase of Caulobacter crescentus CB15: cloning and expression in Escherichia coli and functional identification.

The biochemical properties of the maltodextrin-hydrolyzing enzymes of cold-tolerant proteobacterium Caulobacter crescentus CB15 remain to be elucidated, although whose maltodextrin transport systems were well investigated. We cloned the putative glucoamylase of C. crescentus CB15 (CauloGA) gene. The CauloGA gene product that was expressed in E. coli was prone to forming inclusion bodies; however, most of the gene product was expressed in a soluble and active form when it was expressed as a fusion protein with Staphylococcus Protein A. The fusion protein was purified using an IgG Sepharose column and was identified as the active GA. The optimum temperature and pH for the activity of this GA toward maltotriose as a substrate were approximately 40°C and 5.0, respectively, and a differential scanning fluorimetry (DSF) analysis revealed that the melting temperature (Tm) of CauloGA was 42.9°C. The kinetic analyses with maltotriose and other maltodextrins as the substrates indicated that CauloGA has higher kcat and smaller Km values at 30°C with both substrates compared with other GAs at lower substrate concentration. However, the enzyme activities toward the substrates decreased as the substrate concentrations increased at concentrations higher than approximately 10-fold the Km. The function-based identification of thermolabile Caulobacter GA contributes to the understanding of the maltodextrin-degradation system of C. crescentus as well as the bacterial GA's function-structure relationship.

Basal autophagy is required for the efficient catabolism of sialyloligosaccharides.

Macroautophagy is an essential, homeostatic process involving degradation of a cell's own components; it plays a role in catabolizing cellular components, such as protein or lipids, and damaged or excess organelles. Here, we show that in Atg5(-/-) cells, sialyloligosaccharides specifically accumulated in the cytosol. Accumulation of these glycans was observed under non-starved conditions, suggesting that non-induced, basal autophagy is essential for their catabolism. Interestingly, once accumulated in the cytosol, sialylglycans cannot be efficiently catabolized by resumption of the autophagic process, suggesting that functional autophagy is important for preventing sialyloligosaccharides from accumulating in the cytosol. Moreover, knockdown of sialin, a lysosomal transporter of sialic acids, resulted in a significant reduction of sialyloligosaccharides, implying that autophagy affects the substrate specificity of this transporter. This study thus provides a surprising link between basal autophagy and catabolism of N-linked glycans.

Calystegine B3 as a specific inhibitor for cytoplasmic alpha-mannosidase, Man2C1.

Cytoplasmic α-mannosidase (Man2C1) has been implicated in non-lysosomal catabolism of free oligosaccharides derived from N-linked glycans accumulated in the cytosol. Suppression of Man2C1 expression reportedly induces apoptosis in various cell lines, but its molecular mechanism remains unclear. Development of a specific inhibitor for Man2C1 is critical to understanding its biological significance. In this study, we identified a plant-derived alkaloid, calystegine B(3), as a potent specific inhibitor for Man2C1 activity. Biochemical enzyme assay revealed that calystegine B(3) was a highly specific inhibitor for Man2C1 among various α-mannosidases prepared from rat liver. Consistent with this in vitro result, an in vivo experiment also showed that treatment of mammalian-derived cultured cells with this compound resulted in drastic change in both structure and quantity of free oligosaccharides in the cytosol, whereas no apparent change was seen in cell-surface oligosaccharides. Calystegine B(3) could thus serve as a potent tool for the development of a highly specific in vivo inhibitor for Man2C1.

Evidence for an essential deglycosylation-independent activity of PNGase in Drosophila melanogaster.

BACKGROUND: Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. PRINCIPAL FINDINGS: In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. CONCLUSION: Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function.

Characterization of rice nucleotide sugar transporters capable of transporting UDP-galactose and UDP-glucose.

Using the basic local alignment search tool (BLAST) algorithm to search the Oryza sativa (Japanese rice) nucleotide sequence databases with the Arabidopsis thaliana UDP-galactose transporter sequences as queries, we found a number of sequences encoding putative O. sativa UDP-galactose transporters. From these, we cloned four putative UDP-galactose transporters, designated OsUGT1, 2, 3 and 4, which exhibited high sequence similarity with Arabidopsis thaliana UDP-galactose transporters. OsUGT1, 2, 3 and 4 consisted of 350, 337, 345 and 358 amino acids, respectively, and all of these proteins were predicted to have multiple transmembrane domains. To examine the UDP-galactose transporter activity of the OsUGTs, we introduced the OsUGTs' expression vectors into UDP-galactose transporter activity-deficient Lec8 cells. Our results showed that transfection with OsUGT1, 2 and 3 resulted in recovery of the deficit phenotype of Lec8 cells, but transfection with OsUGT4 did not. The results of an in vitro nucleotide sugar transport assay of OsUGTs, carried out with a yeast expression system, suggested that OsUGT4 is a UDP-glucose transporter rather than a UDP-galactose transporter. Although plants have multiple UDP-galactose transporter genes, phylogenic analysis indicates that plant UDP-galactose transporter genes are not necessarily evolutionary related to each other.

Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.

In eukaryotic cells, N-glycosylation has been recognized as one of the most common and functionally important co- or post-translational modifications of proteins. "Free" forms of N-glycans accumulate in the cytosol of mammalian cells, but the precise mechanism for their formation and degradation remains unknown. Here, we report a method for the isolation of yeast free oligosaccharides (fOSs) using endo-beta-1,6-glucanase digestion. fOSs were undetectable in cells lacking PNG1, coding the cytoplasmic peptide:N-glycanase gene, suggesting that almost all fOSs were formed from misfolded glycoproteins by Png1p. Structural studies revealed that the most abundant fOS was M8B, which is not recognized well by the endoplasmic reticulum-associated degradation (ERAD)-related lectin, Yos9p. In addition, we provide evidence that some of the ERAD substrates reached the Golgi apparatus prior to retrotranslocation to the cytosol. N-Glycan structures on misfolded glycoproteins in cells lacking the cytosol/vacuole alpha-mannosidase, Ams1p, was still quite diverse, indicating that processing of N-glycans on misfolded glycoproteins was more complex than currently envisaged. Under ER stress, an increase in fOSs was observed, whereas levels of M7C, a key glycan structure recognized by Yos9p, were unchanged. Our method can thus provide valuable information on the molecular mechanism of glycoprotein ERAD in Saccharomyces cerevisiae.

Analysis of CMP-sialic acid transporter-like proteins in plants.

It is commonly accepted that sialic acids do not exist in plants. However, putative gene homologs of animal sialyltransferases and CMP-sialic acid transporters have been detected in the genomes of some plants. To elucidate the physiological functions of these genes, we cloned 2 cDNAs from Oryza sativa (Japanese rice), each of which encodes a CMP-sialic acid transporter-like protein designated as OsCSTLP1 and OsCSTLP2. To examine the CMP-sialic acid transporter activity of OsCSTLP1 and OsCSTLP2, we introduced their expression vectors into CMP-sialic acid transporter activity-deficient Lec2 cells. Transfection with OsCSTLP1 resulted in recovery of the deficit phenotype of Lec2 cells, but transfection with OsCSTLP2 did not. We also performed an in vitro nucleotide sugar transport assay using a yeast expression system. Among the nucleotide sugars examined, the OsCSTLP1-containing yeast microsomal membrane vesicles specifically incorporated CMP-sialic acid, indicating that OsCSTLP1 has CMP-sialic acid transporter activity. On the other hand, OsCSTLP2 did not exhibit any nucleotide sugar transporter activity. T-DNA insertion lines of Arabidopsis thaliana targeting the homologs of the OsCSTLP1 and OsCSTLP2 genes exhibited a lethal phenotype, suggesting that these proteins play important roles in plant development and may transport important nucleotide sugars such as CMP-Kdo in physiological conditions.

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans.

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N'-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.