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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.