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Free N -glycans (FNGs) are oligosaccharides that are structurally related to N -linked glycans, and are widely found in nature. The mechanisms responsible for the formation and degradation of intracellular FNGs are well characterized in mammalian cells. More recent analysis in mammalian sera shows that there are various types of extracellular free glycans, including FNGs. However, it is unknown whether these free glycans are widely distributed in vertebrates. In this study, we investigated the occurrence of free glycans in salmonid serum. We found that it contained sialyl or neutral FNGs and sialyl lactose/ N -acetyllactosamine (LacNAc)-type glycans, which was consistent with that found in mammalian sera. Many of the structures of FNGs matched those of N -glycans from serum glycoproteins. This study revealed that various types of free glycans are present in fish serum, demonstrating their wide occurrence among vertebrates. • This study identified free N -glycans (FNGs) in salmonid serum. • This study also identified sialyl lactose/ N -acetyllactosamine-type glycans. • This study highlighted the wide distribution of free glycans in vertebrate serum. • FNG structures in salmonid serum were similar to N -glycans of serum glycoproteins. [ABSTRACT FROM AUTHOR]

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]