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Frameshift variant c.577del in the EPO gene can result in the extension of the amino acid sequence of EPO by invalidating the termination codon. As the molecular weight of its encoded protein EPO (VAR‐EPO) is similar to that of rEPO, the World Anti‐Doping Agency has published Annex B to the TD2022EPO in order to protect athletes with variant c.577del from the suspicion of rEPO administrations. However, it is still necessary to develop a confirmation method for rEPO that can discriminate rEPO from VAR‐EPO. Based on the glycosylated characteristic of EPO, we selected the detection of de‐N‐glycosylated EPO as a complementary confirmation method for rEPO in blood samples. All samples were analyzed for both intact EPO and de‐N‐glycosylated EPO with SDS‐PAGE, including rEPO spiked samples and blank samples. The results showed that, after de‐N‐glycosylation, a single‐band was detected in samples collected from non‐variant carriers, no matter whether the sample was spiked with rEPO. In samples collected from variant carriers, a double‐band was detected. The ratio of lower band to upper band increased significantly corresponding to the concentration of rEPO. We calculated a series of cut‐off values by normality distribution function to identify the presence of rEPO. Neither false positive results in blank samples nor false negative results in spiked samples at the applicable Minimum Required Performance Levels were found. This indicates that this method could be adopted as a complementary confirmation method for rEPO in blood samples. A revised testing strategy was also proposed, which would discriminate rEPO directly without further investigation. [ABSTRACT FROM AUTHOR]

N‐Glycosylated substrates that undergo ER‐associated degradation (ERAD) are often deglycosylated by the cytosolic peptide:N‐glycanase (Ngly1) during their proteasomal degradation in the cytosol. Consequently, the presence of non‐ or deglycosylated forms of such proteins after treatment with proteasome inhibitors is widely used as evidence for cytosolic deglycosylation by Ngly1. However, in this study, the accumulation of nonglycosylated RTA∆m, a model ERAD substrate, was still observed in mouse cells lacking cytosolic de‐N‐glycosylating enzymes, when treated with proteasome inhibitors. It was found that RTA∆m is normally partially N‐glycosylated, while the nonglycosylated form is rapidly degraded by proteasomes in the cytosol. Our results suggest that the occurrence of 'nonglycosylated' ERAD substrates upon treatment with proteasome inhibitors is not necessarily a clue for cytosolic deglycosylation. [ABSTRACT FROM AUTHOR]

Highlights: [•] The Trichoderma atroviride PNGase Png1 is predicted to be an inactive enzyme. [•] The T. atroviride Eng18A ENGase is an N-glycosylated protein. [•] The T. atroviride Eng18A and Eng18B ENGases are active deglycosylating enzymes. [•] The T. atroviride Eng18B ENGase, is proposed to act in the ERAD process. [Copyright &y& Elsevier]

As a part of the study to reveal the biological significance of de-N-glycosylation in plants, we analysed the structural features of free N-glycans (FNGs) accumulated inside cells and secreted to the extracellular space using a rice cell culture system. The structural analysis of FNGs obtained from the intracellular fraction revealed that the high-mannose type N-glycans with one GlcNAc residue (GN1-type) occurred at a concentration of ∼10 nmol/g, while the truncated complex type N-glycans with a N, N′-diacetylchitobiosyl unit (GN2-type) occurred at a concentration of ∼1 nmol/g. This result suggested that two kinds of glycoenzymes, cytosolic endo-β-N-acetylglucosaminidase (ENGase) and intracellular acidic peptide:N-glycanse (PNGase), are involved in the production of FNGs in rice cell as well as in other plant cells. On the other hand, in the culture medium, Lewis a epitope-containing complex and high-mannose type FNGs with the N, N′-diacetylchitobiosyl unit were found, suggesting extracellular acidic PNGase to be involved in the release of N-glycans from folded/processed glycoproteins in extracellular space. Furthermore, in the culture medium, we found unusual GN1-FNGs that have a biantennary complex type structure harbouring the Lewis a epitope, suggesting cytosolic ENGase and golgi N-glycan-processing enzymes to be involved in the production of these plant complex type FNGs. [ABSTRACT FROM PUBLISHER]

The article presents a study on tomato Endo-β-N-acetyglucosaminidase molecular cloning and gene expression analysis. It reveals that gene encoding on tomato ENGase (Endo-LE) does not have any implication on tomato ripening process. It also discusses the analysis made on the amount of messenger ribonucleic acid (mRNA) for Endo-LE found in tomato fruits.

The article reports on the changes in the structural features of free N-Glycan and endoglycosidase activity during tomato fruit ripening. It states that free N-Glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development.

Abstract: Common de-N-glycosylation protocols usually require a lengthy incubation time. Although pressure cycling technology or scientific microwave reactors can accelerate this enzyme reaction, they may not be easily accessible. In this brief report, we employed an alternative strategy using a standard domestic microwave oven to perform the de-N-glycosylation. Model glycoproteins (bovine RNase B, bovine fetuin, and human IgG) and a complex mixture from human plasma were fully deglycosylated in 20min, without any apparent adverse affects on the glycans or protein backbones. This new method provides a simple and inexpensive solution to achieve rapid de-N-glycosylation. [Copyright &y& Elsevier]

The article discusses a study which examines the expression of Endo-β-N-acetylglucosaminidase (ENGase) in Arabidopsis thaliana mutant. Findings reveal that the mutant did not demonstrate any ENGase activity due to suppression. However, it notes that Arabidopsis thaliana produce high-mannose type free N-glycans bearing N,N'-Acetylchitobiosyl unit.