The occurrence of nonglycosylated forms of N‐glycoprotein upon proteasome inhibition does not confirm cytosolic deglycosylation

Abstract

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.

Rat hepatocytes secrete free oligosaccharides

“…To validate this hypothesis, we treated rat primary hepatocytes with NGI-1, a known cellular inhibitor of OST activity ( 33 ). It has previously been shown that this compound inhibits the release of FNGs in Ngly1 –/– Engase –/– mouse embryonic fibroblast cells ( 38 ). Our results showed that the NGI-1 treatment indeed significantly decreased the amount of Gn2-type sialyl FNGs ( Fig.…”

“…The gradient program was as follows: 0 to 0.5 min, 1 to 10% eluent B; 0.5 to 45 min, 10 to 55% eluent B; 45 to 47 min, isocratic 70% eluent B; and 47 to 67 min, isocratic 1% eluent B. The jack bean α-mannosidase-sensitive peaks, collected from dual gradient reversed phase HPLC, were further separated in the same column and eluent A/B using a previously reported program ( 38 ).…”