Free oligosaccharide regulation during mammalian protein N-glycosylation

Abstract

During protein N-glycosylation in mammalian cells, free oligosaccharides (fOS) are generated from lipid-linked oligosaccharides by a pyrophosphatase activity and oligosaccharyltransferase and from misfolded glycoproteins by peptide:N-glycanase in both the ER and cytoplasm. Trafficking machinery comprising oligosaccharide-specific ER and lysosomal transporters, an endo-beta-N-acetyl-glucosaminidase, and the cytosolic M2C1 mannosidase drives a flux of fOS from the ER to cytoplasm and from the cytoplasm into lysosomes where fOS are degraded. Transport of fOS out of the ER is normally efficient and if inhibited causes fOS to be secreted via the Golgi apparatus. By contrast, fOS clearance from the cytosol into lysosomes is less efficient resulting in low micromolar concentrations of fOS in the cytoplasm. Structural analysis of cytosolic fOS reveals oligosaccharide families whose relative abundance highlights the importance of different ER-associated degradation (ERAD) pathways for misfolded glycoproteins and suggests that in liver cells substantial amounts of glycoproteins destined for ERAD may transit early compartments of the Golgi apparatus. Glycoprotein quality control and ERAD are controlled by N-glycan/lectin interactions and the fOS trafficking pathway would seem to ensure that fOS do not interfere with these processes which occur in both the ER and cytoplasm. Although Saccharomyces cerevisiae strains harbouring mutations in genes of the yeast fOS metabolic pathway do not display obvious phenotypes, mammalian fOS are quantitatively more important and the processes leading to their regulation are more complex, raising the possibility that distinct phenotypes will be seen in mammalian cells or animals in which fOS metabolism is modified.

Endoplasmic Reticulum-associated Degradation (ERAD) and Free Oligosaccharide Generation in Saccharomyces cerevisiae

“…Thus, the origin of the differences between our data and those of Hirayama et al (21) remains to be determined. However, our data on fOS structures occurring at steady state during the different yeast growth phases are in agreement with previous metabolic radiolabeling studies in which we show that, after 24-h chase incubations, the major Png1p-dependent [ 3 H]fOS appear to contain only 3-4 mannose residues (19,20). If the role of Ams1p is solely to demannosylate fOS (and other glycoconjugates) in order to recycle mannose into biosynthetic pathways, it makes sense that this process is only activated during postdiauxic growth when external glucose (which can be converted into mannose intracellularly) becomes limiting.…”

“…2C) that lack the capacity to glucosylate the dolichol-linked oligosaccharide that serves as donor for protein N-glycosylation in the ER. occurred normally in the vma1⌬ deletion mutant that is deficient in vacuolar acidification, indicating that fOS processing occurs in the cytosol (20), where Ams1p is synthesized and known to be active (32). In agreement with these data, it has been shown that at least the initial steps of fOS demannosylation are accelerated in atg19⌬ cells in which cytoplasm-to-vacuole targeting of Ams1p is blocked (21).…”

“…Metabolic radiolabeling studies demonstrated that 70 -80% of S. cerevisiae [ 3 H]fOS are generated by Png1p (19,20) and that Ams1p, the vacuolar mannosidase, degrades these compounds (19). In the ams1⌬ strain, although the major [ 3 H]fOS is Mns1p-generated [ 3 H]Man 8 GlcNAc 2 (d1,d3), small amounts of a [ 3 H]Man 7 GlcNAc 2 -like structure, not detected on 3 H-glycoproteins, are generated (19).…”

Nouvelles fonctions de la peptideN-glycanase indépendantes de sa capacité de déglycosylation

“…Dans les lysosomes, une autre activité est portée par la N 4 -(β-Na c é t y l g l u c o s a m i n y l ) -1 -asparaginase qui ne peut agir que si la dégradation de la partie peptidique de la glycoprotéine a eu lieu au préalable (panneau de droite). Certaines données suggèrent également qu'il existerait une activité PNGase à l'intérieur même du réticulum endoplasmique (RE), mais bien que cette activité soit sensible à l'inhibiteur Z-VAD-fmk, la protéine responsable n'a pas encore été caractérisée [32,33]. À ces activités de déglycosylation, il faut ajouter l'endo-β-N-acétylglucosaminidase cytosolique (ENGase) qui est également capable d'hydrolyser les chaînes N-liées en clivant la liaison entre les deux résidus de N-acétylglucosamine (GlcNAc) de l'oligosaccharide (panneau du milieu), et qui pourrait prendre le relais de la PNGase.…”

Generation and Metabolism of Cytosolic Free Oligosaccharides in Caenorhabditis elegans

“…Quality control and degradation of aberrant proteins are considered to be responsible for protecting cells from the stress induced by the accumulation of unfolded or inactive proteins (3). Recently, it has been demonstrated that oligomannose-type free oligosaccharides (FOSs) are generated in the cytosol and ER lumen during protein synthesis and quality control (4,5).…”