Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I

Abstract

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

A complex between peptide: N -glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins

“…An increasing number of reports suggest that the N-glycanase is located in or on ER membrane because, in the presence of proteasome inhibitors, the glycosylated protein and deglycosylated intermediates are either exclusively or predominantly associated with the ER rather than with the cytosol (39). This model is supported by various studies using diverse glycoproteins as substrates, e.g., Ig subunits (40), class I MHC heavy chains (41) a ribophorin-I variant (42), cog thyroglobulin mutant (1), cystic fibrosis transmembrane regulator (43), and T cell receptor ␣-subunit (44). These findings also suggests that N-deglycosylation and proteasome-mediated degradation of proteins may be coupled events.…”

Role for a Zinc Finger Protein (Zfp111) in Transformation of 208F Rat Fibroblasts by Jaagsiekte Sheep Retrovirus Envelope Protein

“…The normal function of ERAD is to transport misfolded proteins (marked by ubiquitination) from the ER back to the cytosol for proteosomal degradation; it seems possible that, like MMTV Rem, JSRV P70 env could be transported back to the cytoplasm by this mechanism without degradation. In mammalian cells, misfolded proteins are targeted to the ERAD pathway following the removal of 3 to 4 mannose residues by ER processing alpha 1,2-mannosidase (ER ManI) (58)(59)(60)(61). P70 env could be a misfolded version of Env (or at least appears to be misfolded to the ERAD machinery) that becomes more abundant in cells that are cotransfected with Zfp111.…”

The Retrotranslocation Protein Derlin-1 Binds Peptide:N-Glycanase to the Endoplasmic Reticulum

“…Conflicting evidence also suggested localization in the ER lumen (Weng and Spiro, 1997). PNGase in higher eukaryotes acts on a diverse range of misfolded glycoprotein substrates, namely immunoglobulin (Ig) subunits (Mancini et al, 2000), MHC class I heavy chains (Kamhi-Nesher et al, 2001), a ribophorin I variant (Kitzmuller et al, 2003), cog thyroglobulin mutant (Tokunaga et al, 2000), cystic fibrosis transmembrane regulator (Xiong et al, 1999), and T-cell receptor (TCR) ␣-subunits (Yu et al, 1997).…”

“…PNGase has been reported to localize in the cytosol (Suzuki et al, 1998) (Suzuki et al, 1997), and the lumen of the ER (Weng and Spiro, 1997). Previous studies showed that glycosylated proteins or deglycosylated intermediates are predominantly associated with the microsomes rather than with the cytosol (Kitzmuller et al, 2003). The observation that PNGase is found in the cytosol and associated with the ER (Katiyar et al, 2004) can be explained by the presence of alternative ERAD routes, with deglycosylation either coupled to or independent of retrotranslocation (Misaghi et al, 2004).…”