Site-specific de-
            <i>N</i>
            -glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide:
            <i>N</i>
            -glycanase as a quality control system for newly synthesized proteins

Suzuki, Tadashi; Kitajima, Ken; Emori, Yasufumi; Inoue, Yasuo; Inoue, Sadako

doi:10.1073/pnas.94.12.6244

Cited by 85 publications

(72 citation statements)

References 51 publications

(48 reference statements)

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“…Localization of these proteins around the ER periphery indicates that they could be involved in the ERAD process (23). PNGase has been reported to be present in the ER (24), microsomes (25), and the cytosol (26). Some studies using immunofluorescence suggest the association of proteasomes with the cytosolic face of ER membrane both in mammalian cells (27,28) and in yeast (29,30).…”

Section: Resultsmentioning

confidence: 99%

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

Katiyar

Lennarz

2004

Proc. Natl. Acad. Sci. U.S.A.

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Peptide:N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs. Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells. Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells. Immunoprecipitation analysis revealed the interaction of h͞mPNGase with the 19S proteasome subunit, hS4, through hHR23B. Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B. Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins. Based on this finding, we propose that after deglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.

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Section: Resultsmentioning

confidence: 99%

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

Katiyar

Lennarz

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The degree of N-glycosylation of component H appeared to be higher than that of component L or hen egg OVA. Alternatively, Suzuki et al 20) have reported that in the oviduct only di-N-glycosylated OVA was cleaved immediately at Asn-311 by PNGase F, and that it is was secreted as the mono-N-glycosylated form at Asn-292. To investigate further the binding site of a carbohydrate chain on components H and L, we attempted to treat the recombinant proteins together with hen egg OVA with PNGase F in the non-denaturing condition without 2-mercaptoethanol and SDS.…”

Section: Deglycosylation and Glycosylation Profilingmentioning

confidence: 99%

Structural Characteristics of Hen Egg Ovalbumin Expressed in YeastPichia pastoris

Ito

Matsudomi

2005

Bioscience, Biotechnology, and Biochemistry

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“…As discussed previously, PNGase has been reported to be localized in the cytosol (Suzuki et al, 1998) and also reported to be associated with the ER membrane (Suzuki et al, 1997;Katiyar et al, 2004). Conflicting evidence also suggested localization in the ER lumen (Weng and Spiro, 1997).…”

Section: Discussionmentioning

confidence: 72%

“…In fact, in the studies of Wiertz et al (1996) only the deglycosylated forms of MHC class I molecules were noted to be associated with the Sec61 complex of the ER membrane, indicating that glycoproteins in this channel must also encounter N-glycanase. Another example of the ER membranelocalized deglycosylation is the protein ovalbumin in which site specific N-deglycosylation was attributed to an ER-situated N-glycanase (Suzuki et al, 1997). In contrast to HCs, TCR␣ apparently utilizes a different dislocation machinery for degradation (Misaghi et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…Reports on the cellular localization of PNGase are conflicting. 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).…”

Section: Introductionmentioning

confidence: 99%

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

Katiyar

Joshi

Lennarz

2005

MBoC

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The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein.Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate. INTRODUCTIONIn eukaryotes proteins are synthesized on membrane-bound ribosomes and undergo a folding process in the lumen of the endoplasmic reticulum (ER; Helenius, 2001, 2003). Misfolded or incompletely assembled multisubunit glycoproteins are recognized by the endoplasmic reticulumassociated degradation (ERAD) pathway regulated largely by their N-linked polymannose oligosaccharides. In this quality control system, lectin-like molecular chaperones, calnexin and calreticulin, recognize the Glc 1 Man 9 GlcNAc 2 oligosaccharide and assist in folding of newly synthesized glycoproteins. Lectin interaction with Glc 1 Man 9 GlcNAc 2 glycans after trimming with ER alpha-glucosidases, and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and degradation by the proteasome (Cresswell and Hughes, 1997;Kopito, 1997;Brodsky and McCracken, 1999;Romisch, 1999;Spiro, 2004).The mannose trimming of N-linked glycans plays an important role in the ERAD of glycoproteins (Spiro, 2004). In both yeast and human cells, it is reported that the misfolded glycoproteins in the ER are degraded through ERAD only after the glycan is trimmed to the Man8B form, while misfolded proteins stay within the ER when they retain the Man9 form of oligosaccharides. Accordingly, it was assumed that there existed a Man8-binding lectin, later identified as EDEM, that recognizes misfolded glycoproteins in the Man8B form in the ER and directs them to the ERAD pathway (Hosokawa et al., 2001). The misfolded glycoproteins exit the ER via a multiprotein complex referred to as a retrotranslocon or dislocon (Plemper et al., 1997;Bebok et al., 1998;de Virgilio et al., 1998;Tsai et al., 2002). In alternative models both PNGase and proteasomes may be either free in the cytosol or ER membrane imbedded or attached (Spiro, 2004).A well-studied model for the ERAD pathway utilizes the human cytomegalovirus, which affects the MHC class I antigen presentation. The vira...

show abstract

Site-specific de- N -glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N -glycanase as a quality control system for newly synthesized proteins

Cited by 85 publications

References 51 publications

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

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

Structural Characteristics of Hen Egg Ovalbumin Expressed in YeastPichia pastoris

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

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