A role for N-glycanase in the cytosolic turnover of glycoproteins

Hirsch, Christian; Blom, Daniël; Ploegh, Hidde L.

doi:10.1093/emboj/cdg107

Cited by 203 publications

(199 citation statements)

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“…One model suggests that N-glycans are re-glucosylated only if positioned close to the folding defect. 21 Again, because the terminals of a polypeptide chain may be relatively easier to be unfolded, UGT1 bound on the terminal N-glycan could detect the uncovered hydrophobic patches more often and resend the protein into the folding cycle in ER. Further studies will be needed to confirm this hypothesis.…”

Section: Discussionmentioning

confidence: 99%

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Liu

Nguyen

Wolfert

et al. 2009

Biotechnology Progress

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N-glycosylation is important for the folding and quality control of membrane and secretory proteins. We used mutagenesis to introduce N-glycosylation sequons in recombinant proteins to improve their secretion in HEK293 cells. Seven recombinant proteins, with or without endogenous N-glycosylation sequons, were tested by this method. Our results indicate that N-glycosylation sequons located at the N-or C-terminal are glycosylated at high rates and thus the N-and C-terminal may be convenient sites for effectively attaching oligosaccharide chains. More importantly, introduction of oligosaccharide chains at such positions has been found to improve the secretion levels for the majority of the recombinant proteins in our studies, regardless of endogenous N-glycosylation, presumably by improving their folding in the endoplasmic reticulum. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1468Prog., 25: -1475Prog., 25: , 2009 Keywords: N-glycosylation, protein expression, secretion, protein folding, mutagenesis, quality control IntroductionAsparagine glycosylation (N-glycosylation) is an important process for the delivery of secretory and membrane proteins to the extracellular space or cell surface. Thus, most secretory and membrane proteins contain asparagine-linked (N-linked) glycans, with one or more oligosaccharide chains attached to the polypeptide backbone. For some proteins, Nglycosylation is absolutely required for their secretion. For others, it may not be necessary. Nevertheless, oligosaccharide chains have been shown to be the important parts of the proteins and may play roles in the folding, transport, degradation, structure, stability, receptor-recognition, cellular localization, or other biological activities of the glycoproteins (reviewed in Refs. 1-3). Protein N-glycosylation takes place at the membrane of the endoplasmic reticulum (ER). The ER plays a primary quality control function to ensure the fidelity and proper folding of proteins before they travel out of the compartment, as incorrectly folded proteins can be toxic to the cell. A presynthesized oligosaccharide chain on a lipid carrier, dolichol, is transferred onto the asparagine residue in the NXS/T sequon of the nascent polypeptide by the oligosaccharyltransferase (OST) complex while translation is in process. 2 The attached oligosaccharide chains continue to be modified along the maturation pathway of the proteins in the ER and the N-glycosylation status will dictate the fate of the synthesized protein. The newly synthesized protein can proceed to secretion, aggregation, or degradation depending on the status and pattern of its N-glycosylation. [4][5][6] In the overexpression and production of recombinant proteins, the efficiency of N-glycosylation will affect the yield of the protein recovery and the circulation life of a pharmaceutical molecule. Although N-glycosylation is important, it is well known that not all potential glycosylation sites are attached with oligosaccharide chains. In our routine expression experi...

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Section: Discussionmentioning

confidence: 99%

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Liu

Nguyen

Wolfert

et al. 2009

Biotechnology Progress

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“…Peptide-N-glycanase (PNGase) catalyzes the deglycosylation of several misfolded N-linked glycoproteins (2,3) by cleaving the bulky glycan chain before the proteins are degraded by the proteasome (4). PNGase is highly conserved in eukaryotes and possesses a catalytic Cys, His, and Asp triad embedded in a transglutaminase fold.…”

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confidence: 99%

“…Moreover, recent studies revealed that PNGase specifically acts on the unfolded form of high-mannose type N-glycosylated proteins (4,12,13). However, how PNGase binds to glycan chains and how it recognizes the high-mannose type substrates is unknown.…”

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confidence: 99%

Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module

Zhou

Zhao

Truglio

et al. 2006

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

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The inability of certain N-linked glycoproteins to adopt their native conformation in the endoplasmic reticulum (ER) leads to their retrotranslocation into the cytosol and subsequent degradation by the proteasome. In this pathway the cytosolic peptide-N-glycanase (PNGase) cleaves the N-linked glycan chains off denatured glycoproteins. PNGase is highly conserved in eukaryotes and plays an important role in ER-associated protein degradation. In higher eukaryotes, PNGase has an N-terminal and a C-terminal extension in addition to its central catalytic domain, which is structurally and functionally related to transglutaminases. Although the N-terminal domain of PNGase is involved in protein-protein interactions, the function of the C-terminal domain has not previously been characterized. Here, we describe biophysical, biochemical, and crystallographic studies of the mouse PNGase C-terminal domain, including visualization of a complex between this domain and mannopentaose. These studies demonstrate that the C-terminal domain binds to the mannose moieties of N-linked oligosaccharide chains, and we further show that it enhances the activity of the mouse PNGase core domain, presumably by increasing the affinity of mouse PNGase for the glycan chains of misfolded glycoproteins.endoplasmic reticulum ͉ N-linked glycoproteins ͉ proteasome ͉ protein degradation ͉ deglycosylation

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“…Misfolded N-linked glycoproteins destined for degradation are recognized by a highly conserved deglycosylating enzyme, peptide:N-glycanase (PNGase), implicated in proteasomal degradation (4). Very recent findings established that PNGase acts upstream of the proteasome and facilitates subsequent degradation of misfolded N-linked glycoproteins by deglycosylating them (5,6).…”

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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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A role for N-glycanase in the cytosolic turnover of glycoproteins

Cited by 203 publications

References 48 publications

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Enhancing the secretion of recombinant proteins by engineering N‐glycosylation sites

Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module

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

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