<i>N</i>-glycan processing in ER quality control

Ruddock, Lloyd W.; Molinari, Maurizio

doi:10.1242/jcs.03225

Cited by 262 publications

(212 citation statements)

References 90 publications

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“…N-glycosylation plays a very important role in protein folding in the ER by the association of the monoglucosylated oligosaccharide chain with the ER lectins, calnexin or calreticulin and ERp57. [4][5][6] The re-monoglucosylation of the oligosaccharide chain is regulated by UDP-glucose-glycoprotein glucosyltransferase (UGT1). One model suggests that N-glycans are re-glucosylated only if positioned close to the folding defect.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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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%

Section: Introductionmentioning

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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“…Since UDP-glucose/-galactose are crucial glycosyl donors, their deficiencies in these cells will undoubtedly impair protein glycosylation reactions [50], and could in turn trigger ER stress [30,31,50]. Indeed, aberrant glycosylation of glycoproteins and glycolipids have been widely reported in patients with Classic Galactosemia [13,[51][52][53][54][55].…”

Section: Discussionmentioning

confidence: 99%

“…The reduction of EGFR, a heavily glycosylated protein with 11 Nlinked glycosylation sites in the GALT-deficient cells observed in this study is likely to be resulted from impaired protein glycosylation, which would have initiated ER stress in this case. At the same time, ER stress evoked in this case might have led to reduced translation of EGFR mRNA and/or increased degradation of EGFR protein through ER stress associated degradation (ERAD) ( Table 1) [50,56], further diminishing the abundance of this membrane protein (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

Slepak

Tang

Slepak

et al. 2007

Molecular Genetics and Metabolism

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Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity in humans leads to a potentially lethal disorder called Classic Galactosemia. It is well known that patients often accumulate high levels of galactose metabolites such as galactose-1-phosphate (gal-1-p) in their tissues. However, specific targets of gal-1-p and other accumulated metabolites remain uncertain. In this study, we developed a new model system to study this toxicity using primary fibroblasts derived from galactosemic patients. GALT activity was reconstituted in these primary cells through lentivirus-mediated gene transfer. Gene expression profiling showed that GALT-deficient cells, but not normal cells, responded to galactose challenge by activating a set of genes characteristic of endoplasmic reticulum (ER) stress. Western Blot analysis showed that the master regulator of ER stress, BiP, was up-regulated at least three-fold in these-cells upon galactose challenge. We also found that treatment of these cells with galactose, but not glucose or hexose-free media reduced Ca 2+ mobilization in response to activation of Gq-coupled receptors. To explore whether the muted Ca 2+ mobilization is related to reduced inositol turnover, we discovered that gal-1-p competitively inhibited human inositol monophosphatase (hIMPase1). We hypothesize that galactose intoxication under GALT deficiency resulted from accumulation of toxic galactose metabolite products, which led to the accumulation of unfolded proteins, altered calcium homeostasis, and subsequently ER stress.

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“…N-glycans also can promote folding and quality control indirectly by serving as recognition "tags," or sorting signals, that allow glycoproteins to interact with a variety of lectins, glycosidases, and glycosyltransferases (37,39,40,77) (Table 2). Immediately after coupling the oligosaccharide core to the asparagine of the N-glycosylation consensus site, the two outermost terminal glucose residues are removed sequentially by glucosidase I and glucosidase II.…”

Section: Role Of N-glycans In Protein Folding Stability and Qualitymentioning

confidence: 99%

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

Vagin

Kraut²,

Sachs³

2009

American Journal of Physiology-Renal Physiology

178

157

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ized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the transGolgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors. apical sorting; apical membrane retention; H-K-ATPase ␤-subunit; lectin EPITHELIAL CELLS CARRY OUT vectorial transport that requires polarized distribution of transporters and receptors to apical or basolateral membrane domains. These domains are separated by tight junctions that connect neighboring cells in the epithelial monolayer and act as diffusion barriers to prevent mixing of apical and basolateral membrane components (22). Asymmetric distribution of plasma membrane proteins is accomplished by their sorting into apical and basolateral containers in the trans-Golgi network (TGN) and/or endosomes followed by vectorial transport of these containers and insertion and retention of the proteins in the appropriate plasma membrane domains. Sorting depends on recognition of apical and basolateral sorting signals within the proteins by cellular sorting machinery (20,58,59,75,76).Numerous studies have indicated that both O-and N-glycans attached to the extracellular domains of some membrane proteins are important for apical location of these proteins. This review will focus on the role of N-glycans in polarized distribution of plasma membrane proteins in epithelia. The role of O-glycans in apical sorting has been described in several recent excellent reviews (18, 71) and will not be discussed further here.A putative role of N-glycans as apical sorting signals was postulated more than 10 years ago (24, 31). However, this hypothesis remains controversial primarily because N-glycans are important for the processes that precede or follow the actual sorting event, such as protein folding, quality control, endoplasmic reticulum (ER)-associated degradation, ER-to-Golgi trafficking, and retention of glycoproteins in the apical membrane. Therefore, merely examining the effect of altering the number or the nature of N-linked glycans on the relative abundance of the glycoprotein in the apical membrane, as has been done in many of the studies reported, does not allow one to distinguish between effects on apica...

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N-glycan processing in ER quality control

Cited by 262 publications

References 90 publications

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

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

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

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