Controlling -linked glycan site occupancy

Jones, Jullian; Krag, Sharon S.; Betenbaugh, Michael J.

doi:10.1016/j.bbagen.2005.07.003

Cited by 156 publications

(125 citation statements)

References 197 publications

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“…Because of the heterogenous nature of the N-glycosylation site occupancy [33], it is possible to produce receptors containing variable degrees of N-glycosylation leading to cleavable and uncleavable conformations. This implies a regulatory mechanism whereby the eventual cellular function of a specific adhesion-GPCR could possibly be determined by the efficiency of site-specific N-glycosylation, and hence the relative amounts of the cleaved and uncleaved receptors.…”

Section: Discussionmentioning

confidence: 99%

Site‐specific N‐glycosylation regulates the GPS auto‐proteolysis of CD97

et al. 2009

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a b s t r a c tAuto-proteolysis at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is a hallmark of adhesion-GPCRs. Although defects in GPS auto-proteolysis have been linked to genetic disorders, information on its regulation remains elusive. Here, we investigated the GPS proteolysis of CD97, a human leukocyte-restricted and tumor-associated adhesion-GPCR. We found that CD97 is incompletely processed, unlike its close homolog, epidermal growth factor-like module-containing mucin-like hormone receptor 2. A unique pattern of N-glycosylation within the GPS motif of related adhesion-GPCRs was identified. The use of N-glycosylation inhibitors and mutants confirm site-specific N-glycosylation is an important determinant of GPS proteolysis in CD97. Our results suggest that N-glycosylation may regulate the processing of adhesion-GPCRs leading to the production of either cleaved or uncleaved molecules.

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Section: Discussionmentioning

confidence: 99%

Site‐specific N‐glycosylation regulates the GPS auto‐proteolysis of CD97

et al. 2009

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“…11,12 The discovery of the quality control system in ER has further elucidated the relationship between the protein N-glycosylation and its secretion. [1][2][3]12 There may be different pathways for proteins to be secreted from cells. 13 The addition of N-glycosylation should increase the chance for proteins to be folded correctly in the ER and therefore will enhance the secretion.…”

Section: Discussionmentioning

confidence: 99%

“…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][2][3]. Protein N-glycosylation takes place at the membrane of the endoplasmic reticulum (ER).…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

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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“…It is the most prevalent co-translational modification that occurs in the lumen of the ER. N-linked glycosylation plays an important role in maintaining protein stability, modulating protein-protein interactions, and protein membrane targeting (19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

Tie¹,

Zheng²,

Pope³

et al. 2006

Biochemistry

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The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In the present study, we identified the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster in SDS-PAGE gel analyses, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site not recoverable by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all the five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by eliminating the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small pentapeptide FLEEL was used as substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but it is important for protein folding and stability.The vitamin K-dependent carboxylase, also known as gamma-glutamyl carboxylase (GGCX) 1 , is an integral membrane protein of the endoplasmic reticulum. It catalyzes the post-translational modification of specific glutamic acid residues of vitamin K-dependent proteins to γ-carboxyglutamic acid residues. This post-translational modification is critical for the biological functions of the vitamin K-dependent proteins involved in blood coagulation, bone metabolism, signal transduction, and cell proliferation (1-3). In addition to its vitamin K-dependent substrate, the carboxylation reaction, which occurs in the lumen of the ER (4, 5), requires the co-substrates carbon dioxide, oxygen, and vitamin K hydroquinone. During the process of carboxylation, the γ-proton of the glutamic acid is abstracted, followed by the addition of carbon dioxide (6-9). Concomitant with carboxylation, vitamin K hydroquinone is oxidized to vitamin K epoxide which must be converted back to vitamin K by the enzyme vitamin K epoxide reductase, thus completing the vitamin K cycle (1, 10). The formation of vitamin K epoxide during the carboxylation reaction has been called an epoxidation reaction (9,11,12 It has been reported that GGCX binds to lectin suggesting that it is a glycoprotein (14, 15). Moreover, Endoglycosidase H treatment of GGCX purifi...

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Controlling -linked glycan site occupancy

Cited by 156 publications

References 197 publications

Site‐specific N‐glycosylation regulates the GPS auto‐proteolysis of CD97

Site‐specific N‐glycosylation regulates the GPS auto‐proteolysis of CD97

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

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

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