The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis

Hebert, Daniel N.; Lamriben, Lydia; Powers, Evan T.; Kelly, Jeffery W.

doi:10.1038/nchembio.1651

Cited by 174 publications

(154 citation statements)

References 99 publications

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“…Endogenous wildtype Chlamydomonas RbcL is acetylated at Pro-3 (Houtz et al, 1992), hydroxylated at Pro-104 and Pro-151, and methylated at Cys-256 and Cys-369 (Taylor et al, 2001), whereas none of these modifications have been reported in Synechococcus RbcL. However, among post-translational modifications, glycosylation is believed to be the most important modification for folding and stability of polypeptides (Hebert et al, 2014). Since glycosylation does not naturally occur in wild-type endogenous Chlamydomonas RbcL, the limited post-translational modification systems in E. coli may ultimately have less of an impact on the ability of the chimeric Rubiscos to fold heterologously.…”

Section: Discussionmentioning

confidence: 97%

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

2016

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

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Section: Discussionmentioning

confidence: 97%

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

2016

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“…Or is the observed essentiality of formation of the C-terminal disulfide favored by sequence-encoded folding information? ATIII appears to rely on the lectin chaperone system comprised of calnexin, calreticulin, as well as the UDP-glucose, glycoprotein glucosyltransferase 1, which directs rebinding and ER retention of nonnative substrates (31,32). The secretion level of ATIII was reduced by two-thirds in the absence of intervention by the lectin chaperone network; however, this diminished secreted fraction was active.…”

Section: Atiiimentioning

confidence: 95%

Cellular folding pathway of a metastable serpin

Chandrasekhar

Wang

et al. 2016

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

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Although proteins generally fold to their thermodynamically most stable state, some metastable proteins populate higher free energy states. Conformational changes from metastable higher free energy states to lower free energy states with greater stability can then generate the work required to perform physiologically important functions. However, how metastable proteins fold to these higher free energy states in the cell and avoid more stable but inactive conformations is poorly understood. The serpin family of metastable protease inhibitors uses large conformational changes that are downhill in free energy to inhibit target proteases by pulling apart the protease active site. The serpin antithrombin III (ATIII) targets thrombin and other proteases involved in blood coagulation, and ATIII misfolding can thus lead to thrombosis and other diseases. ATIII has three disulfide bonds, two near the N terminus and one near the C terminus. Our studies of ATIII in-cell folding reveal a surprising, biased order of disulfide bond formation, with early formation of the C-terminal disulfide, before formation of the N-terminal disulfides, critical for folding to the active, metastable state. Early folding of the predominantly β-sheet ATIII domain in this two-domain protein constrains the reactive center loop (RCL), which contains the proteasebinding site, ensuring that the RCL remains accessible. N-linked glycans and carbohydrate-binding molecular chaperones contribute to the efficient folding and secretion of functional ATIII. The inability of a number of disease-associated ATIII variants to navigate the folding reaction helps to explain their disease phenotypes.

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“…N-glycans are synthesized and transferred to polypeptides containing a signal peptide, by glycosyltransferase, at aspargine residues within the NxS/T sequon on the luminal side of the ER and Golgi (Molinari, 2007;Hebert et al, 2014). However, the mechanism by which HMGB1 becomes glycosylated despite being devoid of signal peptide is still is unknown, as Nglycosylation rarely exists in the cytosol.…”

Section: Discussionmentioning

confidence: 99%

N-linked glycosylation plays a crucial role in the secretion of HMGB1

Kim

Kwak

Park

et al. 2016

Journal of Cell Science

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HMGB1 protein is a delayed mediator of sepsis that is secreted to the extracellular milieu in response to various stimulants, inducing a pro-inflammatory response. HMGB1 is devoid of an endoplasmic reticulum (ER)-targeting signal peptide; hence, the mechanism of extracellular secretion is not completely understood, although HMGB1 is secreted after being subjected to post-translational modifications. Here, we identified the role of N-glycosylation of HMGB1 in extracellular secretion. We found two consensus (N37 and N134) and one non-consensus (N135) residues that were Nglycosylated in HMGB1 by performing liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzing for Nglycan composition and structure. Inhibition of N-glycosylation with tunicamycin resulted in a molecular shift of HMGB1 as assessed by gel electrophoresis. Non-glycosylated double mutant (N→Q) HMGB1 proteins (HMGB1 N37Q/N134Q and HMGB1 N37Q/N135Q ) showed localization to the nuclei, strong binding to DNA, weak binding to the nuclear export protein CRM1 and rapid degradation by ubiquitylation. These mutant proteins had reduced secretion even after acetylation, phosphorylation, oxidation and exposure to pro-inflammatory stimuli. Taken together, we propose that HMGB1 is N-glycosylated, and that this is important for its DNA interaction and is a prerequisite for its nucleocytoplasmic transport and extracellular secretion.

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The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis

Cited by 174 publications

References 99 publications

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Cellular folding pathway of a metastable serpin

N-linked glycosylation plays a crucial role in the secretion of HMGB1

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