scholarly journals N-glycosylation–dependent regulation of hK2P17.1 currents

2019 ◽  
Vol 30 (12) ◽  
pp. 1425-1436 ◽  
Author(s):  
Felix Wiedmann ◽  
Daniel Schlund ◽  
Niels Voigt ◽  
Antonius Ratte ◽  
Manuel Kraft ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Posttranslational Modifications ◽  
Immunoblot Analysis ◽  
Surface Expression ◽  
Genetic Alterations ◽  
Cardiac Conduction ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Glycosylation Sites ◽  
Expression And Activity

Two pore-domain potassium (K2P) channels mediate potassium background currents that stabilize the resting membrane potential and facilitate action potential repolarization. In the human heart, hK2P17.1 channels are predominantly expressed in the atria and Purkinje cells. Reduced atrial hK2P17.1 protein levels were described in patients with atrial fibrillation or heart failure. Genetic alterations in hK2P17.1 were associated with cardiac conduction disorders. Little is known about posttranslational modifications of hK2P17.1. Here, we characterized glycosylation of hK2P17.1 and investigated how glycosylation alters its surface expression and activity. Wild-type hK2P17.1 channels and channels lacking specific glycosylation sites were expressed in Xenopus laevis oocytes, HEK-293T cells, and HeLa cells. N-glycosylation was disrupted using N-glycosidase F and tunicamycin. hK2P17.1 expression and activity were assessed using immunoblot analysis and a two-electrode voltage clamp technique. Channel subunits of hK2P17.1 harbor two functional N-glycosylation sites at positions N65 and N94. In hemi-glycosylated hK2P17.1 channels, functionality and membrane trafficking remain preserved. Disruption of both N-glycosylation sites results in loss of hK2P17.1 currents, presumably caused by impaired surface expression. This study confirms diglycosylation of hK2P17.1 channel subunits and its pivotal role in cell-surface targeting. Our findings underline the functional relevance of N-glycosylation in biogenesis and membrane trafficking of ion channels.

P1601N-glycosylation of TREK-1/hK2P2.1 two-pore-domain (K2P) potassium channels

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
F Wiedmann ◽  
D Schlund ◽  
A Ratte ◽  
H A Katus ◽  
M Kraft ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Posttranslational Modifications ◽  
Enzymatic Digestion ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Function ◽  
Excitable Tissue ◽  
Electrode Voltage ◽  
Channel Surface ◽  
Pore Domain

Abstract Background and purpose Mechanosensitive hTREK-1 (hK2P2.1) two-pore-domain potassium channels give rise to background currents that control resting membrane potential in excitable tissue. Recently TREK-1 currents have been linked to regulation of cardiac rhythm as well as hypertrophy and fibrosis. Even though pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, less is known about its posttranslational modifications. This study aims to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Experimental approach Following pharmacological inhibition of N glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-233T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. Key results TREK-1 channels subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrate that non-glycosylated hTREK-1 channels subunits are able to reach the cell surface in general, but seemingly with reduced efficiency. Conclusion and implications hTREK-1 are glycoproteins and N glycosylation at positions 110 and 134 is involved in channel surface trafficking. These findings extend our view on regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how glycosylation deficiency disorders may promote arrhythmogenesis.

scholarly journals Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

2016 ◽  
Vol 113 (6) ◽  
pp. E705-E714 ◽  
Author(s):  
Akhee S. Jahan ◽  
Maxime Lestra ◽  
Lee Kim Swee ◽  
Ying Fan ◽  
Mart M. Lamers ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Posttranslational Modifications ◽  
Protein Function ◽  
Adaptor Proteins ◽  
Cell Receptor ◽  
Surface Expression ◽  
Jurkat Cells ◽  
Tcr Signaling ◽  
Primary Mouse

Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.

scholarly journals Biochemical and molecular characterization of N66 from the shell ofPinctada mazatlanica

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7212
Author(s):  
Crisalejandra Rivera-Perez ◽  
Catalina Magallanes-Dominguez ◽  
Rosa Virginia Dominguez-Beltran ◽  
Josafat Jehu Ojeda-Ramirez de Areyano ◽  
Norma Y. Hernandez-Saavedra
Keyword(s):  
Posttranslational Modifications ◽  
Protein A ◽  
Pearl Oyster ◽  
Peptide Sequence ◽  
Calcium Carbonate Precipitation ◽  
Base Pairs ◽  
Shell Matrix ◽  
Glycosylation Sites ◽  
Shell Matrix Proteins

Mollusk shell mineralization is a tightly controlled process made by shell matrix proteins (SMPs). However, the study of SMPs has been limited to a few model species. In this study, the N66 mRNA of the pearl oysterPinctada mazatlanicawas cloned and functionally characterized. The full sequence of the N66 mRNA comprises 1,766 base pairs, and encodes one N66 protein. A sequence analysis revealed that N66 contained two carbonic anhydrase (CA) domains, a NG domain and several glycosylation sites. The sequence showed similarity to the CA VII but also with its homolog protein nacrein. The native N66 protein was isolated from the shell and identified by mass spectrometry, the peptide sequence matched to the nucleotide sequence obtained. Native N66 is a glycoprotein with a molecular mass of 60–66 kDa which displays CA activity and calcium carbonate precipitation ability in presence of different salts. Also, a recombinant form of N66 was produced inEscherichia coli, and functionally characterized. The recombinant N66 displayed higher CA activity and crystallization capability than the native N66, suggesting that the lack of posttranslational modifications in the recombinant N66 might modulate its activity.

scholarly journals N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels

2019 ◽  
Vol 20 (20) ◽  
pp. 5193 ◽  
Author(s):  
Felix Wiedmann ◽  
Daniel Schlund ◽  
Francisco Faustino ◽  
Manuel Kraft ◽  
Antonius Ratte ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Cardiac Rhythm ◽  
293T Cell ◽  
Enzymatic Digestion ◽  
Xenopus Laevis Oocytes ◽  
Channel Function ◽  
Cellular Excitability ◽  
K2p Channels ◽  
Electrode Voltage ◽  
Pore Domain

Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

scholarly journals Site-specific deacylation by ABHD17a controls BK channel splice variant activity

2020 ◽  
Vol 295 (49) ◽  
pp. 16487-16496 ◽  
Author(s):  
Heather McClafferty ◽  
Hamish Runciman ◽  
Michael J. Shipston
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Membrane Trafficking ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Site Specific ◽  
And Function ◽  
A Site

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

scholarly journals Expression and purification of human Afamin for structure/function analysis

2014 ◽  
Vol 70 (a1) ◽  
pp. C308-C308
Author(s):  
Alessandra Altamirano ◽  
Linda Fineder ◽  
Ramona Berberich ◽  
Barbara Fürnrorh ◽  
Friedrich Fresser ◽  
...  
Keyword(s):  
Function Analysis ◽  
Protein Crystallization ◽  
Plasma Concentrations ◽  
Immunoblot Analysis ◽  
Free Cell ◽  
Protein Glycosylation ◽  
Hek293 Cells ◽  
Chemical Inhomogeneity ◽  
Fab Fragments ◽  
Glycosylation Sites

Protein glycosylation plays an important role in protein stability, folding, and secretion but presents a major challenge for protein crystallization. Crystallization of highly glycosylated proteins is often difficult because of conformational and chemical inhomogeneity of the glycan decorations. Therefore, it is almost always necessary to modify the material to obtain a conformational homogenous protein that can crystallize. (1) We have explored several avenues in the case of the human 87 kDa glycoprotein afamin. Afamin (AFM), a member of the albumin gene family, is mainly expressed in the liver and secreted into the bloodstream. (2) Elevated afamin plasma concentrations are associated with major diseases such as metabolic syndrome and cancer;(3) however, pathophysiological functions are largely unknown. Therefore, we are pursuing the crystal structure of various forms of recombinantly expressed human afamin (rhAFM) for structure guided exploration and analysis of its function. Multiple variants of rhAFM are pursued: 1) fully glycosylated wild-type rhAFM (expressed in CHO cell lines); 2) rhAFM complexed with Fab fragments of two anti-AFM mAbs, 3) enzymatic deglycosylation of rhAFM with PGNaseF, 4) partially glycosylated rhAFM (expressed in glycosylation-deficient Lec1-CHO cells); and 5) glycosylation-free rhAFM, obtained from HEK293 cells virally transfected with a cDNA mutant lacking all 5 glycosylation sites by replacement of ASN with ASP. Yields of up to 2 mg/mL glycosilation free mutant rhAFM in HEK273 cells have been comparable to native rhAFM. C-terminal His6-tagged rhAFM was captured by Ni-IMAC from serum-free cell culture supernatants. All variants and Fab-AFM complexes were polished via SEC yielding pure products per SDS-PAGE and immunoblot analysis. Crystals have been obtained from Fab fragments and crystalline spherulites which are useful for microseeding from Fab-AFM complexes.

scholarly journals Cell surface expression of murine, rat, and human Fc receptors by Xenopus oocytes.

1984 ◽  
Vol 160 (2) ◽  
pp. 606-611 ◽  
Author(s):  
E Pure ◽  
A D Luster ◽  
J C Unkeless
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Surface Expression ◽  
Gamma Interferon ◽  
Membrane Receptors ◽  
Cell Surface Expression ◽  
Xenopus Laevis Oocytes ◽  
High Avidity ◽  
U937 Cells ◽  
Mouse Igg2a

We report that Xenopus laevis oocytes can efficiently translate and insert heterologous membrane receptors into the oocyte plasma membrane, where they can be detected by the binding of either monoclonal antibodies or ligands. Thus, oocytes injected with mRNA from the mouse J774 macrophage-like cell line, the rat RBL-1 basophilic leukemia, and the U937 promonocyte cell line, bound 2.4G2 Fab, rat IgE, and mouse IgG2a, respectively. The increase in the high avidity Fc gamma R observed after gamma-interferon induction of U937 cells was also observed after injection of mRNA from gamma-interferon-induced U937 cells into oocytes. This suggests either much greater message stability or a greater rate of transcription of Fc gamma Rhi mRNA in the gamma-interferon-induced cells. The assay affords a sensitive method for the detection of rare mRNA species that code for plasma membrane proteins.

Activity of the renal Na+-K+-2Cl− cotransporter is reduced by mutagenesis of N-glycosylation sites: role for protein surface charge in Cl− transport

2006 ◽  
Vol 290 (5) ◽  
pp. F1094-F1102 ◽  
Author(s):  
Anahí Paredes ◽  
Consuelo Plata ◽  
Manuel Rivera ◽  
Erika Moreno ◽  
Norma Vázquez ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Functional Expression ◽  
Site Directed Mutagenesis ◽  
Intrinsic Activity ◽  
Xenopus Laevis Oocytes ◽  
Type I ◽  
Wild Type ◽  
Glycosylation Sites ◽  
Green Fluorescent

The renal-specific Na+-K+-2Cl− cotransporter NKCC2 belongs to the SLC12 gene family; it is the target for loop diuretics and the cause of type I Bartter's syndrome. Because the NKCC2 sequence contains two putative N-linked glycosylation sites, one of which is conserved with the renal Na+-Cl− cotransporter in which glycosylation affects thiazide affinity, we assessed the role of glycosylation on NKCC2 functional properties. One (N442Q or N452Q) or both (N442,452Q) N-glycosylation sites were eliminated by site-directed mutagenesis. Wild-type NKCC2 and mutant clones were expressed in Xenopus laevis oocytes and analyzed by 86Rb+ influx, Western blotting, and confocal microscopy. Inhibition of glycosylation with tunicamycin in wild-type NKCC2-injected oocytes resulted in an 80% reduction of NKCC2 activity. Immunoblot of injected oocytes revealed that glycosylation of NKCC2 was completely prevented in N442,452Q-injected oocytes. Functional activity was reduced by 50% in N442Q- and N452Q-injected oocytes and by 80% in oocytes injected with N442,452Q, whereas confocal microscopy of oocytes injected with wild-type or mutant enhanced green fluorescent protein-tagged NKCC2 clones revealed that surface fluorescence intensity was reduced ∼20% in single mutants and 50% in the double mutant. Ion transport kinetic analyses revealed no changes in cation affinity and a small increase in Cl− affinity by N442Q and N442,452Q. However, a slight decrease in bumetanide affinity was observed. Our data demonstrate that NKCC2 is glycosylated and suggest that prevention of glycosylation reduces its functional expression by affecting insertion into the plasma membrane and the intrinsic activity of the cotransporter.

scholarly journals Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

2018 ◽  
Vol 45 (5) ◽  
pp. 2071-2085 ◽  
Author(s):  
Maria Agthe ◽  
Yvonne Garbers ◽  
Joachim Grötzinger ◽  
Christoph Garbers
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Post Translational Modification ◽  
Glycosylation Sites ◽  
D2 Domain

Background/Aims: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. Methods: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. Results: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. Conclusion: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.

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