Glycosylation of recombinant rabbit immunoglobulins influences protease susceptibility as shown by comprehensive mass spectrometric glycan analysis

Abstract Recombinant immunoglobulins (rIgGs) have become increasingly important as therapeutic agents and diagnostic tools in recent years. Genetic engineering allows the introduction of non-natural features such as the Sortase motif for site-directed labeling. In this study, the enzyme Sortase A (SrtA) was used for the proteolytic cleavage of rIgGs to produce their biotinylated Fab fragments by locating the cleavage site close to the hinge region. However, SrtA cleavage of engineered rabbit IgGs (rRb-IgGs) derived from human embryonic kidney (HEK) 293 cells showed significantly lower yields compared with their mouse counterparts. Non-recombinant Rb-IgGs have N- and O-glycans, and the presence of O-glycans close to the hinge region of the rRb-IgGs might affect the susceptibility of these antibodies to SrtA cleavage. In addition, the glycosylation pattern of rIgGs differs depending on the host cell used for expression. Therefore, we analyzed the N- and O-glycans of various rRb-IgGs expressed in HEK293 cells, detecting and quantifying 13 different N-glycan and 3 different O-glycan structures. The distribution of the different detected glycoforms in our rRb-IgG N-glycan analysis is in agreement with previous studies on recombinant human IgG N-glycans, confirming the hypothesis that the host cell defines the glycosylation of the recombinant produced IgGs. O-glycosylation could be mapped onto the threonine residue within the hinge region sequence XPTCPPPX, as already described previously for non-recombinant Rb-IgGs. Substitution of this threonine allowed an almost complete Fab fragment cleavage. Therefore, we could confirm the hypothesis that the O-glycans affect the SrtA activity, probably due to steric hindrance.

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Abstract 423: Caveolin-3 Enriches and Dynamically Interacts With Swell1

Circulation Research ◽

10.1161/res.127.suppl_1.423 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Daniel G Turner ◽

Leonid Tyan ◽

Sami Stroebel ◽

Frank Deguire ◽

Di Lang ◽

...

Keyword(s):

Protein Expression ◽

Hek293 Cells ◽

Scaffolding Protein ◽

Cell Swelling ◽

Hypotonic Solution ◽

Hek 293 ◽

Dynamic Relationship ◽

293 Cells ◽

Fret Efficiency ◽

Oxide Synthase

Caveolae are small (50-100nm) membrane invaginations formed by caveolin proteins enriched with cholesterol and lipids. Caveolae play a crucial role in mechanoprotection and mechano-electrical transduction by buffering membrane tension and facilitating activation of mechanosensitive ion channels, including a recently discovered swelling-activated chloride channel SWELL1 (also known as LRRC8A). However, the dynamic relationship between the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) and SWELL1 is poorly understood. The objective of this study was to determine how Cav3 interacts with SWELL1 channels and modulates their activity during mechano-electrical transduction. In HEK 293 cells transfected with Cav3, co-immunoprecipitation analysis recapitulated cardiac data showing association between SWELL1 and Cav3. Using transiently expressed Cav3-GFP and SWELL1-mCherry fusion proteins in HEK293 cells, we observed a high FRET efficiency between the two proteins in an isotonic (1T) solution, confirming their close (<5nm) proximity. In a hypotonic solution (0.7T, mimicking cell stretch), FRET efficiency decreased two-fold. Furthermore, FRET efficiency decreased two-fold to control levels when incubated with methyl-beta cyclodextrin, a cholesterol solubilizer. These data suggest that the relationship between Cav3 and SWELL1 is dependent on membrane mechanical tension and caveolae lipid raft integrity. Interestingly, in transfected cells, SWELL1 protein expression and whole cell swelling-activated chloride current ( I Cl,swell ) were increased four-fold and two-fold, respectively, while mRNA expression was reduced two-fold. This may indicate that caveolae formed by Cav3 expression enrich for SWELL1 and increase their half-life, thus requiring lower mRNA availability despite higher protein expression. Our findings indicate a close dynamic interplay between Cav3 and SWELL1, with a strong regulatory action of Cav3 on I Cl,swell activity. Given that I Cl,swell increases and interprotein FRET efficiency decreases in hypotonic solution, it is likely that Cav3 inhibits the activation of SWELL1 similarly to its known inhibition of nitric oxide synthase.

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Bupivacaine inhibits a small-conductance calcium-activated potassium type 2 channel (SK2) in HEK293 cells

10.21203/rs.3.rs-115427/v1 ◽

2020 ◽

Author(s):

Hongfei Chen ◽

Fangfang Xia ◽

Zhousheng Jin ◽

Zhijian Fu

Keyword(s):

Hek293 Cells ◽

Free Calcium ◽

Heart Cells ◽

Dependent Manner ◽

Patch Clamp Technique ◽

Hek 293 ◽

293 Cells ◽

Ic50 Value ◽

Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause severe cardiotoxicity. Small conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unknown. This study investigated the effect of bupivacaine on SK2 channels.Methods: The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch clamp technique was used to study the effect of bupivacaine on SK2 channels. Concentration-response relationship of bupivacaine for inhibiting SK2 current (0 mV) was fitted to a Hill equation, and the half-maximal inhibitory concentration (IC50) value was determined.Results: Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine and lidocaine on the SK2 current was 16.5, 46.5, and 77.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium.Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity.

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Mexiletine rescues the mixed phenotype in SCN5A-1795insD hiPSC-CMs

EP Europace ◽

10.1093/europace/euab116.584 ◽

2021 ◽

Vol 23 (Supplement_3) ◽

Author(s):

G Nasilli ◽

L Yiangou ◽

C Palandri ◽

AO Verkerk ◽

RP Davis ◽

...

Keyword(s):

Sodium Current ◽

Hek293 Cells ◽

Resting Membrane Potential ◽

Loss Of Function ◽

Hek 293 ◽

Hek 293 Cells ◽

Funding Sources ◽

293 Cells ◽

Induced Pluripotent ◽

Mixed Phenotype

Abstract Funding Acknowledgements Type of funding sources: None. Background The sodium channel blocker mexiletine can reduce late sodium current (INa) in patients with LQT3 syndrome, and additionally restore the decreased peak INa associated with SCN5A loss of function mutations. Purpose To investigate whether mexiletinecan rescue the mixed phenotype associated with the SCN5A-1795insD mutation in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs). Methods and Results HEK293 cells transfected with SCN5A-1795insD and SCN5A-WT and hiPSC-CMs from a patient carrying the SCN5A-1795insD mutation were incubated with a therapeutic dose of mexiletine (10 µM) or vehicle (H2O) for 48h. Peak INa, late INa and action potential (AP) properties were assessed by patch-clamp analysis. In HEK-293 cells transfected with SCN5A-1795insD or SCN5A-WT, exposure to mexiletine caused a significant increase in peak INa, in addition to a small increase in late INa in HEK-293 cells transfected with SCN5A-1795insD. In 1795insD hiPSC-CMs, peak INa was significantly increased whereas late INa was unchanged after mexiletine treatment. Accordingly, mexiletine increased AP upstroke velocity in SCN5A-1795insD hiPSC-CMs (indicating a rescue of INa availability), while AP amplitude, resting membrane potential and AP duration were unaffected. Conclusions Chronic treatment with a therapeutic concentration of mexiletine is capable of rescuing the mixed phenotype in SCN5A-1795insD hiPSC-CMs.

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Glycine receptor subunit composition alters the action of GABA antagonists

Visual Neuroscience ◽

10.1017/s0952523807070368 ◽

2007 ◽

Vol 24 (4) ◽

pp. 513-521 ◽

Cited By ~ 22

Author(s):

PING LI ◽

MALCOLM SLAUGHTER

Keyword(s):

Gaba Receptors ◽

Gaba Receptor ◽

Glycine Receptor ◽

Significant Inhibition ◽

Hek293 Cells ◽

Glycine Receptors ◽

Hek 293 ◽

293 Cells ◽

Two Factors ◽

Α Subunits

GABA receptor antagonists produce an unexpectedly significant inhibition of native glycine receptors in retina and in α1 or α2 homomeric glycine receptors (GlyRs) expressed in HEK 293 cells. In this study we evaluate this phenomenon in heteromeric glycine receptors, formed by mixing α1, α2, and β subunits. Picrotoxinin, picrotin, SR95531, and bicuculline are all more effective antagonists at GlyRs containing α2 subunits than α1 subunits. Inclusion of β subunits reduces the inhibitory potency of picrotoxinin and picrotin but increases the potency of SR95531 and bicuculline. As a result of these two factors, bicuculline is particularly poor at discriminating GABA and glycine receptors. Picrotin, which has been reported to be inactive at GABA receptors, blocks glycine currents in retina and in HEK293 cells, suggesting its utility as a selective glycine antagonist. However, picrotin is a more potent inhibitor of GABA than glycine in retinal neurons. We also tested if GABA and glycine receptor subunits can combine to form functional receptors. If GABAAR γ2S subunits are co-expressed with GlyR α subunits, the mixed receptor is glycine-sensitive and GABA-insensitive. But the mixed receptor exhibits a non-competitive picrotoxinin inhibition that is not observed in the homomeric GlyRs. This suggests that glycine and GABA subunits can co-assemble to form functional glycine receptors.

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PT-1 selectively activates AMPK-γ1 complexes in mouse skeletal muscle, but activates all three γ subunit complexes in cultured human cells by inhibiting the respiratory chain

Biochemical Journal ◽

10.1042/bj20141142 ◽

2015 ◽

Vol 467 (3) ◽

pp. 461-472 ◽

Cited By ~ 35

Author(s):

Thomas E. Jensen ◽

Fiona A. Ross ◽

Maximilian Kleinert ◽

Lykke Sylow ◽

Jonas R. Knudsen ◽

...

Keyword(s):

Respiratory Chain ◽

Hek293 Cells ◽

Mouse Muscle ◽

Subcellular Compartment ◽

Α Subunit ◽

Hek 293 ◽

293 Cells ◽

Cultured Human Cells ◽

Direct Binding ◽

Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) occurs as heterotrimeric complexes in which a catalytic subunit (α1/α2) is bound to one of two β subunits (β1/β2) and one of three γ subunits (γ1/γ2/γ3). The ability to selectively activate specific isoforms would be a useful research tool and a promising strategy to combat diseases such as cancer and Type 2 diabetes. We report that the AMPK activator PT-1 selectively increased the activity of γ1- but not γ3-containing complexes in incubated mouse muscle. PT-1 increased the AMPK-dependent phosphorylation of the autophagy-regulating kinase ULK1 (unc-51-like autophagy-activating kinase 1) on Ser555, but not proposed AMPK-γ3 substrates such as Ser231 on TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1 (TBC1D1) or Ser212 on acetyl-CoA carboxylase subunit 2 (ACC2), nor did it stimulate glucose transport. Surprisingly, however, in human embryonic kidney (HEK) 293 cells expressing human γ1, γ2 or γ3, PT-1 activated all three complexes equally. We were unable to reproduce previous findings suggesting that PT-1 activates AMPK by direct binding between the kinase and auto-inhibitory domains (AIDs) of the α subunit. We show instead that PT-1 activates AMPK indirectly by inhibiting the respiratory chain and increasing cellular AMP:ATP and/or ADP:ATP ratios. Consistent with this mechanism, PT-1 failed to activate AMPK in HEK293 cells expressing an AMP-insensitive R299G mutant of AMPK-γ1. We propose that the failure of PT-1 to activate γ3-containing complexes in muscle is not an intrinsic feature of such complexes, but is because PT-1 does not increase cellular AMP:ATP ratios in the specific subcellular compartment(s) in which γ3 complexes are located.

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Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

International Journal of Molecular Sciences ◽

10.3390/ijms22094637 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4637

Author(s):

Daniel Barth ◽

Andreas Lückhoff ◽

Frank J. P. Kühn

Keyword(s):

Amino Acid Residues ◽

Hek 293 ◽

Complete Inactivation ◽

293 Cells ◽

Activation Mechanisms ◽

Specific Regulation ◽

Species Specific ◽

Inside Out ◽

Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

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Preliminary Study on Pasta Samples Characterized in Antioxidant Compounds and Their Biological Activity on Kidney Cells

Nutrients ◽

10.3390/nu13041131 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1131 ◽

Cited By ~ 1

Author(s):

Federico Di Marco ◽

Francesco Trevisani ◽

Pamela Vignolini ◽

Silvia Urciuoli ◽

Andrea Salonia ◽

...

Keyword(s):

Indoleacetic Acid ◽

Antioxidant Properties ◽

Antioxidant Compounds ◽

Kidney Cells ◽

Hek 293 ◽

293 Cells ◽

High Concentrations ◽

Β Carotene ◽

Egg Pasta

Pasta is one of the basic foods of the Mediterranean diet and for this reason it was chosen for this study to evaluate its antioxidant properties. Three types of pasta were selected: buckwheat, rye and egg pasta. Qualitative–quantitative characterization analyses were carried out by HPLC-DAD to identify antioxidant compounds. The data showed the presence of carotenoids such as lutein and polyphenols such as indoleacetic acid, (carotenoids from 0.08 to 0.16 mg/100 g, polyphenols from 3.7 to 7.4 mg/100 g). To assess the effect of the detected metabolites, in vitro experimentation was carried out on kidney cells models: HEK-293 and MDCK. Standards of β-carotene, indoleacetic acid and caffeic acid, hydroalcoholic and carotenoid-enriched extracts from samples of pasta were tested in presence of antioxidant agent to determine viability variations. β-carotene and indoleacetic acid standards exerted a protective effect on HEK-293 cells while no effect was detected on MDCK. The concentrations tested are likely in the range of those reached in body after the consumption of a standard pasta meal. Carotenoid-enriched extracts and hydroalcoholic extracts showed different effects, observing rescues for rye pasta hydroalcoholic extract and buckwheat pasta carotenoid-enriched extract, while egg pasta showed milder dose depending effects assuming pro-oxidant behavior at high concentrations. The preliminary results suggest behaviors to be traced back to the whole phytocomplexes respect to single molecules and need further investigations.

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Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts

Molecules ◽

10.3390/molecules26040973 ◽

2021 ◽

Vol 26 (4) ◽

pp. 973

Author(s):

Tilen Koklič ◽

Alenka Hrovat ◽

Ramon Guixà-González ◽

Ismael Rodríguez-Espigares ◽

Damaris Navio ◽

...

Keyword(s):

Lipid Rafts ◽

Order Parameter ◽

Spin Probe ◽

Gonadotropin Releasing Hormone ◽

Paramagnetic Resonance ◽

Hek 293 ◽

293 Cells ◽

Gonadotropin Releasing Hormone Receptor ◽

Electron Paramagnetic

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Using kICS to Reveal Changed Membrane Diffusion of AQP-9 Treated with Drugs

Membranes ◽

10.3390/membranes11080568 ◽

2021 ◽

Vol 11 (8) ◽

pp. 568

Author(s):

Jakob L. Kure ◽

Thommie Karlsson ◽

Camilla B. Andersen ◽

B. Christoffer Lagerholm ◽

Vesa Loitto ◽

...

Keyword(s):

Diffusion Coefficient ◽

Plasma Membrane ◽

Membrane Proteins ◽

Actin Polymerization ◽

Correlation Spectroscopy ◽

Image Correlation ◽

Membrane Diffusion ◽

Hek 293 ◽

293 Cells ◽

Aquaporin 9

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-β-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

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