CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart.     

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.     

Co‐expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor‐β1 into a biologically active protein

Transforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

The N‐terminal tripeptide of insulin‐like growth factor‐I protects against β‐amyloid‐induced somatostatin depletion by calcium and glycogen synthase kinase 3β modulation

The protective effects of insulin‐like growth factor I on the somatostatin (SRIF) system in the temporal cortex after β‐amyloid (Aβ) injury may be mediated through its N‐terminal tripeptide glycine‐proline‐glutamate (GPE). GPE is cleaved to cyclo[Pro‐Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Aβ25–35‐treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Aβ25–35 accumulation, cytosolic calcium levels ([Ca²⁺]_c) and the intracellular signaling mechanisms involved. GPE and cPG did not change Aβ25–35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Aβ25–35 insult, which was coincident with Akt activation and glycogen synthase kinase 3β inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca²⁺ influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin‐like growth factor I effects on the SRIF system through a mechanism independent of Aβ clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Characterizing the release of bioactive N‐glycans from dairy products by a novel endo‐β‐N‐acetylglucosaminidase

Endo‐β‐N‐acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI‐1) is a novel enzyme that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐glycan core of high mannose, hybrid, and complex N‐glycans. These conjugated N‐glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein ratio (1:3,000–1:333) were evaluated on the release of N‐glycans from bovine colostrum whey by EndoBI‐1. A central composite design was used, including a two‐level factorial design (2⁴) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N‐glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan‐free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI‐1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1331–1339, 2015     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

β-1,4-Galactosyltransferase-catalyzed glycosylation of sugar derivatives: Modulation of the enzyme activity by α-lactalbumin, immobilization and solvent tolerance

The influence of different parameters on the activity of the β-1,4-galactosyltransferase (β-1,4-GalT) from bovine milk has been investigated using various acceptor and donor substrates. It was found that the “specifier” protein α-lactalbumin (α-LA), which interacts with β-1,4-GalT forming the lactose synthase (LS) complex, is not necessary when the acceptors are different glucopyranosides, and, in some cases, it can even have an inhibitory effect, like with the complex glucosides ginsenoside Rg₁ (1) and colchicoside (2). By optimization of the reaction conditions, the galactosylated and glucosylated derivatives of 2 were prepared, using UDP-Gal and UDP-Glc as sugar donors, respectively, and characterized. Moreover, β-1,4-GalT was covalently immobilized on Eupergit C 250 L in the absence of α-LA, and the synthetic performances of this immobilized biocatalyst were evaluated. Finally, the best organic cosolvents to be used both with β-1,4-GalT and the LS complex were identified.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.