Transfected CHO Cells Research Articles

Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100−150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

Point mutations that inactivate MGAT4D-L, an inhibitor of MGAT1 and complex N-glycan synthesis

The membrane-bound, long form of MGAT4D, termed MGAT4D-L, inhibits MGAT1 activity in transfected cells and reduces the generation of complex N-glycans. MGAT1 is the GlcNAc-transferase that initiates complex and hybrid N-glycan synthesis. We show here that Drosophila MGAT1 was also inhibited by MGAT4D-L in S2 cells. In mammalian cells, expression of MGAT4D-L causes the substrate of MGAT1 (Man5GlcNAc2Asn) to accumulate on glycoproteins, a change that is detected by the lectin Galanthus nivalis agglutinin (GNA). Using GNA binding as an assay for the inhibition of MGAT1 in MGAT4D-L transfectants, we performed site-directed mutagenesis to determine requirements for MGAT1 inhibition. Deletion of 25 amino acids (aa) from the C terminus inactivated MGAT4D-L, but deletion of 20 aa did not. Conversion of the five key amino acids (PSLFQ) to Ala, or deletion of PSLFQ in the context of full-length MGAT4D-L, also inactivated MGAT1 inhibitory activity. Nevertheless, mutant, inactive MGAT4D-L interacted with MGAT1 in co-immuno-precipitation experiments. The PSLFQ sequence also occurs in MGAT4A and MGAT4B GlcNAc-transferases. However, neither inhibited MGAT1 in transfected CHO cells. MGAT4D-L inhibitory activity could be partially transferred by attaching PSLFQ or the 25-aa C terminus of MGAT4D-L to the C terminus of MGAT1. Mutation of each amino acid in PSLFQ to Ala identified both Leu and Phe as independently essential for MGAT4D-L activity. Thus, replacement of either Leu-395 or Phe-396 with Ala led to inactivation of MGAT4D-L inhibitory activity. These findings provide new insights into the mechanism of inhibition of MGAT1 by MGAT4D-L, and for the development of small molecule inhibitors of MGAT1.

Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors.

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT–transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.

Molecular basis defining the selectivity of substituted isoquinolinones for the melatonin MT2 receptor

Melatonin MT1 and MT2 receptors represent attractive drug targets for the treatment of various disorders. However, the high conservation of the melatonin binding pocket has hindered the development of subtype-selective compounds. By leveraging on the recently resolved crystal structures of MT1 and MT2 receptors, this study aims to elucidate the structural basis of MT2-selectivity of a panel of isoquinolinone derivatives. Molecular modelling and ligand docking approaches were employed to predict residues involved in forming interactions with the MT2-selective isoquinolinones. Seven conserved residues (Asn175, His208, Trp264, Asn268, Gly271, Tyr294 and Tyr298) were selected as targets for site-directed mutagenesis. Ca2+ mobilization, cAMP inhibition, phosphorylation of extracellular signal-regulated kinase, and ligand binding assays were performed to functionally characterize the receptor mutants in transfected CHO cells. Unlike melatonin, isoquinolinones bearing a 3-methoxybenzyloxyl substituent were unaffected by alanine substitution at His208 of MT2. Although alanine substitutions at Tyr294 or Tyr298 reduced the potency of melatonin and some isoquinolinones on MT2, similar mutations on MT1 allowed five hitherto ineffective isoquinolinones to act as agonists. An isoquinolinone antagonist bearing a 4-methoxybenzyloxyl moiety turned into an agonist at MT2 mutants with alanine substitutions at His208, Tyr294 or Tyr298. A subset of residues is apparently involved in forming a hydrophobic binding cavity to confer selectivity upon the aromatic substituent of isoquinolinone compounds. Two conserved tyrosine residues on transmembrane helix 7 may confer ligand selectivity at MT1 and MT2 receptors, while a conserved histidine on transmembrane helix 5 is apparently involved in receptor activation.

Generation and characterization of human soluble VE‐cadherin (sVE‐cadherin)

Endothelial barrier function is maintained by tight and adherens junctions. Adherens junction protein Vascular Endothelial (VE)‐cadherin plays a critical role for maintenance of endothelial barrier function by providing the mechanical strength between adjacent endothelial cells. Previously, it was shown that inflammation‐induced loss of endothelial barrier function is associated with the formation of soluble VE‐cadherin fragments (sVE‐cadherin) that comprise the extracellular domains (EC) 1‐5. Moreover, it was proposed that sVE‐cadherin could be involved in the loss of endothelial barrier function. Here, we generated a recombinant sVE‐cadherin EC1‐5 construct to test whether the presence of sVE‐cadherin alone and independent of pro‐inflammatory stimuli would affect the integrity of endothelial barrier function.DNA templates of extracellular domains EC1‐5 of human VE‐cadherin were amplified via PCR and ligated into different plasmids. Then, Chinese‐Hamster Ovary cells (CHO) were transfected with these vectors which resulted in CHO cells secreting functional active sVE‐cadherin. This sVE‐cadherin in CHO supernatants was purified and used for biochemical characterization. Thereafter, functional analysis was performed on human microvascular endothelial cells (HDMECs).First, sequencing of the purified products from CHO cells that had been transfected with the plasmids containing EC1‐5 confirmed the sequence of the extracelIular domains EC1‐5 of VE‐cadherin. In the cell culture supernatants of transfected CHO cells the presence of sVE‐cadherin was further verified using Western Blots where a 90 kDa protein in the cell culture supernatant was detected that proved to be sVE‐cadherin as revealed by an antibody directed against the extracellular domain of VE‐cadherin. In addition, by using an ELISA‐based system that specifically recognizes extracellular domains of VE‐cadherin, levels of 100–200 ng/ml of sVE‐cadherin were found in the cell culture supernatants of transfected CHO cells which in summary confirmed the successful generation of human sVE‐cadherin.To further test the hypothesis that sVE‐cadherin compromises endothelial barrier function HDMECs were incubated with different concentrations of sVE‐cadherin followed by measurements of transendothelial electrical resistance (TER) and immunostaining. In TER measurements sVE‐cadherin led to a significant loss of TER compared to controls after 2h hours of incubation beginning at concentrations of 75 ng/ml. In immunostaining sVE‐cadherin application on endothelial monolayers resulted in intercellular gap formation, loss of VE‐cadherin at the cell borders and led to the formation of stress fibers in the actin cytoskeleton. Preliminary experiments using immunoprecipitation assays showed that sVE‐cadherin binds to vascular endothelial protein tyrosine phosphatase (VE‐PTP) pointing towards a potential mechanism by which sVE‐cadherin induces loss of VE‐cadherin.In summary, we successfully generated human sVE‐cadherin. Our data in endothelial cells demonstrate that sVE‐cadherin disrupts endothelial barrier function in a dose‐dependent manner independent of the presence of pro‐inflammatory stimuli.Support or Funding InformationDFG SCHL1962/4‐2

Determining the Effects of Legionella pneumophila SidE Effector Proteins on Host Vesicular Trafficking

Legionella pneumophila is an opportunistic intracellular pathogen responsible for Legionnaires’ disease in humans. Upon infection, L. pneumophila forms the Legionella‐containing vacuole (LCV), shielding itself from the host immune response. From the LCV, L. pneumophila uses a specialized secretion system to translocate over 350 effector proteins into the host cell. Effectors target and manipulate host pathways in order to optimize the host’s cellular environment to support L. pneumophila replication (1). One important cellular process targeted during infection is endocytic recycling. Recycling pathways are generally categorized as slow or fast, depending on the time it takes for endocytosed cargo to return to the plasma membrane. Rab11 is a key regulator of the slow endocytic recycling pathway (2) and it moves along microtubules partly through its interaction with Rab11‐FIP1A. It was previously shown that mutation of Rab11‐FIP1A disrupts transferrin recycling (3). In the context of infection by L. pneumophila, our group has shown that transferrin recycling in human macrophages is inhibited early during infection. We hypothesize that the L. pneumophila effectors protein family SidE is at least partly responsible for this defect. Pull‐down assays revealed that interaction between Rab11 and Rab11‐FIP1A is disrupted in the presence of SdeC. In transiently transfected CHO cells, GFP‐Rab11‐FIP1A is found on vesicles in the perinuclear region. However, when GFP‐Rab11‐FIP1A and mCherry‐SdeC are co‐expressed in CHO cells, we observed that GFP‐Rab11‐FIP1A changed its localization to several enlarged structures. We are investigating whether SdeC’s ubiquitination activity is responsible for disrupting interaction of Rab11 with Rab11‐FIP1A. These results will help establish the effects of ubiquitination by SidE effector proteins on the slow recycling pathway.Support or Funding Information

Trypanosoma cruzi-Infected Human Macrophages Shed Proinflammatory Extracellular Vesicles That Enhance Host-Cell Invasion via Toll-Like Receptor 2.

Extracellular vesicles (EVs) shed by trypomastigote forms of Trypanosoma cruzi have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from T. cruzi or T.cruzi-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that T. cruzi-infected macrophages release higher numbers of EVs (50–300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or T.cruzi-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from T. cruzi during infection to maintain the inflammatory response in the host.

Development of an inexpensive Raman-compatible substrate for the construction of a microarray screening platform.

Biomaterial microarrays are being developed to facilitate identifying the extrinsic cues that elicit stem cell fate decisions to self-renew, differentiate and remain quiescent. Raman microspectroscopy, often combined with multivariate analysis techniques such as partial least square-discriminant analysis (PLS-DA), could enable the non-invasive identification of stem cell fate decisions made in response to extrinsic cues presented at specific locations on these microarrays. Because existing biomaterial microarrays are not compatible with Raman microspectroscopy, here, we develop an inexpensive substrate that is compatible with both single-cell Raman spectroscopy and the chemistries that are often used for biomaterial microarray fabrication. Standard deposition techniques were used to fabricate a custom Raman-compatible substrate that supports microarray construction. We validated that spectra from living cells on functionalized polyacrylamide (PA) gels attached to the custom Raman-compatible substrate are comparable to spectra acquired from a more expensive commercially available substrate. We also showed that the spectra acquired from individual living cells on functionalized PA gels attached to our custom substrates were of sufficient quality to enable accurate identification of cell phenotypes using PLS-DA models of the cell spectra. We demonstrated this by using cells from laboratory lines (CHO and transfected CHO cells) as well as adult stem cells that were freshly isolated from mice (long-term and short-term hematopoietic stem cells). The custom Raman-compatible substrate reported herein may be used as an inexpensive substrate for constructing biomaterial microarrays that enable the use of Raman microspectroscopy to non-invasively identify the fate decisions of stem cells in response to extrinsic cues.

An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals

Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.

Direct Visualization of Platelet Integrins Using ANTI-Transmembrane Domain Peptides Containing a BLUE Fluorescent Amino Acid

Antibodies containing a fluorescent label or recombinant proteins containing a fluorescent reporter are commonly used to visualize proteins in situ. However, antibodies and bulky reporters can perturb protein structure and function. To overcome this problem, we have designed a minimally perturbing blue fluorescent unnatural amino acid that enables the direct imaging of intrinsically-fluorescent proteins with their interaction partners. Specifically, we used a nitrile-derivatized tryptophan, 4-cyanotryptophan (4CNTrp), which differs by only two atoms from native tryptophan. 4CNTrp has unique photophysical properties in the visible blue region: an absorption maximum at ~325 nm, an emission maximum at ~420 nm, a large fluorescence quantum yield (>0.8 in aqueous solution), a long fluorescence lifetime (ca. 13 ns), and good photostability (Hilaire et al. PNAS 2017; 114: 6005-09). Based on these properties, 4CNTrp-labeling was recently used to visualize the binding of a model peptide pHLIP (pH-(Low) Insertion Peptide) to cell membranes via wide-field fluorescence microscopy. 4CNTrp is also a viable FRET donor for acceptor pairs in the green visible region. 4CNTrp can be prepared by a simple, high-yielding cost-effective synthetic route and because it does not inhibit bacterial growth, it may be practical for cellular applications (Zhang et al. Chem. Comm. 2019; 55: 5095-98). Here, we used 4CNTrp-labeled peptides to directly image integrins on cell surfaces. Previously, we described the synthesis of peptides, called CHAMP peptides for Computed Helical Anti-Membrane Protein, that target the transmembrane (TM) domains of integrins in a sequence-specific manner (Yin et al. Science 2007; 315: 1817-22). Thus, the CHAMP peptides anti-αIIb and anti-αv specifically bind to the TM domains of αIIb and αv in platelet membranes, causing separation of the αIIbβ3 and αvβ3 TM domains and αIIbβ3 and αvβ3 activation. Anti-αIIb also caused αIIbβ3 clustering that was visualized using the β3-specific monoclonal antibody SSA6 labeled with Alexa Fluor 488 but only after the platelets were fixed with paraformaldehyde to prevent antibody-induced clustering. To enable the direct visualization of αIIβ3, αvβ3, and α2β1 on cell surfaces, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4CNTrp at their N-termini (CN-αIIb, CN-αv, CN-β1). We found that 4CNTrp labeling did not perturb the ability of the CHAMP peptides to bind to integrins and to cause specific integrin activation. Thus, CN-αIIb caused αIIbβ3-dependent platelet aggregation, CN-αv caused the αvβ3-dependent platelet adhesion to osteopontin, and CN-α1 caused α2β1-mediated platelet adhesion to collagen. We then used high resolution wide-field deconvolution microscopy to image 4CNTrp-containing integrins on the surface of platelets, HEL cells, and transfected CHO cells. Compared to 4CNTrp-containing random peptides that uniformly decorate cell surfaces, the CN-labeled CHAMP peptides were present as a limited number of discrete foci on the cell surface. To confirm that these foci represented CN-peptide containing integrins, we co-stained the cells with Alexa Fluor 488-labeled integrin-specific monoclonal antibodies and found that CN-peptide and antibody fluorescence coincided. Thus, these studies demonstrate the specific and direct imaging of integrins embedded in cell membranes in their activated state using 4CNTrp-labeled anti-integrin TM peptides. Because 4CNTrp can readily be incorporated into proteins and peptides with little if any structural perturbation, 4CNTrp-labeling provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. Disclosures No relevant conflicts of interest to declare.

Methylation-related metabolic effects of D4 dopamine receptor expression and activation

D4 dopamine receptor (D4R) activation uniquely promotes methylation of plasma membrane phospholipids, utilizing folate-derived methyl groups provided by methionine synthase (MS). We evaluated the impact of D4R expression on folate-dependent phospholipid methylation (PLM) and MS activity, as well as cellular redox and methylation status, in transfected CHO cells expressing human D4R variants containing 2, 4, or 7 exon III repeats (D4.2R, D4.4R, D4.7R). Dopamine had no effect in non-transfected CHO cells, but increased PLM to a similar extent for both D4.2R- and D4.4R-expressing cells, while the maximal increase was for D4.7R was significantly lower. D4R expression in CHO cells decreased basal MS activity for all receptor subtypes and conferred dopamine-sensitive MS activity, which was greater with a higher number of repeats. Consistent with decreased MS activity, D4R expression decreased basal levels of methylation cycle intermediates methionine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), as well as cysteine and glutathione (GSH). Conversely, dopamine stimulation increased GSH, SAM, and the SAM/SAH ratio, which was associated with a more than 2-fold increase in global DNA methylation. Our findings illustrate a profound influence of D4R expression and activation on MS activity, coupled with the ability of dopamine to modulate cellular redox and methylation status. These previously unrecognized signaling activities of the D4R provide a unique link between neurotransmission and metabolism.

High cell density transient transfection of CHO cells for TGF-β1 expression.

High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A linear correlation between fluorescence intensity and transfection efficiency was improved. The transfection efficiency of PEI was highly dependent on the transfection conditions and directly related to the level of recombinant protein. Several factors were required to optimize the transient transfection process; these factors included the media type (which is compatible with low or high cell density transfection), the preculture CHO-K1 suspension cell density, and the pDNA to PEI level. Based on design of experiment (DoE) analyses, the optimal transfection conditions for 10×106cells/mL in the CHOMACS CD medium achieved 73% transfection efficiency and a cell viability of over 80%. These results were confirmed for the production of transforming growth factor-beta 1 (TGF-β1) in a shake flask. The purified TGF-β1 protein concentration from 60mL supernatant was 27µg/mL, and the protein was biologically active.

Atypical Chemokine Receptor 3 Generates Guidance Cues for CXCL12-Mediated Endothelial Cell Migration.

Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the ACKR3 zebrafish ortholog ackr3b in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that ackr3b is a CXCL12-binding ortholog of human ACKR3. ackr3b is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of ackr3b becomes restricted to the basal part of the somites. After ackr3b knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker hlx1. In addition, ackr3b morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for ackr3b in endothelial cell guidance, CXCL12 gradient generated by ACKR3 expression in CHO cell transfectants guides human endothelial cell migration in an in vitro cell co-culture chemotaxis assay. Our results demonstrate that ackr3b plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12.

CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.

Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.

Optimizing the production and affinity purification of HIV-1 envelope glycoprotein SOSIP trimers from transiently transfected CHO cells.

We describe methods to improve the efficiency with which HIV-1 Envelope glycoprotein SOSIP trimer immunogens can be produced by transient transfection of ExpiCHO-S cells and then affinity purified using the trimer-specific human monoclonal antibody PGT145. The specificity of PGT145 for properly folded trimers allows for the facile, one-step, isolation of these immunogens in research laboratories. PGT145 columns are also valuable as a component of more complex purification processes in current Good Manufacturing Practice programs. However, we found that PGT145 purification was highly variable and markedly inefficient when used to process supernatants from transiently transfected ExpiCHO-S cells expressing the BG505 SOSIP.664 and other trimeric Env proteins. In contrast, no such problems arose when the same Env proteins derived from a stable CHO cell line were processed on the same PGT145 columns, or with transient transfection supernatants from 293F cells. An investigation of the ExpiCHO-S transfection system identified the presence of polyanions, including but perhaps not limited to dextran sulfate, in the Enhancer component of the transfection system. We hypothesized that these polyanions bound to the cationic PGT145 epitope on the trimers and impeded their ability to bind to the PGT145 affinity column. We found that replacing the Enhancer component with alternative culture medium supplements substantially increased the yield of PGT145-purifiable trimers, and we also confirmed that both dextran sulfate and the Enhancer component were indeed inhibitors of PGT145 binding to BG505 SOSIP.664 trimers in immunoassays. The presence of polyanions, including but not limited to nucleic acids, should be considered in other circumstances where PGT145 columns are less efficient than expected at purifying native-like trimers.

Agonist-induced desensitisation of β3 -adrenoceptors: Where, when, and how?

β3 -Adrenoceptor agonists have proven useful in the treatment of overactive bladder syndrome, but it is not known whether their efficacy during chronic administration may be limited by receptor-induced desensitisation. Whereas the β2 -adrenoceptor has phosphorylation sites that are important for desensitisation, the β3 -adrenoceptor lacks these; therefore, it had been assumed that β3 -adrenoceptors are largely resistant to agonist-induced desensitisation. While all direct comparative studies demonstrate that β3 -adrenoceptors are less susceptible to desensitisation than β2 -adrenoceptors, desensitisation of β3 -adrenoceptors has been observed in many models and treatment settings. Chimeric β2 - and β3 -adrenoceptors have demonstrated that the C-terminal tail of the receptor plays an important role in the relative resistance to desensitisation but is not the only relevant factor. While the evidence from some models, such as transfected CHO cells, is inconsistent, it appears that desensitisation is observed more often after long-term (hours to days) than short-term (minutes to hours) agonist exposure. When it occurs, desensitisation of β3 -adrenoceptors can involve multiple levels including down-regulation of its mRNA and the receptor protein and alterations in post-receptor signalling events. The relative contributions of these mechanistic factors apparently depend on the cell type under investigation. Which if any of these factors is applicable to the human urinary bladder remains to be determined. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Analysis of LPI-causing mutations on y+LAT1 function and localization

Backgroundy+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein.ResultsSystem y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants.ConclusionsThe three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.

Immature surfactant protein-B impairs the antioxidant capacity of HDL

Circulating immature surfactant protein B (proSP-B) forms emerged as the most reliable lung-specific circulating marker for alveolar-capillary membrane (ACM) dysfunction and for the overall clinical status of heart failure (HF). Notably, in terms of HF hospitalization, immature SP-B overwhelms the prognostic role of other most frequently used clinical parameters such as those related to lung dysfunction. The strong prognostic value of circulating proSP-B in HF suggests more widespread and possible systemic effects. Thus, we assessed the plasma distribution of proSP-B evaluating whether it exists in a lipoprotein-bound form and its impact on lipoprotein structure and function.ProSP-B forms were detectable in high-density lipoprotein (HDL) only. To assess the impact of proSP-B on HDL, HDL from healthy subjects were enriched with proSP-B produced by a stably transfected CHO cell line that specifically expresses and releases the human proSP-B. After enrichment, HDL size and lipoprotein electrophoretic mobility, and protein composition did not show apparent differences. HDL antioxidant capacity (HOI), assessed as their ability to inhibit air-induced LDL oxidation, was impaired after proSP-B enrichment. HOI was also higher in HF patients with respect to age-matched control healthy subjects (p = 0.013).Circulating proSP-B, besides its potential role as a specific marker for ACM dysfunction in HF patients with diagnostic and prognostic value, binds to human HDL impairing their antioxidant capacity. These findings shed light on proSP-B as a molecule that contributes to the reduction of the defense against oxidative stress, a key mediator in the pathogenesis of HF.

Chemerin 156F, generated by chymase cleavage of prochemerin, is elevated in joint fluids of arthritis patients

BackgroundChemerin is a chemoattractant involved in immunity that also functions as an adipokine. Chemerin is secreted as an inactive precursor (chem163S), and its activation requires proteolytic cleavages at its C-terminus, involving proteases in coagulation, fibrinolysis, and inflammation. Previously, we found chem158K was the dominant chemerin form in synovial fluids from patients with arthritis. In this study, we aimed to characterize a distinct cleaved chemerin form, chem156F, in osteoarthritis (OA) and rheumatoid arthritis (RA).MethodsPurified chem156F was produced in transfected CHO cells. To quantify chem156F in OA and RA samples, we developed a specific ELISA for chem156F using antibody raised against a peptide representing the C-terminus of chem156F.ResultsCa2+ mobilization assays showed that the EC50 values for chem163S, chem156F, and chem157S were 252 ± 141 nM, 133 ± 41.5 nM, and 5.83 ± 2.48 nM, respectively. chem156F was more active than its precursor, chem163S, but very much less potent than chem157S, the most active chemerin form. Chymase was shown to be capable of cleaving chem163S at a relevant rate. Using the chem156F ELISA we found a substantial amount of chem156F present in synovial fluids from patients with OA and RA, 24.06 ± 5.51 ng/ml and 20.35 ± 5.19 ng/ml (mean ± SEM, n = 25) respectively, representing 20% of total chemerin in OA and 76.7% of chemerin in RA synovial fluids.ConclusionsOur data show that chymase cleavage of chem163S to partially active chem156F can be found in synovial fluids where it can play a role in modulation of the inflammation in joints.