[Show abstract][Hide abstract] ABSTRACT: The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
PLoS ONE 08/2015; 10(8). DOI:10.1371/journal.pone.0136507 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This short commentary examines the factors that led to Food and Drug Administration's approval of the first plant-derived biologic.
In 2012, the first plant-derived protein pharmaceutical (biologic) was approved for commercial use in humans. The product, a recombinant form of human β-glucocerebrosidase marketed as ELELYSO, was developed by Protalix Biotherapeutics (Carmiel, Israel). The foresight to select this particular therapeutic product for development, flawless production pipeline, and serendipity seem to provide the key in explaining how ELELYSO became the first plant-derived biologic to achieve approval by Food and Drug Administration.
While the circumstances that enabled Protalix and its scientists to become the first to arrive at this historic milestone are perhaps unique, it is anticipated that more biologics will follow suit in winning regulatory endorsement.
[Show abstract][Hide abstract] ABSTRACT: The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates non-covalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a pre-hairpin intermediate of gp41. The expression level of MPR-TMTEV6His was improved by fusion to the C-terminus of Mistic protein, yielding ˜1 mg of pure protein per liter. The isolated MPR-TMTEV6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.
Protein Science 11/2014; 23(11). DOI:10.1002/pro.2540 · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The VLPNPV 2014 Conference that was convened at the Salk institute was the second conference of its kind to focus on advances in production, purification, and delivery of virus-like particles (VLPs) and nanoparticles. Many exciting developments were reported and discussed in this interdisciplinary arena, but here we report specifically on the contributions of plant-based platforms to VLP vaccine technology as reported in the section of the conference devoted to the topic as well in additional presentations throughout the meeting. The increasing popularity of plant production platforms is due to their lower cost, scalability, and lack of contaminating animal pathogens seen with other systems. Reports include production of complex VLPs consisting of 4 proteins expressed at finely-tuned expression levels, a prime-boost strategy for HIV vaccination using plant-made VLPs and a live viral vector, and the characterization and development of plant viral nanoparticles for use in cancer vaccines, drug delivery, and bioimaging.
Human Vaccines and Immunotherapeutics 10/2014; 10(10):3068-73. DOI:10.4161/21645515.2014.979693 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
[Show abstract][Hide abstract] ABSTRACT: Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre- and post-exposure treatment. The presence and functional necessity of complex N-glycans (i.e. sialylated structures) is a challenging issue in respect to its recombinant expression. We aim to produce recombinant BChE (rBChE) in plants with a glycosylation profile that largely resembles the plasma-derived counterpart. rBChE was transiently co-expressed in the model plant Nicotiana benthamiana. Site-specific sugar profiling by mass spectrometry of secreted rBChE collected from the intercellular fluid (IF) revealed the presence of mono- and di-sialylated N-glycans, with overall glycosylation profile that is virtually identical to the plasma-derived orthologue. Increase in sialylation content of rBChE was acehived by the over-expression of an additional glycosylation enzyme that generates branched N-glycans, (i.e. GnTIV), which resulted in the production of rBChE decorated with a large fraction of tri-sialylated structures. Sialylated as well as non-sialylated plant-derived rBChE exhibit functional in vitro activity comparable to that of its commercially available equine-derived counterpart. These results demonstrate the ability of plants to generate valuable proteins with designed sialylated glycosylation profiles optimized for therapeutic efficacy. Moreover, the efficient synthesis of carbohydrates present only in minute amounts on the native protein (tri-sialylated N-glycans) facilitates the generation of a product with superior efficacies and/or new therapeutic functions.
[Show abstract][Hide abstract] ABSTRACT: Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.
[Show abstract][Hide abstract] ABSTRACT: Cocaine addiction affects millions of people with disastrous personal and social consequences. Cocaine is one of the most reinforcing of all drugs of abuse, and even those who undergo rehabilitation and experience long periods of abstinence have more than 80% chance of relapse. Yet there is no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts. Recent studies, however, have demonstrated a promising potential treatment option with the help of the serum enzyme butyrylcholinesterase (BChE), which is capable of breaking down naturally occurring (−)-cocaine before the drug can influence the reward centers of the brain or affect other areas of the body. This activity of wild-type (WT) BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against (−)-cocaine. Plants are a promising means to produce large amounts of these cocaine hydrolase variants of BChE, cheaply, safely with no concerns regarding human pathogens and functionally equivalent to enzymes derived from other sources. Here, in expressing cocaine-hydrolyzing mutants of BChE in Nicotiana benthamiana using the MagnICON virus-assisted transient expression system, and in reporting their initial biochemical analysis, we provide proof-of-principle that plants can express engineered BChE proteins with desired properties.
[Show abstract][Hide abstract] ABSTRACT: The transmembrane HIV-1 envelope protein gp41 has been shown to play critical roles in the viral mucosal transmission and infection of CD4+ cells. Gag is a structural protein configuring the enveloped viral particles and has been suggested to constitute a target of the cellular immunity that may control viral load. We hypothesized that HIV enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the membrane proximal external, transmembrane and cytoplasmic domains (dgp41) could be expressed in plants. To this end, plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a Tobamovirus-based expression system or a combination of both. Our results of biophysical, biochemical and electron microscopy characterization demonstrates that plant cells could support not only the formation of enveloped HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These findings provide further impetus for the journey towards a broadly efficacious and inexpensive subunit vaccine against HIV-1.
[Show abstract][Hide abstract] ABSTRACT: Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.
Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.
Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD100 of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.
Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.
PLoS ONE 03/2013; 8(3):e59159. DOI:10.1371/journal.pone.0059159 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family.
[Show abstract][Hide abstract] ABSTRACT: Cholinergic signaling suppresses inflammation in blood and brain and attenuates apoptosis in other tissues, but whether it blocks inflammation in skeletal muscle under toxicant exposure, injuries and diseases remained unexplored. Here, we report nicotinic attenuation of inflammation and alteration of apoptotic protein expression pattern in murine muscle tissue and cultured myotubes, involving the RNA-binding protein, Tristetraprolin, and the anti-apoptotic protein, Mcl-1. In muscles and C2C12 myotubes, cholinergic excitation by exposure to nicotine or the organophosphorous pesticide, Paraoxon, induced Tristetraprolin overproduction while reducing pro-inflammatory transcripts such as IL-6, CXCL1 (KC) and CCL2 (MCP-1). Furthermore, nicotinic excitation under exposure to the bacterial endotoxin LPS attenuated over-expression of the CCL2 and suppressed the transcriptional activity of NF-ĸB and AP-1. Tristetraprolin was essential for this anti-inflammatory effect of nicotine in basal conditions. However, its knockdown also impaired the pro-inflammatory response to LPS. Finally, in vivo administration of Paraoxon or recombinant Acetylcholinesterase, leading respectively to either gain or loss of cholinergic signaling, modified muscle expression of key mRNA processing factors and several of their apoptosis-related targets. Specifically, cholinergic imbalances enhanced the kinase activators of the Serine-Arginine splicing kinases, Clk1 and Clk3. Moreover, Paraoxon raised the levels of the anti-apoptotic protein, Mcl-1, through a previously unrecognized polyadenylation site selection mechanism, producing longer, less stable Mcl-1 mRNA transcripts. Together, our findings demonstrate that in addition to activating muscle function, acetylcholine regulates muscle inflammation and cell survival, and point to Tristetraprolin and the choice of Mcl-1 mRNA polyadenylation sites as potential key players in muscle reactions to insults.
[Show abstract][Hide abstract] ABSTRACT: The membrane-proximal region spanning residues 649-684 of the HIV-1 envelope protein gp41 (MPR₆₄₉₋₆₈₄) is an attractive vaccine target for humoral immunity that blocks viral transcytosis across the mucosal epithelia. However, induction of high-titer MPR₆₄₉₋₆₈₄-specific antibodies remains a challenging task. To explore potential solutions for this challenge, we tested a new translational fusion protein comprising the plague F1-V antigen and MPR₆₄₉₋₆₈₄ (F1-V-MPR₆₄₉₋₆₈₄). We employed systemic immunization for initial feasibility analyses. Despite strong immunogenicity demonstrated for the immunogen, repeated systemic immunizations of mice with F1-V-MPR₆₄₉₋₆₈₄ hardly induced MPR₆₄₉₋₆₈₄-specific IgG. In contrast, a single immunization with F1-V-MPR₆₄₉₋₆₈₄ mounted a significant anti-MPR₆₄₉₋₆₈₄ IgG response in animals that were primed with another MPR₆₄₉₋₆₈₄ fusion protein based on the cholera toxin B subunit. Additional boost immunizations with F1-V-MPR₆₄₉₋₆₈₄ recalled and maintained the antibody response and expanded the number of specific antibody-secreting B cells. Thus, while F1-V-MPR₆₄₉₋₆₈₄ alone was not sufficiently immunogenic to induce detectable levels of MPR₆₄₉₋₆₈₄-specific antibodies, these results suggest that prime-boost immunization using heterologous antigen-display platforms may overcome the poor humoral immunogenicity of MPR₆₄₉₋₆₈₄ for the induction of durable humoral immunity. Further studies are warranted to evaluate the feasibility of this strategy in mucosal immunization. Lastly, our findings add to a growing body of evidence in support of this strategy for immunogen design for poorly immunogenic epitopes besides the MPR of HIV-1's transmembrane envelope protein.
[Show abstract][Hide abstract] ABSTRACT: The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.
Proceedings of the National Academy of Sciences 11/2010; 107(47):20251-6. DOI:10.1073/pnas.1009021107 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge.
[Show abstract][Hide abstract] ABSTRACT: The essential role of acetylcholinesterase (AChE) in the neuromuscular junction (NMJ) predicts severe consequences following loss of function by its irreversible inhibition. The vital role of the acetylcholine-hydrolyzing enzyme AChE in terminating cholinergic neurotransmission has been recognized for almost as many years. Anticholinesterases can be commonly encountered as industrial pesticides, weapons of war, Alzheimer's medications as well as the natural toxins of many organisms including fungi, plants, and animals. Exposure to clinically relevant doses of anticholinesterases results in immediate and multisystem physiological disturbances that underscore the broad anatomical distribution of the mammalian cholinergic system. The body adapts to the insult and attempts to compensate for the cholinergic dysregulation by inhibitor–enzyme interactions, NMJ remodeling, and changes in circulating cytokine profiles. In addition the loss of synaptic cholinergic regulation by AChE inhibition has immediate negative consequences for mammalian physiology. The severe multisystem clinical presentation of anti-ChE intoxication demonstrates the essential and ubiquitous nature of the mammalian cholinergic system. Current medical intervention in the case of acute exposure to anticholinesterase agents includes use of the muscarinic receptor antagonist atropine to block overstimulation, and oximes to reactivate the OP-modified AChE. The reversible carbamate inhibitor, pyridostigmine bromide, is also used for prophylaxis.
Handbook of Toxicology of Chemical Warfare Agents, 12/2009: pages 691-717; , ISBN: 9780123744845
[Show abstract][Hide abstract] ABSTRACT: A major focus of biotechnology is the improvement of human health around the globe. It is anticipated that the genomic revolution will greatly expand our knowledge of the molecular basis of many diseases and pathological states. Combining this knowledge with powerful screening techniques will be used in the development of safe and efficacious biologics and drugs for the prevention and treatment of disease. Unfortunately, the availability of these new biologics and drugs for use by all those who need them greatly depends on economic considerations such as the cost of their development, production, and delivery. Therefore, a major challenge of biotechnology is to translate clinical innovations to economically viable practice. The production of plant-derived vaccines for mucosal delivery is a step toward that goal.
New Generation Vaccines, 4th edited by MM Levine, 12/2009: chapter 30: pages 306-315; Informa Healthcare., ISBN: 978-1-4200-6073-7
[Show abstract][Hide abstract] ABSTRACT: Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.
As a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4 degrees C for up to 5 months.
Our results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.