Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Plant‐based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C‐terminal fused to the heavy chain of 14D9 (vac‐Abs) and compared with secreted and ER‐retained variants (sec‐Ab, ER‐Ab, respectively). Accumulation of ER‐ and vac‐Abs was 10‐ to 15‐fold higher than sec‐Ab. N‐glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec‐Ab while vac‐Abs carried mainly oligomannosidic (Man 7‐9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec‐Ab‐RFP localized in the apoplast while vac‐Abs‐RFP were exclusively detected in the central vacuole. The data suggest that vac‐Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N‐glycans). Importantly, vac‐Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post‐translational modifications, but also point to a reconsideration of current concepts in plant glycan processing.     

Volatile exposure within the honeybee hive and its effect on olfactory discrimination

Honeybees of different ages and reproductive castes cohabit in the hive where they are exposed to many odors that might affect associative learning. Our aim was to analyze the role of odors pre-exposed as volatiles on appetitive learning in honeybees of different ages and search for their long-term effect both under natural and laboratory conditions. By evaluating memory acquisition and retention through a differential proboscis extension response conditioning, we found that hive-exposed odors offered as a reinforced conditioned stimulus during training promoted a learning-reduced effect [latent inhibition (LI)]. On the other hand, no effect was found when the non-reinforced conditioned stimulus was pre-exposed. The LI effect varied with the odor identity. However, only slight differences were found with the age of the bees. Exposure-conditioning intervals longer than 24 h did not show an LI effect unless the odor concentration was increased or exposure was prolonged. Our results show that pre-exposed volatiles could either reduce learning performance, if this odor is later associated with food, or be irrelevant in the case that alternative scented resources circulate within the colony. The differential effects found according to the olfactory exposure characteristics could strongly influence the propagation of chemosensory information within the hive.     

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

Assessment of Environmental Hazards to Public Health in Temperate Urban Argentina

EcoHealth - Human health risk in urban areas depends on multiple environmental features. We performed a year-round survey in a highly urbanized district located in temperate Argentina (General San...     

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.     

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4 ng/mL, AOH 2 and 3.3 ng/mL; red wine: AME 0.1 and 0.2 ng/mL, AOH 4.5 and 7.5 ng/mL; apple juice: AME 1.7 and 2.8 ng/mL, AOH 5 and 9 ng/mL; other juices: AME 2.0 and 3.1 ng/mL, AOH 6 and 10 ng/mL). Recoveries in all cases were greater than 80 %. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18 ng/mL, 6 of 56 samples of red wine had a maximum of 13 ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225 ng/mL) did the toxin level exceed the LOQ.