Efficiency Comparative Approach of Plant-Produced Monoclonal Antibodies against Rabies Virus Infection

Rabies encephalitis is a fatal zoonotic viral disease caused by the neurotropic rabies virus. It remains a major public health concern as it causes almost 100% fatality and has no effective medication after the onset of the disease. However, this illness is preventable with the timely administration of effective post-exposure prophylaxis (PEP) consisting of the rabies vaccine and passive immune globulins (HRIG and ERIG). Recently, conventional PEP has been shown to have many limitations, resulting in little support for these expensive and heterologous globulins. Monoclonal antibody (mAb) production via recombinant technology in animal and human cell cultures, as well as a plant-based platform, was introduced to overcome the costly and high-tech constraints of former preparations. We used transient expression technology to produce two mAbs against the rabies virus in Nicotiana benthamiana and compared their viral neutralizing activity in vitro and in vivo. The expression levels of selective mAbs E559 and 62-71-3 in plants were estimated to be 17.3 mg/kg and 28.6 mg/kg in fresh weight, respectively. The plant-produced mAbs effectively neutralized the challenge virus CVS-11 strain in a cell-based RFFIT. In addition, the combination of these two mAbs in a cocktail protected hamsters from rabies virus infection more effectively than standard HRIG and ERIG. This study suggests that the plant-produced rabies antibody cocktail has promising potential as an alternative biological to polyclonal RIG in rabies PEP

Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile’s innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile’s innate immune system

Identification of Fatty Acid Glucose Esters as Os9BGlu31 Transglucosidase Substrates in Rice Flag Leaves

Rice Os9BGlu31 transglucosidase transfers glucosyl moieties between various carboxylic acids and alcohols, including phenolic acids and flavonoids, in vitro. The role of Os9BGlu31 transglucosidase in rice plant metabolism has only been suggested to date. Methanolic extracts of rice bran and leaves were found to contain oleic acid and linoleic acid to which Os9BGlu31 could transfer glucose from the 4-nitrophenyl β-d-glucoside (4NPGlc) donor to form 1-<i>O</i>-acyl glucose esters. Os9BGlu31 showed higher activity with oleic acid (18:1) and linoleic acid (18:2) than with stearic acid (18:0) and had both a higher <i>k</i>_cat and a higher <i>K</i>_m for linoleic than oleic acid in the presence of 8 mM 4NPGlc donor. <i>Os9BGlu31</i> knockout mutant rice lines were found to have significantly larger amounts of fatty acid glucose esters than wild-type control lines. Because the transglucosylation reaction is reversible, these data suggest that fatty acid glucose esters act as glucosyl donor substrates for Os9BGlu31 transglucosidase in rice

Rice Os9BGlu31 is a transglucosidase with the capacity to equilibrate phenylpropanoid, flavonoid, and phytohormone glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu¹⁶⁹), catalytic nucleophile residues (Glu³⁸⁷), and His³⁸⁶, the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.Sukanya Luang, Jung-Il Cho, Bancha Mahong, Rodjana Opassiri, Takashi Akiyama, Kannika Phasai, Juthamath Komvongsa, Nobuhiro Sasaki, Yan-ling Hua, Yuki Matsuba, Yoshihiro Ozeki, Jong-Seong Jeon, and James R. Ketudat Cairn

Recombinant Expression and Characterization of the Cytoplasmic Rice β-Glucosidase Os1BGlu4

<div><p>The Os1BGlu4 β-glucosidase is the only glycoside hydrolase family 1 member in rice that is predicted to be localized in the cytoplasm. To characterize the biochemical function of rice Os1BGlu4, the <i>Os</i>1<i>bglu</i>4 cDNA was cloned and used to express a thioredoxin fusion protein in <i>Escherichia coli</i>. After removal of the tag, the purified recombinant Os1BGlu4 (rOs1BGlu4) exhibited an optimum pH of 6.5, which is consistent with Os1BGlu4's cytoplasmic localization. Fluorescence microscopy of maize protoplasts and tobacco leaf cells expressing green fluorescent protein-tagged Os1BGlu4 confirmed the cytoplasmic localization. Purified rOs1BGlu4 can hydrolyze <i>p</i>-nitrophenyl (<i>p</i>NP)-<i>β</i>-d-glucoside (<i>p</i>NPGlc) efficiently (<i>k</i>_cat/<i>K</i>_m  =  17.9 mM⁻¹·s⁻¹), and hydrolyzes <i>p</i>NP-<i>β</i>-d-fucopyranoside with about 50% the efficiency of the <i>p</i>NPGlc. Among natural substrates tested, rOs1BGlu4 efficiently hydrolyzed β-(1,3)-linked oligosaccharides of degree of polymerization (DP) 2–3, and β-(1,4)-linked oligosaccharide of DP 3–4, and hydrolysis of salicin, esculin and <i>p</i>-coumaryl alcohol was also detected. Analysis of the hydrolysis of <i>p</i>NP-<i>β</i>-cellobioside showed that the initial hydrolysis was between the two glucose molecules, and suggested rOs1BGlu4 transglucosylates this substrate. At 10 mM <i>p</i>NPGlc concentration, rOs1BGlu4 can transfer the glucosyl group of <i>p</i>NPGlc to ethanol and <i>p</i>NPGlc. This transglycosylation activity suggests the potential use of Os1BGlu4 for <i>p</i>NP-oligosaccharide and alkyl glycosides synthesis.</p></div

The pH optimum and pH stability of rOs1BGlu4 hydrolysis activity.

<p>A. pH optimum determination: rOs1BGlu4 (0.25 µg) was assayed with 1 mM <i>p</i>NPGlc in different 50 mM pH buffers (formate, pH 4.0; sodium acetate, pH 4.5–5.5; sodium phosphate, pH 6.0–7.5; Tris, pH 8.0–9.5; CAPS, pH 10.0–11.0) at 30°C for 10 min. B. pH stability evaluation: rOs1BGlu4 (20 µg) was incubated in the buffers described above for 10 min, 1, 3, 6, 12 and 24 h, then diluted 40-fold in 50 mM phosphate buffer, pH 6.5, and the activity was determined. The data are provided as mean + SE.</p

Evaluation of laminaritriose hydrolysis.

<p>A. Thin layer chromatographic evaluation of products of laminaritriose hydrolysis at different time points. rOs1BGlu4 (0.125 µg) was incubated with 1 mM laminaritriose in 50 mM sodium phosphate, pH 6.5, at 30 °C from 5 to 30 min (5 m–30 m). Samples were incubated with (+) and without (-) enzyme, then evaluated by silica gel TLC with sulfuric acid staining. The positions of glucose (G); laminaribiose (L2); and laminaritriose (L3) are marked. B: Kinetic data for laminaritriose hydrolysis. The Michaelis–Menten curve and inset Lineweaver-Burk plot are shown, along with the derived kinetic parameters and standard errors.</p

TLC of hydrolysis products of rOs1BGlu4 with cello-oligosaccharides and laminari-oligosaccharides.

<p>In each 50 µl reaction, 0.125 µg rOs1BGlu4 was incubated with 1 mM oligosaccharide in 50 mM sodium phosphate, pH 6.5, at 30 °C for 20 min. Samples were incubated with (+) and without (−) enzyme. Then, 2 µl of the reaction was spotted onto the TLC plate. Standards and substrates are: G, glucose; C2, cellobiose; C3, cellotriose; C4, cellotetraose; C5, cellopentaose; C6, cellohexaose; L2, laminaribiose; L3, laminaritriose; L4, laminaritetraose and L5, laminaripentaose.</p

Kinetic parameters of rOs1BGlu4 in the hydrolysis of <i>p</i>NP glycosides and oligosaccharides.

<p>Kinetic parameters of rOs1BGlu4 in the hydrolysis of <i>p</i>NP glycosides and oligosaccharides.</p

The effects of the different incubation times and substrate concentrations on the transglycosylation activity of Os1BGlu4.

<p>TLC analysis of products are shown. The standards are marked as M: marker, including <i>p</i>NPGlc (pG), <i>p</i>NP-<i>β</i>-cellobioside (pC2). <i>p</i>NP marks the position of <i>p</i>-nitrophenol. For the reactions, con is a control reaction without rOs1BGlu4, and 0.5–40, stand for reactions including 0.5 mM <i>p</i>NPGlc, 5 mM <i>p</i>NPGlc, 10 mM <i>p</i>NPGlc, 20 mM <i>p</i>NPGlc, and 40 mM <i>p</i>NPGlc, respectively, while 1, 2 and 3 hours are the incubation times. A: TLC plate visualized by the carbohydrate staining method. B: TLC plate visualized by UV light.</p