Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

Vitamin B6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B6 vitamers were detected as compared with the control.

Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc.

Enzymatic conversion from pyridoxal to pyridoxine caused by microorganisms within tobacco phyllosphere.

Vitamin B6 (VB6) comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate (PLP) is a coenzyme involved in a high diversity of biochemical reactions. In plants, PLP is de novo synthesized, and pyridoxine (PN) is usually maintained as the predominant B6 vitamer. Although the conversion from pyridoxal (PL) to PN catalyzed by PL reductase in plants has been confirmed, the enzyme itself remains largely unknown. We previously found pre-incubation at 35 °C dramatically enhanced PL reductase activity in tobacco leaf homogenate. In this study, we demonstrated that the increase in the reductase activity was a consequence of phyllosphere microbial proliferation. VB6 was detected from tobacco phyllosphere, and PL level was the highest among three non-phosphorylated B6 vitamers. When the sterile tobacco rich in PL were kept in an open, warm and humid environment to promote microorganism proliferation, a significant change from PL to PN was observed. Our results suggest that there may be a plant-microbe interaction in the conversion from PL to PN within tobacco phyllosphere.

Enzymatic transamination of pyridoxamine in tobacco plants.

Vitamin B6 (VB6) comprises a group of pyridine compounds that are involved in a surprisingly high diversity of biochemical reactions. Humans and animals depend largely on plants for their VB6 nutrition. Many studies have focused on biosynthesis of VB6 and comparatively little is known about VB6 metabolic conversion in plants. Recently, we have found that an efficient conversion pathway between pyridoxal (PL) and pyridoxamine (PM) is present in tobacco, but the catalytic enzyme remains an unsolved mystery. In this study, enzymes catalyzing the transamination of PM were purified from tobacco leaves and characterized. Our results suggest that a specific PM-pyruvate aminotranferase dominates the reversible transamination of PM in tobacco, and also show that the apo form of glutamic-oxaloacetic aminotranferase from tobacco, but not the holoenzyme, is able to catalyze the analogous transamination reaction between PM and either oxaloacetate or α-ketoglutarate. PM-pyruvate aminotranferase is involved in a degradation pathway for VB6 compounds in bacteria. Therefore, our study raises questions about whether the degradation pathway of VB6 exists in plants.

Effect of abiotic stress on the abundance of different vitamin B6 vitamers in tobacco plants.

There are six different vitamin B6 (VB6) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP), and pyridoxine 5'-phosphate (PNP), of which PLP is the active form. Although transcriptional regulation of the genes involved in the de novo and salvage pathways of PLP syntheses after stress treatments has been described for Arabidopsis thaliana and tobacco plants, it remains open as to whether this in turn affects VB6 levels. In this study, the effects of chilling, UV radiation, intensity of illumination, osmotic pressure, oxidative and drought stresses on the abundance of different B6 vitamers in tobacco plants were examined by using high performance liquid chromatography (HPLC). The abiotic stressors resulted in significant increase of PLP, and caused some corresponding changes with PL and PN. The highest increase of PLP was 2.5-fold compared to the control plants, followed by a continuous decline back to the control levels. These changes are presumably caused by the regulation and control mechanism on the VB6 metabolism in plants.

Development of hybrid viral vectors for gene therapy.

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.

Characterization of an acid phosphatase responsible for hydrolysis of pyridoxal 5'-phosphate in tobacco plants.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B(6), is an important cofactor for many enzymatic reactions. PLP is also a very reactive molecule, and the hydrolysis of PLP is crucial for controlling intracellular PLP concentrations. However, little is known about the enzymatic hydrolysis of PLP in plants. In this study, a novel acid phosphatase was purified from tobacco leaves and characterized by using PLP as a substrate. This phosphatase was purified 180-fold with a yield of 28% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose FF, Sephadex G-100 and SP Sephadex C-25. Our data revealed that the purified enzyme was a dimer with a molecular mass of approximately 50 kDa. The purified phosphatase had maximum catalytic activity at pH 5.5, and displayed optimal activity at 50 °C. The enzyme required divalent metal ion for activity, and Mg(2+), among a few tested cations, was the most effective for catalysis under saturating substrate concentrations. The activity of the purified phosphatase was inhibited by molybdate, fluoride and EDTA, but was not inhibited by levamisole and tartrate. The phosphatase hydrolyzed a broad range of substrates at different rates, and the hydrolysis of PLP was competitively inhibited by ATP, pNPP, and by the reaction products, PL and inorganic phosphate. The phosphatase had a Km of 0.24 mM and a Vmax of 2.76 µmol/min/mg with PLP. When pyridoxamine 5'-phosphate or pyridoxine 5'-phosphate was tested as a substrate, the phosphatase activity was reduced by 50%. Our study suggests that the enzyme is a nonspecific acid phosphatase responsible for hydrolysis of all three phosphorylated B(6) vitamers in tobacco plants.

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19.

Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

Characterization of enzymes involved in the interconversions of different forms of vitamin B(6) in tobacco leaves.

There are six different vitamin B(6) (VB(6)) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP) and pyridoxine 5'-phosphate (PNP). PLP is a coenzyme required by more than 100 cellular enzymes. In spite of the importance of this vitamin, the understanding of VB(6) metabolic conversion in plants is limited. In this study, we developed a sensitive and reliable method to assay VB(6)-metabolizing enzyme activities by monitoring their products visually using high-performance liquid chromatography. With this method, the reactions catalyzed by PL/PM/PN kinase, PMP/PNP oxidase, PM-pyruvate aminotransferase, PL reductase and PLP phosphatase were all nicely detected using crude protein extracts of tobacco leaves. Under optimal in vitro conditions, specific activities of those enzymes were 0.15 ± 0.03, 0.10 ± 0.03, 0.08 ± 0.02, 0.64 ± 0.13 and 23.08 ± 1.98 nmol product/min/mg protein, respectively. This is the first report on the conversion between PM and PL catalyzed by PM-pyruvate aminotransferase in plants. Furthermore, the PL reductase activity was found to be heat inducible. Our study sheds light on the VB(6) metabolism taking place in plants.

Interconversions of different forms of vitamin B6 in tobacco plants.

There are six different vitamin B(6) (VB(6)) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5'-phosphate (PLP), pyridoxamine 5'-phosphate (PMP), and pyridoxine 5'-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB(6) in the human diet, and VB(6) plays an important role in plants, the mechanisms underlying the interconversions of different VB(6) forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100mg/L of PM, PL or PN and the abundance of the different B(6) vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB(6) was about 3.9 µg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB(6) by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B(6) vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.

Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector.

Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors.