Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an alpha-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-alpha-D-glucopyranoside with a pH optimum of 5.5-5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-alpha-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable alpha-glucosidase and alpha-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed.     

Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.     

Enhanced Accumulation of BiP in Transgenic Plants Confers Tolerance to Water Stress

The binding protein (BiP) is an important component of endoplasmic reticulum stress response of cells. Despite extensive studies in cultured cells, a protective function of BiP against stress has not yet been demonstrated in whole multicellular organisms. Here, we have obtained transgenic tobacco (Nicotiana tabacum L. cv Havana) plants constitutively expressing elevated levels of BiP or its antisense cDNA to analyze the protective role of this endoplasmic reticulum lumenal stress protein at the whole plant level. Elevated levels of BiP in transgenic sense lines conferred tolerance to the glycosylation inhibitor tunicamycin during germination and tolerance to water deficit during plant growth. Under progressive drought, the leaf BiP levels correlated with the maintenance of the shoot turgidity and water content. The protective effect of BiP overexpression against water stress was disrupted by expression of an antisense BiP cDNA construct. Although overexpression of BiP prevented cellular dehydration, the stomatal conductance and transpiration rate in droughted sense leaves were higher than in control and antisense leaves. The rate of photosynthesis under water deficit might have caused a degree of greater osmotic adjustment in sense leaves because it remained unaffected during water deprivation, which was in marked contrast with the severe drought-induced decrease in the CO(2) assimilation in control and antisense leaves. In antisense plants, the water stress stimulation of the antioxidative defenses was higher than in control plants, whereas in droughted sense leaves an induction of superoxide dismutase activity was not observed. These results suggest that overexpression of BiP in plants may prevent endogenous oxidative stress.     

Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity

Expression and stability of immunoglobulins in transgenic plants have been investigated and optimized by accumulation in different cellular compartments as cytosol, apoplastic space and endoplasmic reticulum (ER) as will be discussed in this review. In several cases described the highest accumulation of complete active antibodies was achieved by targeting into the apoplastic space. High-level expression of active recombinant single-chain Fv antibodies (scFv's) was obtained by retention of these proteins in the lumen of the endoplasmic reticulum. This has been shown for leaves and seeds of transgenic tobacco as well as for potato tubers. Transgenic tobacco seeds, potato tubers and tobacco leaves can facilitate stable storage of scFv's accumulated in the ER over an extended (seeds, tubers) or a short (leaves) period of time. The expression of specific scFv's in different plant species, plant organs and cellular compartments offers the possibility of blocking regulatory factors or pathogens specifically. Examples are scFv's expressed in the cytosol and the apoplastic space of transgenic plant cells modulating the infection process of plant viruses and a cytosolically expressed scFv that influenced the activity of phytochrome A protein. The immunomodulation approach has been shown to be also applicable for investigating the action of the phyto-hormone abscisic acid (ABA). High-level accumulation of specific anti-ABA scFv's in the ER of all leaf cells has been used to block the influence of ABA on the stomatal functions. Seed-specific expression of high amounts of anti-ABA-scFv's at a defined time of seed-development induced a developmental switch from seed ripening to vegetative growth. It has been demonstrated that ER retention is essential for the accumulation of sufficient scFv to bind high concentrations of ABA in the transgenic seeds.     

Endoplasmic reticulum stress underlying the pro-apoptotic effect of epigallocatechin gallate in mouse hepatoma cells

It has been recently reported that tea flavanols, including epigallocatechin gallate (EGCG), efficiently inhibit glucosidase II in liver microsomes. Since glucosidase II plays a central role in glycoprotein processing and quality control in the endoplasmic reticulum we investigated the possible contribution of endoplasmic reticulum stress and unfolded protein response (UPR) to the pro-apoptotic activity of EGCG in mouse hepatoma cells. The enzyme activity measurements using 4-methylumbelliferyl-alpha-d-glucopyranoside substrate confirmed the inhibition of glucosidase II in intact and alamethicin-permeabilized cells. EGCG treatment caused a progressive elevation of apoptotic activity as assessed by annexin staining. The induction of CHOP/GADD153, the cleavage of procaspase-12 and the increasing phosphorylation of eIF2alpha were revealed in these cells by Western blot analysis while the induction of endoplasmic reticulum chaperones and foldases was not observed. Time- and concentration-dependent depletion of the endoplasmic reticulum calcium stores was also demonstrated in the EGCG-treated cells by single-cell fluorescent detection. The massive alterations in the endoplasmic reticulum morphology revealed by fluorescent microscopy further supported the development of UPR. Collectively, our results indicate that EGCG interferes with protein processing in the endoplasmic reticulum presumably due to inhibition of glucosidase II and that the stress induces an incomplete unfolded protein response with dominantly pro-apoptotic components.     

Down-Regulation of OsSPX1 Causes High Sensitivity to Cold and Oxidative Stresses in Rice Seedlings

Rice SPX domain gene, OsSPX1, plays an important role in the phosphate (Pi) signaling network. Our previous work showed that constitutive overexpression of OsSPX1 in tobacco and Arabidopsis plants improved cold tolerance while also decreasing total leaf Pi. In the present study, we generated rice antisense and sense transgenic lines of OsSPX1 and found that down-regulation of OsSPX1 caused high sensitivity to cold and oxidative stresses in rice seedlings. Compared to wild-type and OsSPX1-sense transgenic lines, more hydrogen peroxide accumulated in seedling leaves of OsSPX1-antisense transgenic lines for controls, cold and methyl viologen (MV) treatments. Glutathione as a ROS scavenger could protect the antisense transgenic lines from cold and MV stress. Rice whole genome GeneChip analysis showed that some oxidative-stress marker genes (e.g. glutathione S-transferase and P450s) and Pi-signaling pathway related genes (e.g. OsPHO2) were significantly down-regulated by the antisense of OsSPX1. The microarray results were validated by real-time RT-PCR. Our study indicated that OsSPX1 may be involved in cross-talks between oxidative stress, cold stress and phosphate homeostasis in rice seedling leaves.     

Inhibitory effect of pseudo-aminosugars on oligosaccharide glucosidases I and II and on lysosomal alpha-glucosidase from rat liver

We examined the inhibitory effect of three pseudo-aminosugars (validamine, valienamine, and valiolamine), which were isolated from the broth of Streptomyces hygroscopicus, on the oligosaccharide-processing glucosidases I and II involved in glycoprotein biosynthesis in rat liver. Both glucosidases I and II were inhibited to the same extent by the pseudoaminosugars, and valiolamine had a more potent inhibitory activity than validamine or valienamine. A 50% inhibition of valiolamine was observed at 12 microM for glucosidase I and glucosidase II activities acting respectively on the substrates Glc3Man9GlcNAc2 and p-nitrophenyl alpha-D-glucopyranoside. Further, in order to investigate further the ability of valiolamine to inhibit glucosidase I, reaction products were analyzed by gel filtration on a Bio-Gel P-4 column. We also compared the inhibitory action of these pseudo-aminosugars on the acid alpha-glucosidase of rat liver lysosomes. They competitively inhibited the hydrolysis of both substrates, maltose and glycogen. Valiolamine again had a more potent lysosomal alpha-glucosidase inhibitory activity than the other two. The Ki values of valiolamine for the hydrolysis of maltose and glycogen were 8.1 and 11 microM, respectively. Valiolamine is a particularly effective inhibitor of oligosaccharide glucosidases I and II and of lysosomal alpha-glucosidase. Hence valiolamine might be useful as a research tool in investigations of carbohydrate metabolism.     

Control of potato tuber dormancy and sprouting by expression of sense and antisense genes of pyrophosphatase in potato

Inorganic pyrophosphate (PPi) is an enzyme involved in sugar metabolism in potato tubers. In our previous study, we isolated an inorganic pyrophosphatase (PPase) gene from potato and obtained the transgenic potato plants transformed with the sense and antisense PPase genes respectively. In the present experiment, the physiological indexes, tuber dormancy, and sprouting characteristics of the transgenic potatoes were analyzed and evaluated. The result showed that the PPase activity and the inorganic phosphate content of tubers were lower in the antisense transgenic plant lines but were higher in the sense transgenic plant lines, compared with wild-type tubers. Soluble sugars, such as glucose, fructose and sucrose increased in transgenic plants that had overexpression of the sense PPase gene, but decreased in the antisense transgenic plant lines, compared with wild-type tubers. Tuber sprouting time of the antisense transgenic plants were delayed for 2 and 3 weeks and reached the 100 % sprouting rate only after 14 and 16 weeks storage compared with the wild-type when tubers are stored under 25 and 4 °C, respectively. In contrast, tuber sprouting time of the sense transgenic plants was earlier by approximately 2 weeks than that of wild-type tubers under these storage temperatures.     

Chemical modification of the glucosidase inhibitor 1-deoxynojirimycin. Structure-activity relationships.

The ability of the glucosidase inhibitor 1-deoxynojirimycin (dNM) and a series of N-alkylated dNM derivatives to interfere with biosynthesis, transport, and maturation of the glycoprotein alpha 1-antitrypsin in HepG2 cells was investigated. Inhibition of endoplasmic reticulum glucosidase I and II by dNM and its derivatives resulted in an intracellular accumulation of alpha 1-antitrypsin with glucose-containing high mannose type oligosaccharides (precursor). N-alkylation of dNM increased its potency in inhibiting endoplasmic reticulum glucosidases, as determined from the concentration required for half maximal inhibition. N-Alkylated derivatives of dNM were better able to inhibit glucosidase I than glucosidase II (deduced from the number of glucose residues retained in Endo H-releasable oligosaccharides). The inhibition of glucosidase activity imposed by alkylated dNM derivatives was less easily reversed than that by dNM, an effect most pronounced for N-methyl-dNM. Branching of the alkyl group of dNM derivatives decreased the inhibitory potency. Although dNM and its derivatives interfered strongly with intracellular oligosaccharide processing, they did not completely block N-glycan maturation of alpha 1-antitrypsin even at the highest concentrations tested.     

Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II

Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II contained species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.     

Cell wall 1,6-beta-glucan synthesis in Saccharomyces cerevisiae depends on ER glucosidases I and II, and the molecular chaperone BiP/Kar2p.

The role of glucose trimming in the endoplasmic reticulum of Saccharomyces cerevisiae was investigated using glucosidase inhibitors and mutant strains devoid of glucosidases I and II. These glucosidases are responsible for removing glucose residues from the N-linked core oligosaccharides attached to newly synthesized polypeptide chains. In mammalian cells they participate together with calnexin, calreticulin and UDP-glucose:glycoprotein glucosyltransferase in the folding and quality control of newly synthesized glycoproteins. In S.cerevisiae, glucosidase II is encoded by the GLS2 gene, and glucosidase I, as suggested here, by the CWH41 gene. Using castanospermine (an alpha-glucosidase inhibitor) and yeast strains defective in glucosidase I, glucosidase II and BiP/Kar2p, it was demonstrated that cell wall synthesis depends on the two glucosidases and BiP/Kar2p. In double mutants with defects in both BiP/Kar2p and either of the glucosidases the phenotype was particularly clear: synthesis of 1,6-beta-glucan_a cell wall component_was reduced; the cell wall displayed abnormal morphology; the cells aggregated; and their growth was severely inhibited. No defects in protein folding or secretion could be detected. We concluded that glucose trimming in S.cerevisiae is necessary for proper cell wall synthesis, and that the glucosidases function synergistically with BiP/Kar2p in this process.     

Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells.

Previously isolated lysosomal alpha-glucosidase cDNA clones were ligated to full-length constructs for expression in vitro and in mammalian cells. One of these constructs (pSHAG1) did not code for functional enzyme, due to an arginine residue instead of a tryptophan residue at amino acid position 402. The mutation does not affect the rate of enzyme synthesis, but interferes with post-translational modification and intracellular transport of the acid alpha-glucosidase precursor. Using immunocytochemistry it is demonstrated that the mutant precursor traverses the endoplasmic reticulum and the Golgi complex, but does not reach the lysosomes. Pulse-chase experiments suggest premature degradation. The Trp-402-containing enzyme (encoded by construct pSHAG2) is processed properly, and has catalytic activity. A fraction of the enzyme is localized at the plasma membrane. It is hypothesized that membrane association of the acid alpha-glucosidase precursor, as demonstrated by Triton X-114 phase separation, is responsible for transport to this location. Transiently expressed acid alpha-glucosidase also enters the secretory pathway, since a catalytically active precursor is found in the culture medium. This precursor has the appropriate characteristics for use in enzyme replacement therapy. Efficient uptake via the mannose 6-phosphate receptor results in degradation of lysosomal glycogen in cultured fibroblasts and muscle cells from patients with glycogenosis type II.     

Stabilising normal and mis-sense variant alpha-glucosidase

alpha-Glucosidase (EC 3.2.1.3) is a lysosomal enzyme that hydrolyses alpha-1,4- and alpha-1,6-linkages of glycogen to produce free glucose. A deficiency in alpha-glucosidase activity results in glycogen storage disorder type II (GSD II), also called Pompe disease. Here, d-glucose was shown to be a competitive inhibitor of alpha-glucosidase and when added to culture medium at 6.0 g/L increased the production of this protein by CHO-K1 expression cells and stabilised the enzyme activity. D-Glucose also prevented alpha-glucosidase aggregation/precipitation and increased protein yield in a modified purification scheme. In fibroblast cells, from adult-onset GSD II patients, D-glucose increased the residual level of alpha-glucosidase activity, suggesting that a structural analogue of d-glucose may be used for enzyme enhancement therapy.     

两个反义基因在番茄工程植株中的生理抑制效应分析

用番茄乙烯形成酶（ＥＦＥ）和多聚半乳糖醛酸酶（ＰＧ）反义ｃＤＮＡ转化番茄子叶,获得两个转基因系统。分别比较了两个基因系统果实和叶片的乙烯生成速率、果实中ＥＦＥ酶活性和果胶酶活性,表明反义ＥＦＥ基因在番茄工程植株中能显著抑制ＥＦＥ酶活性和乙烯生成;反义ＰＧ基因则主要是抑制其ＰＧ酶活性。     

Synthesis of andrographolide derivatives: a new family of alpha-glucosidase inhibitors

Andrographolide (1), the cytotoxic agent of the plant Andrographis paniculata, was subjected to semi-synthetic studies leading to a series of new derivatives, a novel family of glucosidase inhibitors. Nicotination of 3,19-hydroxyls in 15-alkylidene andrographolide derivatives (9) was favorable to alpha-glucosidase inhibition activity. Among them, 15-p-chlorobenzylidene-14-deoxy-11,12-didehydro-3,19-dinicotinateandrographolide (11c) was a very potent inhibitor against alpha-glucosidase with an IC50 value of 6 microM. However, all compounds concerned for beta-glucosidase showed no inhibition. All compounds synthesized were characterized by the analysis of NMR, IR, HRMS spectra and the stereochemistry of 2 was confirmed by X-ray analysis.     

Characterization of a histidine- and alanine-rich protein showing interaction with calreticulin in rice

Calreticulin (CRT) is a major calcium-sequestering protein in the endoplasmic reticulum and has been implicated in a variety of cellular functions. To analyze the function of CRT in rice, a yeast two-hybrid protein interaction assay was used for identifying interacting proteins. Fourteen of 17 interacting cDNA clones found coded for a novel histidine- and alanine-rich protein (OsHARP) of 342 amino acid residues. The mRNA expression level of OsHARP was up-regulated in rice seedlings treated with gibberellin (GA), but not ABA and showed a similar pattern as OsCRT mRNA. Rice plants transformed with the OsHARP promoter-GUS construct showed GUS staining in the basal parts of leaf sheaths, and although GUS activity increased when treated with GA₃, it was not as high an increase as when mRNA was analyzed. To elucidate the role of OsHARP in leaf sheath elongation, antisense OsHARP transgenic rice lines were constructed. The antisense OsHARP transgenic rice plants were consistently shorter than the vector control under normal conditions. To examine whether OsHARP expression would affect other proteins, basal leaf sheaths from antisense OsHARP transgenic rice plants were analyzed using proteomic techniques. In antisense transgenic-rice OsHARP plants, OsCRT was down-regulated and the levels of 20 other proteins were changed compared to the pattern of the vector control. These results signify an important role of HARP in rice leaf sheath cell division or elongation and suggest that CRT may interact with HARP during certain stages of development.     

Induction of the activity of glycolytic enzymes correlates with enhanced hydrolysis of sucrose in the cytosol of transgenic potato tubers

The aim of this work was to define the metabolic factors which regulate the respiratory pathways in trangenic potato tubers. We previously found that respiration is enhanced in transgenic tubers which express a yeast invertase and a glucokinase from Zymomonas mobilis . In this study we investigated glycolysis in three further transgenic potato lines with profound changes in the mobilization of sucrose. We studied antisense ADPglucose pyrophosphorylase lines which are characterized by a reduction in starch accumulation and a significant build up of sucrose and related metabolic intermediates. We also report the generation of two novel double transgenic lines where the yeast invertase is expressed specifically in tubers of the ADPglucose pyrophosphorylase antisense line, targeted to either the cytosol or apopolast. We evaluated whether the localization of sucrose cleavage had an impact on the glycolytic induction, and assessed if invertase expression in the high-sucrose background had any further effects on glycolysis. We found that induction of the glycolytic enzymes only occurs when the invertase is targeted to the cytosol, and that the extent of this induction was comparable in the wild type and antisenseADPglucose pyrophosphorylase backgrounds. We conclude that the signal regulating glycolysis is directly linked to cytosolic sucrose hydrolysis.     

Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells

To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a "rocket" which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection.