Enzyme enhancement therapeutics for lysosomal storage diseases: Current status and perspective.

Small-molecule- enzyme enhancement therapeutics (EETs) have emerged as attractive agents for the treatment of lysosomal storage diseases (LSDs), a broad group of genetic diseases caused by mutations in genes encoding lysosomal enzymes, or proteins required for lysosomal function. The underlying enzyme deficiencies characterizing LSDs cause a block in the stepwise degradation of complex macromolecules (e.g. glycosaminoglycans, glycolipids and others), such that undegraded or partially degraded substrates progressively accumulate in lysosomal and non-lysosomal compartments, a process leading to multisystem pathology via primary and secondary mechanisms. Missense mutations underlie many of the LSDs; the resultant mutant variant enzyme hydrolase is often impaired in its folding and maturation making it subject to rapid disposal by endoplasmic reticulum (ER)-associated degradation (ERAD). Enzyme deficiency in the lysosome is the result, even though the mutant enzyme may retain significant catalytic functioning. Small molecule modulators - pharmacological chaperones (PCs), or proteostasis regulators (PRs) are being identified through library screens and computational tools, as they may offer a less costly approach than enzyme replacement therapy (ERT) for LSDs, and potentially treat neuronal forms of the diseases. PCs, capable of directly stabilizing the mutant protein, and PRs, which act on other cellular elements to enhance protein maturation, both allow a proportion of the synthesized variant protein to reach the lysosome and function. Proof-of-principle for PCs and PRs as therapeutic agents has been demonstrated for several LSDs, yet definitive data of their efficacy in disease models and/or in downstream clinical studies in many cases has yet to be achieved. Basic research to understand the cellular consequences of protein misfolding such as perturbed organellar crosstalk, redox status, and calcium balance is needed. Likewise, an elucidation of the early in cellulo pathogenic events underlying LSDs is vital and may lead to the discovery of new small molecule modulators and/or to other therapeutic approaches for driving proteostasis toward protein rescue.

New Irreversible α-l-Iduronidase Inhibitors and Activity-Based Probes.

Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity-based glycosidase probes (ABPs). Direct 3-amino-2-(trifluoromethyl)quinazolin-4(3H)-one-mediated aziridination of l-ido-configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α-l-iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity-based manner with human recombinant α-l-iduronidase (rIDUA, Aldurazyme® ). The structures of IDUA when complexed with the inhibitors in a non-covalent transition state mimicking form and a covalent enzyme-bound form provide insights into its conformational itinerary. Inhibitors 1-3 adopt a half-chair conformation in solution (4 H3 and 3 H4 ), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1-3 may need to overcome an energy barrier in order to switch from the 4 H3 conformation to the transition state (2, 5 B) binding conformation before reacting and adopting a covalent 5 S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients.

N-glycan structures and downstream mannose-phosphorylation of plant recombinant human alpha-L-iduronidase: toward development of enzyme replacement therapy for mucopolysaccharidosis I.

KEY MESSAGE: Arabidopsis N-glycan processing mutants provide the basis for tailoring recombinant enzymes for use as replacement therapeutics to treat lysosomal storage diseases, including N-glycan mannose phosphorylation to ensure lysosomal trafficking and efficacy. Functional recombinant human alpha-L-iduronidase (IDUA; EC 3.2.1.76) enzymes were generated in seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) C5 background, which is deficient in the activity of N-acetylglucosaminyl transferase I, and in seeds of the Arabidopsis gm1 mutant, which lacks Golgi α-mannosidase I (GM1) activity. Both strategies effectively prevented N-glycan maturation and the resultant N-glycan structures on the consensus sites for N-glycosylation of the human enzyme revealed high-mannose N-glycans of predominantly Man5 (cgl-IDUA) or Man6-8 (gm1-IDUA) structures. Both forms of IDUA were equivalent with respect to their kinetic parameters characterized by cleavage of the artificial substrate 4-methylumbelliferyl-iduronide. Because recombinant lysosomal enzymes produced in plants require the addition of mannose-6-phosphate (M6P) in order to be suitable for lysosomal delivery in human cells, we characterized the two IDUA proteins for their amenability to downstream in vitro mannose phosphorylation mediated by a soluble form of the human phosphotransferase (UDP-GlcNAc: lysosomal enzyme N-acetylglucosamine [GlcNAc]-1-phosphotransferase). Gm1-IDUA exhibited a slight advantage over the cgl-IDUA in the in vitro M6P-tagging process, with respect to having a better affinity (i.e. lower K m) for the soluble phosphotransferase. This may be due to the greater number of mannose residues comprising the high-mannose N-glycans of gm1-IDUA. Our elite cgl- line produces IDUA at > 5.7% TSP (total soluble protein); screening of the gm1 lines showed a maximum yield of 1.5% TSP. Overall our findings demonstrate the relative advantages and disadvantages associated with the two platforms to create enzyme replacement therapeutics for lysosomal storage diseases.

AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings.

AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and ß-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.

Seed dormancy cycling in Arabidopsis: chromatin remodelling and regulation of DOG1 in response to seasonal environmental signals.

The involvement of chromatin remodelling in dormancy cycling in the soil seed bank (SSB) is poorly understood. Natural variation between the winter and summer annual Arabidopsis ecotypes Cvi and Bur was exploited to investigate the expression of genes involved in chromatin remodelling via histone 2B (H2B) ubiquitination/de-ubiquitination and histone acetylation/deacetylation, the repressive histone methyl transferases CURLY LEAF (CLF) and SWINGER (SWN), and the gene silencing repressor ROS1 (REPRESSOR OF SILENCING1) and promoter of silencing KYP/SUVH4 (KRYPTONITE), during dormancy cycling in the SSB. ROS1 expression was positively correlated with dormancy while the reverse was observed for CLF and KYP/SUVH4. We propose ROS1 dependent repression of silencing and a sequential requirement of CLF and KYP/SUVH4 dependent gene repression and silencing for the maintenance and suppression of dormancy during dormancy cycling. Seasonal expression of H2B modifying genes was correlated negatively with temperature and positively with DOG1 expression, as were histone acetyltransferase genes, with histone deacetylases positively correlated with temperature. Changes in the histone marks H3K4me3 and H3K27me3 were seen on DOG1 (DELAY OF GERMINATION1) in Cvi during dormancy cycling. H3K4me3 activating marks remained stable along DOG1. During relief of dormancy, H3K27me3 repressive marks slowly accumulated and accelerated on exposure to light completing dormancy loss. We propose that these marks on DOG1 serve as a thermal sensing mechanism during dormancy cycling in preparation for light repression of dormancy. Overall, chromatin remodelling plays a vital role in temporal sensing through regulation of gene expression.

Descriptive and hedonic analyses of low-Phe food formulations containing corn (Zea mays) seedling roots: toward development of a dietary supplement for individuals with phenylketonuria.

BACKGROUND: Seedling roots of anthocyanin-rich corn (Zea mays) cultivars contain high levels of phenylalanine ammonia lyase (PAL) activity. The development of a natural dietary supplement containing corn roots could provide the means to improve the restrictive diet of phenylketonuria (PKU) patients by increasing their tolerance to dietary phenylalanine (Phe). Therefore this research was undertaken to explore the sensory characteristics of roots of four corn cultivars as well as to develop and evaluate food products (cereal bar, beverage, jam-like spread) to which roots had been added. RESULTS: Sensory profiles of corn roots were investigated using ten trained judges. Roots of Japanese Striped corn seedlings were more bitter, pungent and astringent than those of white and yellow cultivars, while roots from the Blue Jade cultivar had a more pronounced earthy/mushroom aroma. Consumer research using 24 untrained panelists provided hedonic (degree-of-liking) assessments for products with and without roots (controls). The former had lower mean scores than the controls; however, the cereal bar had scores above 5 on the nine-point scale for all hedonic assessments compared with the other treated products. CONCLUSION: By evaluating low-Phe food products containing corn roots, this research ascertained that the root-containing low-Phe cereal bar was an acceptable 'natural' dietary supplement for PKU-affected individuals.

Fluorescence-quenched substrates for live cell imaging of human glucocerebrosidase activity.

Deficiency of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) leads to abnormal accumulation of glucosyl ceramide in lysosomes and the development of the lysosomal storage disease known as Gaucher's disease. More recently, mutations in the GBA1 gene that encodes GCase have been uncovered as a major genetic risk factor for Parkinson's disease (PD). Current therapeutic strategies to increase GCase activity in lysosomes involve enzyme replacement therapy (ERT) and molecular chaperone therapy. One challenge associated with developing and optimizing these therapies is the difficulty in determining levels of GCase activity present within the lysosomes of live cells. Indeed, visualizing the activity of endogenous levels of any glycoside hydrolases, including GCase, has proven problematic within live mammalian cells. Here we describe the successful modular design and synthesis of fluorescence-quenched substrates for GCase. The selection of a suitable fluorophore and quencher pair permits the generation of substrates that allow convenient time-dependent monitoring of endogenous GCase activity within cells as well as localization of activity within lysosomes. These efficiently quenched (∼99.9%) fluorescent substrates also permit assessment of GCase inhibition in live cells by either confocal microscopy or high content imaging. Such substrates should enable improved understanding of GCase in situ as well the optimization of small-molecule chaperones for this enzyme. These findings also suggest routes to generate fluorescence-quenched substrates for other mammalian glycoside hydrolases for use in live cell imaging.

Changes in hormone flux and signaling in white spruce (Picea glauca) seeds during the transition from dormancy to germination in response to temperature cues.

BACKGROUND: Seeds use environmental cues such as temperature to coordinate the timing of their germination, allowing plants to synchronize their life history with the seasons. Winter chilling is of central importance to alleviate seed dormancy, but very little is known of how chilling responses are regulated in conifer seeds. White spruce (Picea glauca) is an important conifer species of boreal forests in the North American taiga. The recent sequencing and assembly of the white spruce genome allows for comparative gene expression studies toward elucidating the molecular mechanisms governing dormancy alleviation by moist chilling. Here we focused on hormone metabolite profiling and analyses of genes encoding components of hormone signal transduction pathways, to elucidate changes during dormancy alleviation and to help address how germination cues such as temperature and light trigger radicle emergence. RESULTS: ABA, GA, and auxin underwent considerable changes as seeds underwent moist chilling and during subsequent germination; likewise, transcripts encoding hormone-signaling components (e.g. ABI3, ARF4 and Aux/IAA) were differentially regulated during these critical stages. During moist chilling, active IAA was maintained at constant levels, but IAA conjugates (IAA-Asp and IAA-Glu) were substantially accumulated. ABA concentrations decreased during germination of previously moist-chilled seeds, while the precursor of bioactive GA1 (GA53) accumulated. We contend that seed dormancy and germination may be partly mediated through the changing hormone concentrations and a modulation of interactions between central auxin-signaling pathway components (TIR1/AFB, Aux/IAA and ARF4). In response to germination cues, namely exposure to light and to increased temperature: the transfer of seeds from moist-chilling to 30 °C, significant changes in gene transcripts and protein expression occurred during the first six hours, substantiating a very swift reaction to germination-promoting conditions after seeds had received sufficient exposure to the chilling stimulus. CONCLUSIONS: The dormancy to germination transition in white spruce seeds was correlated with changes in auxin conjugation, auxin signaling components, and potential interactions between auxin-ABA signaling cascades (e.g. the transcription factor ARF4 and ABI3). Auxin flux adds a new dimension to the ABA:GA balance mechanism that underlies both dormancy alleviation by chilling, and subsequent radicle emergence to complete germination by warm temperature and light stimuli.

Insights into mucopolysaccharidosis I from the structure and action of α-L-iduronidase.

Mucopolysaccharidosis type I (MPS I), caused by mutations in the gene encoding α-L-iduronidase (IDUA), is one of approximately 70 genetic disorders collectively known as the lysosomal storage diseases. To gain insight into the basis for MPS I, we crystallized human IDUA produced in an Arabidopsis thaliana cgl mutant. IDUA consists of a TIM barrel domain containing the catalytic site, a ß-sandwich domain and a fibronectin-like domain. Structures of IDUA bound to iduronate analogs illustrate the Michaelis complex and reveal a (2,5)B conformation in the glycosyl-enzyme intermediate, which suggest a retaining double displacement reaction involving the nucleophilic Glu299 and the general acid/base Glu182. Unexpectedly, the N-glycan attached to Asn372 interacts with iduronate analogs in the active site and is required for enzymatic activity. Finally, these IDUA structures and biochemical analysis of the disease-relevant P533R mutation have enabled us to correlate the effects of mutations in IDUA to clinical phenotypes.

A conifer ABI3-interacting protein plays important roles during key transitions of the plant life cycle.

ABI3 (for ABSCISIC ACID INSENSITIVE3), a transcription factor of the abscisic acid signal transduction pathway, plays a major role during seed development, dormancy inception, and dormancy maintenance. This protein appears to also function in meristematic and vegetative plant tissues and under certain stress conditions. We have isolated the ABI3 gene ortholog (CnABI3) from yellow cedar (Callitropsis nootkatensis) and found that it was functionally similar to other ABI3 genes of angiosperms. Here, we report that using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we have identified another protein of yellow cedar (CnAIP2; for CnABI3 INTERACTING PROTEIN2) that physically interacts with CnABI3. Functional analyses revealed that CnAIP2 plays important roles during key transitions in the plant life cycle: (1) CnAIP2 impaired seed development and reduced seed dormancy; (2) CnAIP2 promoted root development, particularly the initiation of lateral roots, and the CnAIP2 gene promoter was exquisitely auxin sensitive; and (3) CnAIP2 promoted the transition from vegetative growth to reproductive initiation (i.e. flowering). The nature of the effects of CnAIP2 on these processes and other evidence place CnAIP2 in the category of a "global" regulator, whose actions are antagonistic to those of ABI3.

Demethylesterification of cell wall pectins in Arabidopsis plays a role in seed germination.

The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor to investigate PME involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans, but there were no changes in the neutral sugar and uronic acid composition of the cell walls. As compared with wild-type seeds, the PME activities of the overexpressing lines were greatly reduced throughout germination, and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology but to alterations of the micropylar endosperm cells, evident by environmental scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.

Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation.

KEY MESSAGE: Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent. Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of ß1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid ß-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein's N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.

Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: a potential oral therapeutic to treat phenylketonuria.

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (∼50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions.

Characterization and downstream mannose phosphorylation of human recombinant α-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seeds.

Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme™). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells-UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine (GlcNAc)-1-phosphotransferase-and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (the 'uncovering enzyme'). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.

Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants.

There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid ß-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, half-maximal inhibitory concentration of isofagomine and thermal stability of the cgl-derived GCase were comparable with those of imiglucerase, a commercially available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose terminated. Additional purification was required to remove ∼15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including the use of a null cgl mutant.

Influence of an ER-retention signal on the N-glycosylation of recombinant human α-L-iduronidase generated in seeds of Arabidopsis.

Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. ß1,2 xylose and α1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme α-L-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5'-, signal-peptide-encoding-, and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific.

A plant cell-based system that predicts aß42 misfolding: potential as a drug discovery tool for Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid ß (Aß) peptides and the failure of mechanisms to clear toxic aggregates. The Aß42 peptide is considered to be a causative factor that underlies the pathophysiology of AD, in part due to its propensity for misfolding and aggregation; the small oligomers that result represent toxic species. Thus agents that prevent Aß42 misfolding/aggregation or, alternatively improve Aß42 oligomer clearance, may have significant therapeutic value. We have developed the basis for a drug screening system based on transgenic plant cells that express Aß42 fusion proteins to serve as the reliable indicators of the general conformational status of Aß42. Within cells of transgenic tobacco and Nicotiana benthamiana, misfolding of Aß42 causes the misfolding of a GFP fusion partner, and consequently there is a loss of fluorescence associated with the native GFP protein. In a similar fusion consisting of Aß42 linked to hygromycin phosphotransferase II (Hpt II), a hygromycin-resistance marker, misfolding of Aß42 leads to a misfolded Hpt II, and consequently the transgenic cells are unable to grow on media containing hygromycin. Importantly, substitution of the 'aggregation-prone' Aß42 with a missense mutant of Aß42 (F19S/L34F) that is not prone to misfolding/aggregation, 'rescues' both fusion partners. Several 'positive control' chemicals that represent inhibitors of Aß42 aggregation, including curcumin, epigallocatechin-3-gallate (EGCG), and resveratrol show efficacy in preventing the Aß42-fusion proteins from misfolding/aggregating in the transgenic plant cells. We discuss the potential of the two fusion protein systems to serve as the basis for an inexpensive, selective, and efficient screening system in which a plant cell can fluoresce or survive only in the presence of drug candidates that are able to prevent Aß42 misfolding/aggregation.

Pharmacological chaperones facilitate the post-ER transport of recombinant N370S mutant ß-glucocerebrosidase in plant cells: evidence that N370S is a folding mutant.

Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding lysosomal acid ß-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a N370S missense mutation in the GCase protein. As part of a larger endeavor to study the fate of mutant human proteins expressed in plant cells, the N370S mutant protein along with the wild-type- (WT)-GCase, both equipped with a signal peptide, were synthesized in transgenic tobacco BY2 cells, which do not possess lysosomes. The enzymatic activity of plant-recombinant N370S GCase lines was significantly lower (by 81-95%) than that of the WT-GCase lines. In contrast to the WT-GCase protein, which was efficiently secreted from tobacco BY2 cells, and detected in large amounts in the culture medium, only a small proportion of the N370S GCase was secreted. Pharmacological chaperones such as N-(n-nonyl) deoxynojirimycin and ambroxol increased the steady-state mutant protein levels both inside the plant cells and in the culture medium. These findings contradict the assertion that small molecule chaperones increase N370S GCase activity (as assayed in treated patient cell lysates) by stabilizing the enzyme in the lysosome, and suggest that the mutant protein is impaired in its ability to obtain its functional folded conformation, which is a requirement for exiting the lumen of the ER.

Role of a respiratory burst oxidase of Lepidium sativum (cress) seedlings in root development and auxin signalling.

Reactive oxygen species are increasingly perceived as players in plant development and plant hormone signalling pathways. One of these species, superoxide, is produced in the apoplast by respiratory burst oxidase homologues (rbohs), a family of proteins that is conserved throughout the plant kingdom. Because of the availability of mutants, the focus of research into plant rbohs has been on Arabidopsis thaliana, mainly on AtrbohD and AtrbohF. This study investigates: (i) a different member of the Atrboh family, AtrbohB, and (ii) several rbohs from the close relative of A. thaliana, Lepidium sativum ('cress'). Five cress rbohs (Lesarbohs) were sequenced and it was found that their expression patterns were similar to their Arabidopsis orthologues throughout the life cycle. Cress plants in which LesarbohB expression was knocked down showed a strong seedling root phenotype that resembles phenotypes associated with defective auxin-related genes. These transgenic plants further displayed altered expression of auxin marker genes including those encoding the auxin responsive proteins 14 and 5 (IAA14 and IAA5), and LBD16 (LATERAL ORGAN BOUNDARIES DOMAIN16), an auxin-responsive protein implicated in lateral root initiation. It is speculated that ROS produced by rbohs play a role in root development via auxin signalling.

Programmed cell death of the megagametophyte during post-germinative growth of white spruce (Picea glauca) seeds is regulated by reactive oxygen species and the ubiquitin-mediated proteolytic system.

The megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death following seed germination. This process is characterized by distinct morphological and biochemical features, such as DNA fragmentation and the induction of proteases. Biphasic production of hydrogen peroxide was detected in the megagametophyte following seed germination. ROS scavengers or inhibitors of ROS production decreased caspase-like protease activity and slowed the progression of cell death. One catalase (CAT) of white spruce reacted with antibodies directed against cotton-seed CAT. The corresponding CAT gene was cloned and compared with the catalase genes of other plant species. The activity of the white spruce CAT enzyme was stimulated by tyrosine phosphorylation. The phosphorylated CAT was subjected to ubiquitination and degraded by the proteasome. Furthermore, the proteasome inhibitor MG132 inhibited the degradation of CAT and delayed cell death. These results suggest that the interplay of CAT and the ubiquitin-mediated proteolytic system is critical in the control of ROS production and subsequent cell death.