Class I α-Mannosidases Are Required for N-Glycan Processing and Root Development in Arabidopsis thaliana

In eukaryotes, class I α-mannosidases are involved in early N-glycan processing reactions and in N-glycan–dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type α-mannosidase I (MNS3) and the two Golgi-α-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected α1,2-mannose residue from Man₉GlcNAc₂, MNS1/2 readily removed three α1,2-mannose residues from Man₈GlcNAc₂. Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man₈GlcNAc₂ accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man₉GlcNAc₂, demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I α-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I α-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.N-glycosylation is a major co- and posttranslational modification of proteins in eukaryotic cells. The biosynthesis of protein N-linked glycans starts in the endoplasmic reticulum (ER) when the oligosaccharyltransferase complex catalyzes the transfer of the Glc₃Man₉GlcNAc₂ oligosaccharide from the lipid-linked precursor to Asn residues (N-X-S/T) of nascent polypeptide chains. Subsequent N-glycan processing involves a series of highly coordinated step-by-step enzymatic conversions occurring in the ER and Golgi apparatus (Kornfeld and Kornfeld, 1985). In the first trimming reactions, α-glucosidases I (GCSI) and GCSII cleave off three glucose residues from Glc₃Man₉GlcNAc₂ to generate Man₉GlcNAc₂ (Figure 1A). The next steps of the pathway are the removal of four α1,2-linked mannose residues to provide the Man₅GlcNAc₂ substrate for the formation of complex N-glycans in the Golgi apparatus. In mammals, these mannose trimming reactions are catalyzed by class I α-mannosidases (glycosyl hydrolase family 47 of the Carbohydrate Active Enzymes database; http://www.cazy.org/). These enzymes are inverting glycosyl hydrolases that are highly specific for α1,2-mannose residues, require Ca²⁺ for catalytic activity, and are sensitive to inhibition by pyranose analogs such as 1-deoxymannojirimycin and kifunensine (Lipari et al., 1995; Gonzalez et al., 1999). Class I α-mannosidases are conserved through eukaryotic evolution and do not share sequence homology with class II α-mannosidases, such as Golgi α-mannosidase II and the catabolic lysosomal and cytoplasmic α-mannosidases (Gonzalez et al., 1999; Herscovics, 2001).Open in a separate windowFigure 1.Cartoon of Important Oligosaccharide Structures.(A) Man₉GlcNAc₂ oligosaccharide (Man9): the substrate for ER-MNSI.(B) Man₈GlcNAc₂ isomer Man8.1 according to Tomiya et al. (1991): the product of ER-MNSI and substrate for Golgi-MNSI.(C) Man₅GlcNAc₂ (Man5.1): the product of the mannose trimming reactions.The linkage of the sugar residues is indicated.[See online article for color version of this figure.]The mammalian class I α-mannosidase family consists of three protein subgroups, which have been distinguished based on their sequence similarity and proposed function: ER-α1,2-mannosidases I (ER-MNSIs), Golgi-α-mannosidases I (Golgi-MNSIs), and ER degradation-enhancing α-mannosidase (EDEM)-like proteins (Mast and Moremen, 2006). In humans, there is a single ER-MNSI, which cleaves the terminal mannose residue from the b-branch of the Man₉GlcNAc₂ oligosaccharide to create the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B). Subsequently, Golgi-MNSI (three isoforms, Golgi-MNSIA, Golgi-MNSIB, and Golgi-MNSIC, are present in humans) catalyze the removal of the remaining three α1,2-linked mannose residues to generate Man₅GlcNAc₂ (Figure 1C). The three human EDEM proteins are not directly involved in N-glycan processing but play a role in ER-associated degradation of glycoproteins (Mast et al., 2005; Hirao et al., 2006; Olivari et al., 2006).The formation of the Man₈GlcNAc₂ isomer (Man8.1), which is catalyzed by ER-MNSI, is the last N-glycan processing step that is conserved in yeast and mammals. Apart from its N-glycan processing function, ER-MNSI plays a key role in ER-mediated quality control of glycoproteins in yeasts and mammals (Mast and Moremen, 2006; Lederkremer, 2009). It has been proposed that ER-MNSI cooperates with mammalian EDEM1 to 3 or the yeast α1,2-mannosidase HTM1 to generate the signal that marks misfolded glycoproteins for degradation through the ER-associated protein degradation (ERAD) pathway. This quality control process, which finally leads to retrotranslocation to the cytoplasm and hydrolysis by the 26S proteasome, serves to prevent the secretion of aberrantly folded cargo proteins and is required to maintain protein homeostasis in the ER. Initially it was proposed that the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B) flags aberrantly folded glycoproteins for degradation; however, recent evidence suggests that further mannose trimming to Man₇GlcNAc₂ in yeast and Man_5-6GlcNAc₂ in mammals is required to trigger ERAD (Avezov et al., 2008; Clerc et al., 2009). In addition, these mannose cleavage reactions serve also to release glycoproteins from the calnexin/calreticulin quality control cycle (Caramelo and Parodi, 2008).Unlike for animals and yeast, much less is known about the biological function of plant class I α-mannosidases. Processing mannosidases have been purified and characterized from mung bean (Vigna radiata) seedlings and castor bean (Ricinus communis) cotyledons (Forsee, 1985; Szumilo et al., 1986; Kimura et al., 1991). These preparations were a mixture of different α-mannosidases, and no evidence for ER-MNSI-like activity was provided. A putative Golgi-α-mannosidase I has been cloned from soybean (Glycine max) (Nebenführ et al., 1999). A green fluorescent protein (GFP)-tagged fusion protein of the soybean enzyme has been shown to reside in the cis-stacks of the Golgi apparatus (Nebenführ et al., 1999; Saint-Jore-Dupas et al., 2006), but its role in N-glycan processing and its enzymatic properties have not been reported so far. Thus, the involvement of class I α-mannosidases in N-glycan processing as well as in glycoprotein quality control in plants is still unclear, and the existence of a plant ER-MNSI has so far been inferred only from the presence of Man₈GlcNAc₂ oligosaccharides on ER-resident glycoproteins (Pagny et al., 2000).Here, we report the molecular cloning and biochemical characterization of the enzymes accounting for ER-MNSI and Golgi-MNSI activities in Arabidopsis thaliana. We also demonstrate that disruption of these genes leads to severe cell expansion defects in roots as well as to distinct cell wall alterations. Hence, the identification of the Arabidopsis ER-type and Golgi class I α-mannosidases not only establishes the molecular basis for the missing steps in the plant N-glycan processing pathway but also provides unprecedented insights into the role of N-glycans in plant development.     

Superfamilies SDR and MDR: From early ancestry to present forms. Emergence of three lines, a Zn-metalloenzyme, and distinct variabilities

Two large gene and protein superfamilies, SDR and MDR (short- and medium-chain dehydrogenases/reductases), were originally defined from analysis of alcohol and polyol dehydrogenases. The superfamilies contain minimally 82 and 25 genes, respectively, in humans, minimally 324 and 86 enzyme families when known lines in other organisms are also included, and over 47,000 and 15,000 variants in existing sequence data bank entries. SDR enzymes have one-domain subunits without metal and MDR two-domain subunits without or with zinc, and these three lines appear to have emerged in that order from the universal cellular ancestor. This is compatible with their molecular architectures, present multiplicity, and overall distribution in the kingdoms of life, with SDR also of viral occurrence. An MDR-zinc, when present, is often, but not always, catalytic. It appears also to have a structural role in inter-domain interactions, coenzyme binding and substrate pocket formation, as supported by domain variability ratios and ligand positions. Differences among structural and catalytic zinc ions may be relative and involve several states. Combined, the comparisons trace evolutionary properties of huge superfamilies, with partially redundant enzymes in cellular redox functions.     

Ovarian Cyst Fluid of Serous Ovarian Tumors Contains Large Quantities of the Brain Amino Acid N-acetylaspartate

Background

In humans, N-acetyl L-aspartate (NAA) has not been detected in other tissues than the brain. The physiological function of NAA is yet undefined. Recently, it has been suggested that NAA may function as a molecular water pump, responsible for the removal of large amounts of water from the human brain. Ovarian tumors typically present as large cystic masses with considerable fluid accumulation.

Methodology and Principal Findings

Using Gas Chromatography-Mass Spectrometry, we demonstrated that NAA was present in a high micromolar concentration in oCF of epithelial ovarian tumors (EOTs) of serous histology, sometimes in the same range as found in the extracellular space of the human brain. In contrast, oCF of EOTs with a mucinous, endometrioid and clear cell histological subtype contained a low micromolar concentration of NAA. Serous EOTs have a cellular differentiation pattern which resembles the lining of the fallopian tube and differs from the other histological subtypes. The NAA concentration in two samples of fluid accumulation in the fallopian tube (hydrosalpinx) was in the same ranges as NAA found in oCF of serous EOTs. The NAA concentration in oCF of patients with serous EOTs was mostly 10 to 50 fold higher than their normal serum NAA concentration, whereas in patients with other EOT subtypes, serum and cyst fluid NAA concentration was comparable.

Conclusions and Significance

The high concentration of NAA in cyst fluid of serous EOTs and low serum concentrations of NAA in these patients, suggest a local production of NAA in serous EOTs. Our findings provide the first identification of NAA concentrations high enough to suggest local production outside the human brain. Our findings contribute to the ongoing research understanding the physiological function of NAA in the human body.     

The effect of application techniques on field‐scale efficacy: can the use of entomopathogenic nematodes reduce damage by western corn rootworm larvae?

• 1 Field studies on the efficacy of entomopathogenic nematodes as biological pest control agents in field crops and over multiple seasons are rare.
• 2 In the present study, we investigated the efficacy of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) at reducing maize root damage caused by the larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). The study consisted of 21 field‐scale experiments in Hungary conducted between 2004 and 2007. Key factors behind successful control, such as application techniques, nematode dosage, soil environment and rainfall, were analyzed.
• 3 Five of the six tested techniques for applying the nematodes using readily available farming machinery resulted in a reduction of root damage, as well as subsequent plant lodging.
• 4 According to damage rating with the Iowa 1–6 scale, damage was reduced by 3–18%. According to the 0.00–3.00 node injury scale, damage was reduced by 14–54%. Approximately 7–62 % of plant lodging, and thus direct yield losses as a result of in‐harvestability, was prevented.
• 5 An increase in nematode dosage and/or an increased rainfall during the period of applications increased the efficacy of H. bacteriophora at reducing root damage. Except for the obvious effect of moisture, the soil environment had little measurable influence on nematode efficacy.
• 6 The present study demonstrates the potential for field‐scale nematode application to significantly reduce damage caused by D. v. virgifera larvae. It is recommended that the method of agent application should be selected on a case‐by‐case basis considering the efficacy data presented here, the available application machinery and its associated costs.

     

Extraction forces in bovine obstetrics: An in vitro study investigating alternate and simultaneous traction modes

Whether extraction of a calf in longitudinal anterior presentation should be carried out by simultaneous or alternate traction on the forelimbs remains controversial. Because most recommendations are based on empirical observations rather than on scientific studies, the aim of this study was to develop an in vitro model to objectively compare the forces occurring during alternate and simultaneous traction. In a biomechanical in vitro model, 12 dead Holstein-Friesian (Bos taurus) calves were pulled through the prepared pelvic specimen of a cow at a controlled speed using two electric motors. Traction was applied simultaneously (ST) to both legs or alternately (AT) to one leg at a time to advance the calf 5 cm (AT 5) or 10 cm (AT 10). Forces on each limb were measured digitally using load cells. In all cases, two peaks of maximum force occurred during the extraction of the cranial part of the body. The first peak was observed when the elbows were pulled into the pelvis, and the second peak occurred when the chest emerged from the pelvis. Up to and including entry of the elbows into the pelvis, the maximum force on a single limb (341 ± 106 N) was lowest (P < 0.01) using AT10. The maximum traction forces acting on a single limb using AT5 (411 ± 86 N) and ST (431 ± 127 N) did not differ (P > 0.05). During extraction of the thorax, the maximum force acting on a single limb was lower (P < 0.0001) using ST (352 ± 98 N) compared with AT5 (432 ± 79 N) and AT10 (547 ± 115 N). Based on these findings, alternate-limb traction, 10 cm at a time, should be used until both elbows have entered the pelvis. Simultaneous traction should then be applied to both forelimbs to complete extraction of the chest.     

Light-induced reduction of bovine adrenodoxin via the covalently bound ruthenium(II) bipyridyl complex: intramolecular electron transfer and crystal structure

Bovine adrenodoxin (Adx) plays an important role in the electron-transfer process in the mitochondrial steroid hydroxylase system of the bovine adrenal cortex. Using electron paramagnetic resonance (EPR) spectroscopy, we showed that photoreduction of the [2Fe-2S] cluster of Adx via (4'-methyl-2,2'-bipyridine)bis(2,2'-bipyridine)ruthenium(II) [Ru(bpy)2(mbpy)] covalently attached to the protein surface can be used as a new approach to probe the "shuttle" hypothesis for the electron transfer by Adx. The 1.5 A resolution crystal structure of a 1:1 Ru(bpy)2(mbpy)-Adx(1-108) complex reveals the site of modification, Cys95, and allows to predict the possible intramolecular electron-transfer pathways within the complex. Photoreduction of uncoupled Adx, mutant Adx(1-108), and Ru(bpy)2(mbpy)-Adx(1-108) using safranin T as the mediating electron donor suggests that two electrons are transferred from the dye to Adx. The intramolecular photoreduction rate constant for the ruthenated Adx has been determined and is discussed according to the predicted pathways.     

Improved enumeration of lactic acid bacteria in mesophilic dairy starter cultures by using multiplex quantitative real-time PCR and flow cytometry-fluorescence in situ hybridization

Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.     

Occurrence of Atractolytocestus huronensis (Cestoda, Caryophyllaeidae) in German pond-farmed common carp Cyprinus carpio

Up to now, the caryophyllid cestode Atractolytocestus huronensis Anthony, 1958, a parasite of common carp, has attracted little attention in Germany. Based on recent publications from the Czech Republic and Hungary, it appears probable that this cestode may be increasingly common in Germany. There is a strong connection between the occurrence of A. huronensis and imports of common carp from the Czech Republic and southern Germany. Although in most cases no clinical alterations in parasitized carp have been observed, care should be taken to avoid further dissemination and to prevent possible losses in commercial pond farming.     

DFT study of alpha- and beta-D-galactopyranose at the B3LYP/6-311++G** level of theory

Forty-one conformations of alpha- and beta-d-galactopyranose were geometry optimized using the B3LYP density functional and 6-311++G** basis set. Full geometry optimization was performed on different ring geometries and different hydroxymethyl rotamers (gg/gt/tg). Analytically derived Hessians were used to calculate zero point energy, enthalpy, and entropy. The lowest energy and free-energy conformation found is the alpha-gg-(4)C(1)-c chair conformation, which is of lower electronic and free energy than the lowest energy alpha-d-glucopyranose conformer because of favorable hydrogen-bonding interactions. The in vacuo calculations showed considerable ( approximately 2.2kcal/mol) energetic preference for the alpha over the beta anomer for galactopyranose in both the (4)C(1) and (1)C(4) chair conformations. Results are compared to glucopyranose and mannopyranose calculations in vacuo. Boat and skew-boat forms were found that remained stable upon gradient optimization, although many starting conformations moved to other boat forms upon optimization. As with glucopyranose and mannopyranose, the orientation and interaction of the hydroxyl groups make the most significant contributions to the conformation-energy relationship in vacuo.