Molecular structure for microbial polysaccharides: conformation of the Klebsiella serotype K9 capsular polysaccharide

Fibre type X-ray diffraction patterns have been obtained from oriented, semicrystalline films prepared from the sodium salt form of the bacterial capsular polysaccharide of Klebsiella serotype K9. The molecule has a pentasaccharide repeating sequence, with four neutral residues in the backbone and a glucoronic acid side chain. A novel feature of the molecule is the incorporation of α-l-rhamnose residues, one 1,2 linked and two 1,3 linked in the backbone. Analysis of the X-ray diffraction results indicate an extended three-fold helical conformation with an axially projected chemical repeat of 1.377 nm. Both left and right handed helices have been examined using linked atom least squares techniques to optimize the stereochemistry while simultaneously meeting the observed helical parameters.     

Molecular structures for microbial polysaccharides: conformation of the Klebsiella serotype K25 capsular polysaccharide

Fibre type X-ray diffraction patterns have been obtained from oriented, semi-crystalline films prepared from the sodium salt of the capsular polysaccharide of Klebsiella serotype K25. This molecule has a tetrasaccharide repeating structure consisting of a disaccharide backbone and a disaccharide side chain. The backbone contains a di-equatorially 1,4 linked β-d-glucose residue followed by a di-equatorially 1,3 linked β-d-galactose residue. The side chain is attached to the axial O(4) position of the galactose residue and consists of a di-equaltorially 1,2 linked β-d-glucoronic acid with a β-d-glucose residue attached terminally. An interesting feature of the backbone linkage geometry of this polysaccharide is its similarity with those of the animal connective tissue polydisaccharides. Analysis of diffraction patterns gives rise to an extended three fold helical conformation with an axially projected advance per chemical repeat of 0.97 nm. Molecular models have been computer generated using least squares techniques to optimize interatomic contacts and simultaneously meet the observed helical parameters. A left handed helix with inter-residue stabilizing hydrogen bonds was found to be most favourable and comparison of this model with other relevant polysaccharide structures is male.     

Synthesis of P 1-(11-phenoxyundecyl)-P 2-(��-D-galactopyranosyl) diphosphate and P 1-(11-phenoxyundecyl)-P 2-(��-D-glucopyranosyl) diphosphate and investigation on their acceptor properties in the reaction of mannosyl residue transfer catalyzed by mannosyltransferase from Salmonella newport

P ¹-(11-phenoxyundecyl)-P ²-(??-D-galactopyranosyl) diphosphate and P ¹-(11-phenoxyundecyl)-P ²-(??-D-glucopyranosyl) diphosphate have been synthesized for the first time, and their ability to serve as a mannosyl residue substrate-acceptors in the enzymatic reaction, catalyzed by mannosyltransferase membrane preparation from Salmonella newport cells, was investigated. It was demonstrated that the derivative containing galactopyranose residue is able to accept the mannosyl residue from GDP-Man, while the derivative containing glucopyranose residue does not have such an ability.     

Host-Pathogen Interactions: XI. Composition and Structure of Wall-released Elicitor Fractions

The structures of the four wall-released elicitor fractions isolated from the Phytophthora megasperma var. sojae mycelial walls have been examined. The results demonstrate that fraction I is primarily composed of a branched β-1,3-glucan, similar in structure to the extracellular elicitors described previously (Ayers, A., J. Ebel, F. Finelli, N. Burger, and P. Albersheim. 1976. Plant Physiol. 57: 751-759). Fractions II and IV are primarily composed of a highly branched mannan-containing glycoprotein, with fraction IV richer in protein than fraction II. Fraction III contains, attached to protein, a mixture of the two polysaccharide types found in fraction I and in fractions II and IV. The structural data presented here, in concert with the biological data presented in the previous two papers (Ayers et al. 1976. Plant Physiol. 57: 751-759; 760-765), demonstrate that the only compound produced by P. megasperma var. sojae which contains elicitor activity is the glucan. Evidence is presented that the terminal glycosyl residues of the glucan are required for elicitor activity. In addition, it is demonstrated that 90% of the glucan can be removed enzymically without any loss of biological activity. The active residue of the enzymic digestion is a highly branched 3- and 3,6-linked glucan containing about 4% mannosyl residues. The results presented suggest that the mannosyl residues of the glucan, which represent only about 1% of the undegraded glucan, are likely to participate in the active site of this molecule. The role of elicitors and phytoalexins in host-pathogen interactions is discussed. Evidence for the existence of and possible identity of another factor, which determines race specificity of host-pathogen interactions, is summarized.     

Effect of acetylation on he molecular interactions and gelling properties of a bacterial polysaccharide

Two chemically similar microbial polysaccharides, XM6 and K54, exhibit noticeably different rheological properties. Polysaccharide K54 differs from XM6 by mono-O-acetylation on alternate repeat units. Fibre X-ray diffraction is used to show that similar helix structures exist for both polysaccharides but that O-acetyl decoration of K54 inhibits association between molecules. This offers an explanation for the ability of XM6 to gel in aqueous solution whereas K54 does not gel.     

Introduction of O-glycosidically linked mannose into proteins via mannosyl phosphoryl dolichol by microsomes from Fusarium soani f. pisi

Cutinase, a glycoprotein containing O-glycosidically linked carbohydrates, is induced in glucose-grown Fusarium solani f. pisi by cutin hydrolysate. Microsomal preparations from the induced cells catalyzed mannosyl transfer from GDP-mannose to glycolipid and glycoprotein fractions but not into oligosaccharide lipids. Maximal rates of mannosyl transfer into glycolipids and glycoproteins were obtained with 5 mm Mg²⁺ and 10 mm Mn²⁺, respectively. Mannosyl transfer into glycolipids and glycoproteins showed pH optima of 8.0 and 7.0, respectively, and both transfers showed an apparent K_m of about 2 μm for GDP-mannose. The mannosyl lipid was identified as β-d-mannosyl phosphoryl dolichol by thinlayer and ion-exchange chromatography, as well as by analyses of the products derived from it by acid and base treatments. The fungal microsomal preparation also catalyzed mannosyl transfer from GDP-mannose to exogenous dolichol phosphate. This transfer was stimulated maximally by 0.09% Triton X-100 and showed a pH optimum at pH 8.0. The apparent K_m values for dolichol phosphate and GDP-mannose were 120 and 2.3 μm, respectively. The product derived from exogenous dolichol phosphate was identified as β-d-mannosyl phosphoryl dolichol as indicated above. The endogenous mannosyl acceptor lipid from this fungus was isolated by DEAE-cellulose chromatography. Analysis of the p-nitrobenzoyl derivatives of the base hydrolysis products of this acceptor lipid by highperformance liquid chromatography showed that the major components of this dolichol were C₉₅ and C₁₀₀. The microsomal preparation also catalyzed the transfer of mannose from exogenous mannosyl phosphoryl dolichol to glycoproteins with a pH optimum of 7.5 and an apparent K_m of 1.7 μm. Analyses of the β-elimination products of the glycoproteins generated from both GDP-mannose and dolichol phosphoryl mannose showed that single mannosyl residues were transferred to hydroxyl groups of the endogenous proteins. Exogenous cutinase was not glycosylated even after denaturation, sulfitolysis, or removal of carbohydrates by HF hydrolysis. Sodium dodecyl sulfate electrophoresis indicated that cutinase and its possible precursors were among the in vitro glycosylation products. Bacitracin and amphomycin but not tunicamycin inhibited the mannosyl transfer reactions.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Triterpenic acids-enriched fraction from Cyclocarya paliurus attenuates insulin resistance and hepatic steatosis via PI3K/Akt/GSK3β pathway

BackgroundNon-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver diseases. Cyclocarya paliurus (C. paliurus), an edible and medicinal plant in Chinese folk, has been demonstrated to ameliorate diabetes, obesity and lipid metabolism disorders. However, its effects on NAFLD and its potential molecular mechanism have not been clearly expounded.PurposeThe present study was designed to explore the therapeutic potential of triterpenic acids-enriched fraction from C. paliurus (CPT), as well as its underlying mechanism in vivo and in vitro models of NAFLD.MethodsThe metabolic effects and possible molecular mechanism of CPT were examined using HepG2 cells and primary hepatocytes (isolated from C57BL/6 J mice) models of fatty liver induced by palmitic acid (PA) and a high fat diet mouse model.ResultsIn high fat diet-induced C57BL/6 J mice, CPT significantly reduced liver weight index, serum alanine transaminase (ALT), aspartate transaminase (AST), triacylglycerol (TG), total cholesterol (TC) and hepatic TG, TC levels. Moreover, CPT dramatically decreased the contents of blood glucose, insulin, and insulin resistance (HOMA-IR) index. Meanwhile, CPT significantly increased the tyrosine phosphorylation level of IRS and the uptake of 2-deoxyglucose (2DG) in PA-induced HepG2 cells and primary hepatocytes fatty liver models. Furthermore, in PA-induced HepG2 cells and primary hepatocytes, CPT significantly decreased the number of lipid droplets and intracellular TG content. In addition, mechanism investigation showed that CPT increased the phosphorylation of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) and glycogen synthase-3β (GSK3β) in vivo and in vitro models, which were abrogated by PI3K inhibitor LY294002 in vitro models.ConclusionThese findings indicate that CPT may exert the therapeutic effects on NAFLD via regulating PI3K/Akt/GSK3β pathway.     

Subcellular Localization of Glycosyl Transferases Involved in Glycoprotein Biosynthesis in the Cotyledons of Pisum sativum L

Subcellular membrane fractions from 21-day-old pea (Pisum sativum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of lipid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.

Other mannosyl transferases are found at a density of 1.123 g/cc and 1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis.

     

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed “starch-mannose-fermentation” biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

Some phytotoxic glycopeptides from Ceratocystis ulmi, the Dutch Elm Disease pathogen

Ceratocystis ulmi, the causal agent of Dutch Elm Disease, produces phytotoxic glycopeptides in culture. A mixture of phytotoxic glycopeptides has been prepared by affinity chromatography on a concanavalin A-Sepharose column and collectively they have been termed the toxin. The polydisperse component that makes up the majority of the toxin (80%) by weight has a molecular weight of about 2.7·10⁵. The large molecular weight component (<5%) elutes at the void volume of a Bio-Gel A50 m column. The other component (15%) appears as a trailing peak on the edge of the major component and has an approximate molecular weight of 7 · 10⁴. The toxin is composed of 38% sugar residues, primarily rhamnose and mannose, and 7% amino acid residues. Methylation analysis coupled with mild acid hydrolysis indicates that the backbone of the polysaccharide portion of the toxin is composed of α-1,6-linked mannosyl residues with a 3-linked terminal rhamnosyl residue linked to C-3 of almost every mannosyl residue. The carbohydrate portion of the molecule is linked to the peptide via O-glycosidic linkages to both threonyl and seryl residues. All three components of the toxin are capable of causing wilt in stem cuttings of American elm.     

Properties of Rhizopus stolonifer Polygalacturonase, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings

Some properties of the polygalacturonase-elicitor from the filtrates of Rhizopus stolonifer cultures have been examined in an attempt to understand its mode of action as an elicitor of casbene synthetase activity in castor bean seedlings. Both the polygalacturonase activity and the elicitor activity are heat-labile with similar heat-sensitivity profiles. Also, the catalytic activity of the enzyme is lost on treatment with sodium periodate, as had been shown previously for the elicitor activity. The pH optimum of the enzyme activity with polygalacturonic acid as the substrate is 4.9. Exposures of germinating castor bean seedlings to the elicitor for short-term periods of 1 to 10 minutes followed by washing and incubation in sterile, distilled water are partially effective in elicitation in comparison with the continuous exposure of the seedlings over 11 hours to the same amount of the elicitor. The initial rate of reaction catalyzed by the enzyme is about 3 times faster with polygalacturonic acid as a substrate than with partially (50%) methylated polygalacturonic acid (pectin). The K_m value of the enzyme for polygalacturonic acid is about 4.2 millimolar in terms of monomeric units and about 0.07 millimolar in terms of polymer concentration. Examination of the types of products formed by the action of the enzyme suggests that it is an endo-hydrolase. The amino acid composition of this enzyme is similar to those of other extracellular fungal proteins reported. The carbohydrate moiety of the glycoprotein polygalacturonase-elicitor is composed of 92% mannose and 8% glucosamine by gas chromatography-mass spectrometry analysis. The linkage group analysis of the carbohydrate moiety showed that mannosyl residues which are 1,2-linked comprise about 70% of the total glycosyl residues and demonstrated the presence of some 1,3,6- and 1,2,6-linked branching mannosyl residues.     

Structural organization of the phospholipid component of Streptomyces hygroscopicus cell membranes as determined from neutron diffraction data

The nanoparameters of an actinobacterial membrane have been estimated by neutron diffraction on multilamellar lipid membranes. The lamellar repeat distance in a partly hydrated membrane prepared with the phospholipid fraction from Streptomyces hygroscopicus is 85.8 ± 0.5°C and decreases to 83.5 ± 0.5 0°C. Some lipids are not incorporated into the bilayer and form a liquid phase of micelles 54.2 ± 0.2     

Cardiomyopathic mutations in essential light chain reveal mechanisms regulating the super relaxed state of myosin

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K–associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively.     

The centromeric K-type repeat and the central core are together sufficient to establish a functional Schizosaccharomyces pombe centromere.

The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system. Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat. The S. pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes. Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy. Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences. Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S. pombe.     

A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate,Lipoarabinomannan

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5–7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10–12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.     

Stages and Conformations of the Tau Repeat Domain during Aggregation and Its Effect on Neuronal Toxicity

Several neurodegenerative diseases are characterized by the aggregation and posttranslational modifications of Tau protein. Its “repeat domain” (TauRD) is mainly responsible for the aggregation properties, and oligomeric forms are thought to dominate the toxic effects of Tau. Here we investigated the conformational transitions of this domain during oligomerization and aggregation in different states of β-propensity and pseudo-phosphorylation, using several complementary imaging and spectroscopic methods. Although the repeat domain generally aggregates more readily than full-length Tau, its aggregation was greatly slowed down by phosphorylation or pseudo-phosphorylation at the KXGS motifs, concomitant with an extended phase of oligomerization. Analogous effects were observed with pro-aggregant variants of TauRD. Oligomers became most evident in the case of the pro-aggregant mutant TauRDΔK280, as monitored by atomic force microscopy, and the fluorescence lifetime of Alexa-labeled Tau (time-correlated single photon counting (TCSPC)), consistent with its pronounced toxicity in mouse models. In cell models or primary neurons, neither oligomers nor fibrils of TauRD or TauRDΔK280 had a toxic effect, as seen by assays with lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. However, oligomers of pro-aggregant TauRDΔK280 specifically caused a loss of spine density in differentiated neurons, indicating a locally restricted impairment of function.     

Regulation of substrate specificity of plant alpha-mannosidase by cobalt ion: in vitro hydrolysis of high-mannose type N-glycans by Co2+-activated Ginkgo alpha-mannosidase

In our previous study (Woo, K. K., et al., Biosci. Biotechnol. Biochem., 68, 2547-2556 (2004), we purified an alpha-mannosidase from Ginkgo biloba seeds; it was activated by cobalt ions and highly active towards high-mannose type free N-glycans occurring in plant cells. In the present study, we have found that the substrate specificity of Ginkgo alpha-mannosidase is significantly regulated by cobalt ions. When pyridylamino derivative of Man9GlcNAc2 (M9A) was incubated with Ginkgo alpha-mannosidase in the absence of cobalt ions, Man5GlcNAc2-PA (M5A) having no alpha1-2 mannosyl residue was obtained as a major product. On the other hand, when Man9GlcNAc2-PA was incubated with alpha-mannosidase in the presence of Co2+ (1 mM), Man3-1GlcNAc2-PA were obtained as major products releasing alpha1-3/6 mannosyl residues in addition to alpha1-2 mannosyl residues. The structures of the products (Man8-5GlcNAc2-PA) derived from M9A by enzyme digestion in the absence of cobalt ions were the same as those in the presence of cobalt ions. These results clearly suggest that the trimming pathway from M9A to M5A is not affected by the addition of cobalt ions, but that hydrolytic activity towards alpha1-3/6 mannosyl linkages is stimulated by Co2+. Structural analysis of the products also showed clearly that Ginkgo alpha-mannosidase can produce truncated high-mannose type N-glycans, found in developing or growing plant cells, suggesting that alpha-mannosidase might be involved in the degradation of high-mannose type free N-glycans.