Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Distinct patterns of expression of the β-1,4-galactosyltransferases during testicular development in the mouse

Glycosylation is one of the most important post-translational modifications and it is clear that the single step of -1,4-galactosylation is performed by a family of -1,4-galactosyltransferases (4-GalTs) and that each member of this family may play a distinct role in different tissues and cells. In this study, we characterized the gene expression of six 4-GalTs in mouse testis and analyzed the changes of galactosylation of testis glycoproteins during postnatal development. Northern blot analysis revealed that 4-GalT-I and 4-GalT-IV were expressed mainly in newborn mouse testis and that the expression of 4-GalT-II increased markedly and persisted at the highest levels in adult mouse testis. The expression of 4-GalT-III and 4-GalT-V, however, remained relatively at low levels during mouse testicular development. In contrast, the expression of 4-GalT-VI was undetectable in mouse testis. The gene expression of 4-GalT-II in mouse testis was further analyzed by in situ hybridization due to its unique expression pattern. Strong hybridization signals were detected in the seminiferous tubules and the expression varied among the different stages of spermatogenic differentiation. The distinct gene expression patterns of 4-GalTs in mouse testis could affect the differential galactosylation of testis glycoproteins, as revealed by lectin histochemistry analysis.     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

Properties of Aspergillus japonicus β-fructofuranosidase immobilized on porous silica

-Fructofuranosidase from Aspergillus japonicus MU-2, which produces fructo-oligosaccharides (1-kestose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside); and nystose: O--D-fructofuranosyl-(2 1)--D-fructofuranosyl-(2 1)--D-fructofuranosyl -D-glucopyranoside) from sucrose, was immobilized, covalently with glutaraldehyde onto alkylamine porous silica, at high efficiency (64%). Optimum pore diameter of porous silica for immobilization of the enzyme was 91.7 nm. After immobilization, the enzyme's stabilities to temperature, metal ions and proteolysis were improved, while its optimum pH and temperature were unchanged. The highest efficiency of continuous production of fructo-oligosaccharides (more than 60%), using a column packed with the immobilized enzyme, was obtained at 40% to 50% (w/v) sucrose. The half-life of the column during long-term continuous operation at 55°C was 29 days.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Comparison of wild-type and UV-mutant β-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications

A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal -glucans. Activity against -(1, 3)(1, 4)-d-glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less -glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley -glucan (13.0–16.9%), but were more active against crude -glucan from barley (16.0–24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash -glucan. Finally, TC2 and TC5 produce more efficient -glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.     

Models for glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation

2-(4-Methoxybenzyl)-1-cyclohexyl--d-glucopyra nosides (1b and 2b) and 2-(4-methoxybenzyl)-1-cyclohexyl--d-galactopy ranosides (1c and 2c), models for glycosidic juvenogens, were synthesized using either D-glucose or D-galactose [in their natural form (3 and 5) or activated form (4 and 6)], and the respective racemic cis or trans isomers of 2-(4-methoxybenzyl)-1-cyclohexanol (1a and 2a) by either enzymic reverse hydrolysis or transglycosylation under both standard heating and microwave irradiation. Commercially available almond -glucosidase (EC 3.2.1.21) or -galactosidase (EC 3.2.1.23) from Escherichia coli were employed using different acetonitrile/water mixtures [9/1 (v/v) for the reverse hydrolysis, and 4/1 (v/v) for the transglycosylation].     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Physicochemical characterization of β-crystallins from bovine lenses: Hydrodynamic and aggregation properties

A detailed investigation of the hydrodynamic and aggregation behaviors has been made on the -crystallins of bovine lens. Results from this study indicated that H (high-molecular-weight -crystallin) and L (low-molecular-weight -crystallin) exhibited considerable heterogeneity in their native structures and subunit polypeptides. Low-speed sedimentation equilibrium showed a heterogeneous paucidisperse system in each -crystallin fraction. Viscosity and circular dichroism studies pointed to a compact and globular shape and the presence of -sheet and -turns in these crystallins. Dissociation of H by urea and guanidinium HCl followed by reassociation during gel-filtration chromatography produced an elution pattern with two fractions corresponding to L crystallin and high-molecular-weight aggregates without the formation of native H. By contrast, under similar treatment, about 60% L reassociated into the correct native structure and the rest into high-molecular-weight fractions. Amino acid analyses of H and L and their corresponding subunit polypeptides demonstrated the close similarity of these crystallins. Trace element analyses indicated that both Ca and Mg are present in H and L crystallins and may be involved in maintaining the native quarternary structures of these proteins.