β-Glucan hydrolases from Aspergillus niger. Isolation of a β-(1→4)-glucan hydrolase and some properties of the β-(1→3)-glucan-hydrolase components

1. The components of an enzyme preparation from Aspergillus niger, which hydrolysed substrates containing beta-(1-->3)- and beta-(1-->4)-glucosidic linkages, were separated by calcium phosphate and Dowex 1 column chromatography. 2. The hydrolytic activity of each fraction from both types of column towards laminaribiose, laminarin, carboxymethylpachyman, pachydextrins, salicin, cellobiose, cellopentaose and swollen cellulose was tested. 3. The activity towards the beta-(1-->3)-glucosidic substrates was found in three well-separated groups of fractions. The differences in action pattern of these groups is discussed. 4. Preparative-scale chromatography that enabled the separation of a beta-(1-->4)-glucan-glucanohydrolase component substantially free of activity towards beta-(1-->3)-glucosidic substrates is described. Residual beta-(1-->3)-glucan-hydrolase activity was removed by adsorption on to insoluble laminarin at pH3.5.     

Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

1. A barley glucan with 68% of beta-(1-->4)-linkages and 32% of beta-(1-->3)-linkages was exhaustively hydrolysed with an Aspergillus niger beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1.4%; cellobiose, 11.9%; 3(2)-O-beta-glucosylcellobiose, 45.0%; a tetrasaccharide(s), which was a substituted cellobiose, 16.4%. A series of unidentified higher-molecular-weight products (26.5%) were also found. 3. The identity of the products suggests that the A. niger beta-(1-->4)-glucan hydrolase hydrolyses beta-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the beta-(1-->4)-glucan hydrolase and an exo-beta-(1-->3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by beta-(1-->4)-glucan hydrolase but are partially hydrolysed by the exo-beta-(1-->3)-glucan hydrolase and therefore possess one or more (1-->3)-linked glucose residues at their non-reducing end.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

Syntheses of α- and β-Glycosyl Donors with a Disaccharide β-D-Gal-(1→3)-D-GalNAc Backbone

The synthesis of thioglycoside glycosyl donors with a disaccharide -D-Gal-(1 3)-D-GalNAc backbone was studied using the glycosylation of a series of suitably protected 3-monohydroxy- and 3,4-dihydroxyderivatives of phenyl 2-azido-2-deoxy-1-thio-- and 1-thio--D-galactopyranosides by galactosyl bromide, fluoride, and trichloroacetimidate. In the reaction with the monohydroxylated glycosyl acceptor, the process of intermolecular transfer of thiophenyl group from the glycosyl acceptor onto the cation formed from the molecule of glycosyl donor dominated. When glycosylating 3,4-diol under the same conditions, the product of the thiophenyl group transfer dominated or the undesired (1 4), rather than (1 3)-linked, disaccharide product formed. The aglycon transfer was excluded when 4-nitrophenylthio group was substituted for phenylthio group in the galactosyl acceptor molecule. This led to the target disaccharide, 4-nitrophenyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio--D-galactopyranoside, in 57% yield. This disaccharide product bears nonparticipating azido group in position 2 of galactosamine and can hence be used to form -glycoside bond. Azido group and the aglycon nitro group were simultaneously reduced in this product and then trichloroacetylated, which led to the -glycosyl donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in 62% yield. The resulting glycosyl donor was used in the synthesis of tetrasaccharide asialo-GM₁.     

Effect of molecular size and modification pattern on the internalization of water soluble β-(1 → 3)-(1 → 4)-glucan by primary murine macrophages

It has been shown that β-(1 → 3)-(1 → 4)-glucans (BG34) from barley and oats can trigger recognition and internalization by murine and human macrophages. Increasing evidence has suggested that macrophage recognition and internalization of BG34 are dramatically affected by the purity of BG34, the molecular weight and chemical modification. In this study, we investigated the structural features of BG34 for macrophage recognition and internalization. We prepared homogeneous BG34s of 10 kDa (BG34-10), 200 kDa (BG34-200) and 500 kDa (BG34-500) with high purity, and then introduced green fluorescence FITC to the reducing ends (Re) or main chain (Mc). The results of size exclusion chromatography, ¹³C NMR, fluorescence microscopy, FACS analyses and MTS assay demonstrated that non-toxic BG34 of 10 kDa (BG34-10) effectively trigger macrophage internalization. The internalization was adversely affected by modifying the main chain of BG34-10 but not the reducing end. Studies using blocking antibodies on several CD11b⁺ and CD11b^? cells suggested that CD11b may play an important role in mediating macrophage internalization of BG34-10. Quantitative RT-PCR and intracellular cytokine stain revealed that macrophages generate increased level of CD11b and TNF-α in response to BG34-10. This study for the first time demonstrated the molecular size (10 kDa) and pattern of modification (reducing end modification) for BG34-10 to mediate macrophage internalization. Since BG34 is water soluble, biocompatible and biodegradable FDA-approved material, this mechanism of BG34-10 can be used to design drug delivery system targeting macrophages.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Biosynthesis of a β-(1 → 4)-mannan in fenugreek seedlings

A particulate enzymatic preparation, extracted from fenugreek seedlings (Trigonella foenum-graecum) catalyses the transfer of mannose from guanosine diphosphate-[U-¹⁴C]mannose and its incorporation into an alkali-soluble polysaccharide. Chemical and enzymatic study of this polysaccharide reveals the presence of only one type of osidic linkage, namely β-(1 → 4)-s-mannopyranosyl. The influence of some factors on this biosynthesis was studied, as well as the MW of the polysaccharide and the existence of an endogenous acceptor.     

Synthetic Studies on Nephritogenic Glycosides. Synthesis of β-Glc-(1→6)-α-Glc-(l→6)-α-Glc-(1→Asn)

Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

Glycoside hydrolase family 1 (GH1) β-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice β-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-β-d-fucoside (k_cat/K_m = 67 mm⁻¹ s⁻¹), pNP-β-d-glucoside (k_cat/K_m = 6.2 mm⁻¹ s⁻¹), and pNP-β-d-galactoside (k_cat/K_m = 1.6 mm⁻¹s⁻¹) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-β-d-glucoside and β-(1→3)- and β-(1→2)-linked disaccharides and was able to hydrolyze apigenin β-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-β-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 Å resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended β-(1→4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-β-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.β-Glucosidases (EC 3.2.1.21) have a wide range of functions in plants, including acting in cell wall remodeling, lignification, chemical defense, plant-microbe interactions, phytohormone activation, activation of metabolic intermediates, and release of volatiles from their glycosides (Esen, 1993). They fulfill these roles by hydrolyzing the glycosidic bond at the nonreducing terminal glucosyl residue of a glycoside or an oligosaccharide, thereby releasing Glc and an aglycone or a shortened carbohydrate. The aglycone released from the glycoside may be a monolignol, a toxic compound, or a compound that further reacts to release a toxic component, an active phytohormone, a reactive metabolic intermediate, or a volatile scent compound (Brzobohatý et al., 1993; Dharmawardhama et al., 1995; Reuveni et al., 1999; Lee et al., 2006; Barleben et al., 2007; Morant et al., 2008). Indeed, the wide range of glucosides of undocumented functions found in plants suggests that many β-glucosidase functions may remain to be discovered.Plant β-glucosidases fall into related families that have been classified as glycosyl hydrolase (GH) families GH1, GH3, and GH5 (Henrissat, 1991; Coutinho and Henrissat, 1998, 1999). Of these, GH1 has been most thoroughly documented and shown to comprise a gene family encoding 40 putative functional GHs in Arabidopsis (Arabidopsis thaliana) and 34 in rice (Oryza sativa) in addition to a few pseudogenes (Xu et al., 2004; Opassiri et al., 2006). In addition to β-glucosidases, plant GH1 members include β-mannosidases (Mo and Bewley, 2002), β-thioglucosidases (Burmeister et al., 1997), and disaccharidases such as primeverosidase (Mizutani et al., 2002) as well as hydroxyisourate hydrolase, which hydrolyzes the internal bond in a purine ring rather than a glycosidic bond (Raychaudhuri and Tipton, 2002). The specificity for the glycone in GH1 enzymes varies. Some enzymes are quite specific for β-d-glucosides or β-d-mannosides, while many accept either β-d-glucosides or β-d-fucosides, and some also hydrolyze β-d-galactosides, β-d-xylosides, and α-l-arabinoside (Esen, 1993). However, most GH1 enzymes are thought to hydrolyze glucosides in the plant, and it is the aglycone specificity that determines the functions of most GH1 enzymes.Aglycone specificity of GH1 β-glucosidases ranges from rather broad to absolutely specific for one substrate and is not obvious from sequence similarity. For instance, maize (Zea mays) ZmGlu1 β-glucosidase hydrolyzes a range of glycosides, including its natural substrate, 2-O-β-d-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one (DIMBOAGlc), but not dhurrin, whereas sorghum (Sorghum bicolor) Dhr1, which is 72% identical to ZmGlu1, only hydrolyzes its natural cyanogenic substrate dhurrin (Verdoucq et al., 2003). Similarly, despite sharing over 80% amino acid sequence identity, the legume isoflavonoid β-glucosidases dalcochinase from Dalbergia cochinchinensis and Dnbglu2 from Dalbergia nigrescens hydrolyze each other''s natural substrate very poorly (Chuankhayan et al., 2007). Thus, small differences in the amino acid sequence surrounding the active site may be expected to account for significant differences in substrate specificity.GH1 is classified in GH clan A, which consists of GH families whose members have a (β/α)₈-barrel structure with the catalytic acid/base on strand 4 of the β-barrel and the catalytic nucleophile on strand 7 (Henrissat et al., 1995; Jenkins et al., 1995). As such, all GH1 enzymes have similar overall structures, but it has been noted that four variable loops at the C-terminal end of the β-barrel strands, designated A, B, C, and D, account for much of the differences in the active site architecture (Sanz-Aparicio et al., 1998). The similar structures with great diversity in substrate specificity make plant GH1 enzymes an ideal model system to investigate the structural basis of substrate specificity. To date, seven plant β-glucosidase structures have been reported, including three closely related chloroplastic enzymes from maize (Czjzek et al., 2000, 2001), sorghum (Verdoucq et al., 2004), and wheat (Triticum aestivum; Sue et al., 2006), the cytoplasmic strictosidine β-glucosidase from Rauvolfia serpentine (Barleben et al., 2007), and the secreted enzymes white clover (Trifolium repens) cyanogenic β-glucosidase (Barrett et al., 1995), white mustard (Sinapsis alba) myrosinase (thioglucosidase; Burmeister et al., 1997), and rice Os3BGlu7 (BGlu1; Chuenchor et al., 2008). These enzymes hydrolyze substrates with a range of structures, but they cannot account for the full range of β-glucosidase substrates available in plants, and determining the structural differences that bring about substrate specificity differences in even closely related GH1 enzymes has proven tricky (Verdoucq et al., 2003, 2004; Sue et al., 2006; Chuenchor et al., 2008).Amino acid sequence-based phylogenetic analysis of GH1 enzymes encoded by the rice genome showed that there are eight clusters containing both rice and Arabidopsis proteins that are more closely related to each other than they are to enzymes from the same plants outside the clusters (Fig. 1; Opassiri et al., 2006). In addition, there are a cluster of sixteen putative β-glucosidases and a cluster of myrosinases in Arabidopsis without any closely related rice counterparts. Comparison with characterized GH1 enzymes from other plants reveals other clusters of related enzymes not found in rice or Arabidopsis, including the chloroplastic enzymes, from which the maize, sorghum, and wheat structures are derived, and the cytoplasmic metabolic enzymes, from with the strictosidine hydrolase structure is derived (Fig. 1). Therefore, although the known structures provide good tools for molecular modeling of plant enzymes, most rice and Arabidopsis GH1 enzymes lack a close correspondence in sequence and functional evolution to these structures, suggesting that the variable loops that determine the active site may be different. It would be useful, therefore, to know the structures and substrate specificities of representative members of each of the eight clusters seen in rice and Arabidopsis. To begin to acquire this information, we have expressed Os3BGlu6, a member of cluster At/Os 1 in Figure 1, characterized its substrate specificity, and determined its structure alone and in complex with a glycosyl intermediate and a nonhydrolyzable substrate analog.Open in a separate windowFigure 1.Simplified phylogenetic tree of the amino acid sequences of eukaryotic GH1 proteins with known structures and those of rice and Arabidopsis GH1 gene products. The protein sequences of the eukaryotic proteins with known structures are marked with four-character PDB codes for one of their structures, including Trifolium repens cyanogenic β-glucosidase (1CBG; Barrett et al., 1995), Sinapsis alba myrosinase (1MYR; Burmeister et al., 1997), Zea mays ZmGlu1 β-glucosidase (1E1F; Czjzek et al., 2000), Sorghum bicolor Dhr1 dhurrinase (1V02; Verdoucq et al., 2004), Triticum aestivum β-glucosidase (2DGA; Sue et al., 2006), Rauvolfia serpentina strictosidine β-glucosidase (2JF6; Barleben et al., 2007), and Oryza sativa Os3BGlu7 (BGlu1) β-glucosidase (2RGL; Chuenchor et al., 2008) from plants, along with Brevicoryne brassicae myrosinase (1WCG; Husebye et al., 2005), Homo sapiens cytoplasmic (Klotho) β-glucosidase (2E9M; Hayashi et al., 2007), and Phanerochaete chrysosporium (2E3Z; Nijikken et al., 2007), while those encoded in the Arabidopsis and rice genomes are labeled with the systematic names given by Xu et al. (2004) and Opassiri et al. (2006), respectively. One or two example proteins from each plant are given for each of the eight clusters of genes shared by Arabidopsis (At) and rice (Os) and the Arabidopsis-specific clusters At I (β-glucosidases) and At II (myrosinases), with the number of Arabidopsis or rice enzymes in each cluster given in parentheses. These sequences were aligned with all of the Arabidopsis and rice sequences in ClustalX (Thompson et al., 1997), the alignment was manually edited, all but representative sequences were removed, and the tree was calculated by the neighbor-joining method, bootstrapped with 1,000 trials, and then drawn with TreeView (Page, 1996). The grass plastid β-glucosidases, which are not represented in Arabidopsis and rice, are shown in the group marked “Plastid.” Percentage bootstrap reproducibility values are shown on internal branches where they are greater than 60%. Except those marked by asterisks, all external branches represent groups with 100% bootstrap reproducibility. To avoid excess complexity, those groups of sequences marked with asterisks are not monophyletic and represent more branches within the designated cluster than are shown. For a complete phylogenetic analysis of Arabidopsis and rice GH1 proteins, see Opassiri et al. (2006).     

Properties of a β-(1→4)-glucan hydrolase from Aspergillus niger

1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.     

Subcellular localization and topology of β(1→4)galactosyltransferase that elongates β(1→4)galactan side chains in rhamnogalacturonan I in potato

The subcellular localization and topology of rhamnogalacturonan I (RG-I) (14)galactosyltransferase(s) ([14]GalTs) from potato (Solanum tuberosum L.) were investigated. Using two-step discontinuous sucrose step gradients, galactosyltransferase (GalT) activity that synthesized 70%-methanol-insoluble products from UDP-[¹⁴C]Gal was detected in both the 0.5 M sucrose fraction and the 0.25/1.1 M sucrose interface. The former fraction contained mainly soluble proteins and the latter was enriched in Golgi vesicles that contained most of the UDPase activity, a Golgi marker. By gel-filtration analysis, products of 180–2,000 Da were found in the soluble fraction, whereas in the Golgi-enriched fraction the products were larger than 80 kDa and could be digested with rhamnogalacturonan lyase and (1,4)endogalactanase to yield smaller rhamnogalacturonan oligomers, galactobiose and galactose. The endogalactanase requires (14)galactans with at least three galactosyl residues for cleavage, indicating that the enzyme(s) present in the 0.25/1.1 M Suc interface transferred one or more galactosyl residues to pre-existing (14)galactans producing RG-I side chains in total longer than a trimer. Thus, the (14)GalT activity that elongates (14)-linked galactan on RG-I was located in the Golgi apparatus. This (14)GalT activity was not reduced after treatment of the Golgi vesicles with proteinase, but approximately 75% of the activity was lost after treatment with proteinase in the presence of Triton X-100. In addition, the (14)GalT activity was recovered in the detergent phase after treatment of Golgi vesicles with Triton X-114. Taken together, these observations supported the view that the RG-I (14)GalT that elongates (14)galactan was mainly located in the Golgi apparatus and integrated into the membrane with its catalytic site facing the lumen.Abbreviations GalT Galactosyltransferase - (14)GalT (14)-Galactosyltransferase - H ⁺ -ATPase Proton ATPase - HG Homogalacturonan - HSP70 ER resident Bip - mMDH Mitochondrial malate dehydrogenase - RG-I Rhamnogalacturonan I - RG-II Rhamnogalacturonan II - RGP Reversibly glycosylated polypeptide - RG-Lyase Rhamnogalacturonan lyase - Suc Sucrose - UDPase Uridine-5-diphosphatase     

Synthesis of β-(1→6)-linked N-acetyl-D-glucosamine oligosaccharide substrates and their hydrolysis by Dispersin B

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.     

The structure of a β-(1→3)-d-glucan from yeast cell walls

Yeast glucan as normally prepared by various treatments of yeast (Saccharomyces cerevisiae) cell walls to remove mannan and glycogen is still heterogeneous. The major component (about 85%) is a branched beta-(1-->3)-glucan of high molecular weight (about 240000) containing 3% of beta-(1-->6)-glucosidic interchain linkages. The minor component is a branched beta-(1-->6)-glucan. A comparison of our results with those of other workers suggests that different glucan preparations may differ in the degree of heterogeneity and that the major beta-(1-->3)-glucan component may vary considerably in degree of branching.     

Interaction of (1→4)- and (1→6)-linked disaccharides with the fenton reagent under physiological conditions

Reactions of (1→4)- and (1→6)-linked disaccharides, mainly of maltose and isomaltose, with the Fenton reagent under physiological conditions were studied. Chemical characterization of oxidation products was conducted by g.l.c. and g.l.c.-m.s. of their trimethylsilyl derivatives, and the results demonstrated that (1→6)-linked disaccharides are more reactive with the hydroxyl radical (·OH) generated by the Fenton reagent than (1→4)-linked disaccharides. About 35–40% of (1→6)-and 15–20% of (1→4)-linked disaccharides were oxidatively degraded to smaller molecules after incubation for 24 h. Of the four disaccharides examined, namely, maltose, isomaltose, cellobiose, and gentiobiose, the α-(1→6)-linked disaccharide isomaltose exhibited the highest reactivity, whereas the β-(1→4)-linked disaccharide cellobiose showed the lowest. These results suggest the existence of a relationship between the configuration of the glycosidic linkage and the reactivity with ·OH in aqueous solution.     

The structure of a β-(1→6)-d-glucan from yeast cell walls

By selective enzymolysis, or chemical fractionation, a minor polysaccharide component has been isolated from yeast (Saccharomyces cerevisiae) glucan. This minor component has a degree of polymerization of about 130-140, a highly branched structure, and a high proportion of beta-(1-->6)-glucosidic linkages. The molecules also contain a smaller proportion of beta-(1-->3)-glucosidic linkages that serve mainly as interchain linkages, but some may also be inter-residue linkages.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.