One-pot enzymatic synthesis of the Galα1→3Galβ1→4GlcNAc sequence within situ UDP-Gal regeneration

The trisaccharide Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4-epimerase, UDP-Gal:GlcNAc 4-galactosyltransferase and UDP-Gal:Gal14GlcNAc 3-galactosyltransferase, Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was formed in a 2.2 µmol ml^–1 yield starting from the acceptor GlcNAc1O-(CH₂)₈COOCH₃. This is an efficient and convenient method for the synthesis of the Gal13Gal14GlcNAc epitope which plays an important role in various biological and immunological processes.     

Characterization of d-galactosyl-β1→4-l-rhamnose phosphorylase from Opitutus terrae

We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45 °C and at pH 6.0–7.0. The values of k_cat and K_m of the phosphorolytic activity of the enzyme on GalRha were 60 s^?1 and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms.     

Studies on the effect of the hepatocyte-stimulating factor on galactose-β1 → 4-N-acetylglucosamine α2 → 6-sialyltransferase in cultured hepatocytes

Rat hepatic Galβ1 → 4GlcNAcα2 → 6 sialyltransferase is released into the blood at elevated levels following an inflammatory challenge: this is a typical response of the group of plasma proteins known as acute-phase reactants. In the present study, primary cultures of liver parenchymal cells are used to demonstrate that the same hepatic cell type that produces plasma proteins such as fibrinogen also produces and releases sialyltransferase. Hepatic production of sialyltransferase is stimulated by a major regulator of hepatic acute-phase reactant production, the hepatocyte-stimulating factor (HSF), while another monokine, interleukin-1, does not affect hepatocyte sialyltransferase production. The maximum increase in sialyltransferase occurs 48 h after exposure to HSF which is considerably later than the fibrinogen response. The sialyltransferase that is stimulated by HSF is the Galβ1 → 4GlcNAcα2 → 6 isozyme.     

Hydrolysis of oligosaccharides of the β-(1→4)-linked d-xylose series by an endo(1→4)-β-d-xylanase from the anaerobic rumen fungus Neocallimastix frontalis

An endo-(14)--d-xylanase from Neocallimastix frontalis was purified by anion-exchange chromatography. The enzyme had an apparent molecular mass of 30 kDa on SDS-PAGE and exhibited maximum activity at 50°C and at pH values between 6.0 and 6.6. Kinetic studies on the hydrolysis of xylo-oligosaccharides, ranging from xylobiose to xylodecaose, showed that xylohexaose and xyloheptaose were the preferred substrates for the enzyme and that xylobiose, xylotriose and xylotetraose were not hydrolysed. Xylose was not a product of the hydrolysis of any of the xylo-oligosaccharide substrates tested. The enzyme appeared to have a strong preference for the hydrolysis of the internal glycosidic bonds of the oligosaccharides, which is typical of endo-(14)--d-xylanase activity, but it differed from other fungal endo-(14)--d-xylanases in that it had uniform action on the various internal linkages in the xylo-oligosaccharides.V. Garcia-Campayo, S.I. McCrae and T.M. Wood are with The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK     

Fine structure of (1→3),(1→4)-β-d-glucan from Zea shoot cell-walls

The insoluble material that remains after extraction of Zea shoots with cold buffer was treated successively with 3m LiCl and hot water. The polysaccharides solubilized by these treatments were mostly (1→3),(1→4)-β-d-glucans. The β-d-glucan from the hot-water-soluble fraction was hydrolyzed by Bacillus subtilis (1→3),(1→4)-β-d-glucan 4-glucanohydrolase. The oligosaccharides were characterized by methylation analysis of the enzymic fragments and by methylation analysis of secondary fragments generated by treatment of the isolated oligosaccharides with Streptomyces QM B814 cellulase. The results demonstrate that the native polysaccharide consists mainly of cellotriosyl and cellotetraosyl residues joined by single (1→3) linkages. Evidence is presented to show that certain other glucosyl sequences are also present in the native polysaccharide including (a) two, three, or four contiguous (1→3)-linkages; (b) blocks of more than four (1→4)-linked glucose residues; (c) regions having alternating (1→3)- and (1→4)-linkages.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

β-D-(1→4), β-D-(1→3) 'mixed linkage' xylans from red seaweeds of the order Nemaliales and Palmariales

Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The β-D-(1→4), β-D-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of β-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.     

Mode of action of endo-(1→3)-β-d-glucanases from marine molluscs on the laminarin from Laminaria cichorioides: The structure and the inhibitory effect of the resulting (1→3;1→6)-β-d-gluco-oligosaccharides

The oligosaccharides released by the action of endo-(1→3)-β-d-glucanases from the marine molluscs Chlamys albidus (laminarinase Lo) and Spisula sachalinensis (laminarinase LIV) on Laminaria laminarin have been studied. For laminarinase Lo, the branched products were shown to be 6²-β-d-glucopyranosyl-laminaribiose and 6³- and 6²-β-d-glucopyranosyl-laminaritrioses by methylation analysis and ¹³C-n.m.r. spectroscopy. It is suggested that one or two (1→3) linkages adjacent to (1→6) branch-points result in resistance to enzymic attack. 6³-β-d-Glucopyranosyl-laminaritriose inhibited laminarinases Lo and LIV (I₅₀ 1.2 × 10⁻³m and 1.5 × 10⁻³m, respectively).     

The Maize Mixed-Linkage (1→3),(1→4)-β-d-Glucan Polysaccharide Is Synthesized at the Golgi Membrane

With the exception of cellulose and callose, the cell wall polysaccharides are synthesized in Golgi membranes, packaged into vesicles, and exported to the plasma membrane where they are integrated into the microfibrillar structure. Consistent with this paradigm, several published reports have shown that the maize (Zea mays) mixed-linkage (1→3),(1→4)-β-d-glucan, a polysaccharide that among angiosperms is unique to the grasses and related Poales species, is synthesized in vitro with isolated maize coleoptile Golgi membranes and the nucleotide-sugar substrate, UDP-glucose. However, a recent study reported the inability to detect the β-glucan immunocytochemically at the Golgi, resulting in a hypothesis that the mixed-linkage β-glucan oligomers may be initiated at the Golgi but are polymerized at the plasma membrane surface. Here, we demonstrate that (1→3),(1→4)-β-d-glucans are detected immunocytochemically at the Golgi of the developing maize coleoptiles. Further, when maize seedlings at the third-leaf stage were pulse labeled with [¹⁴C]O₂ and Golgi membranes were isolated from elongating cells at the base of the developing leaves, (1→3),(1→4)-β-d-glucans of an average molecular mass of 250 kD and higher were detected in isolated Golgi membranes. When the pulse was followed by a chase period, the labeled polysaccharides of the Golgi membrane diminished with subsequent transfer to the cell wall. (1→3),(1→4)-β-d-Glucans of at least 250 kD were isolated from cell walls, but much larger aggregates were also detected, indicating a potential for intermolecular interactions with glucuronoarabinoxylans or intermolecular grafting in muro.An overwhelming body of evidence accumulated has established that the (1→4)-β-d-glucan chains of cellulose microfibrils are synthesized and assembled at the plasma membrane surface (Delmer, 1999; Saxena and Brown, 2005), whereas, with the lone exception of the (1→3)-β-d-glucan, callose, all noncellulosic pectin and cross-linking glycan polysaccharides are synthesized in Golgi membranes (Northcote and Pickett-Heaps, 1966; Ray et al., 1969, 1976; Harris and Northcote, 1971; Zhang and Staehelin, 1992). Using several plant systems, including grass species, autoradiography and membrane fractionation showed that monosaccharides from ¹⁴C-labeled substrates accumulated in cell wall polysaccharides in Golgi vesicles during a pulse were subsequently transferred to the cell wall when chased with unlabeled substrates (Northcote and Pickett-Heaps, 1966; Pickett-Heaps, 1967; Jilka et al., 1972). Early studies showed that labeled sugars from nucleotide-sugar substrates could be incorporated into alcohol-insoluble polysaccharides using microsomal membranes, and later refined by isolation of Golgi membranes and the synthesis of defined polysaccharides with combinations of nucleotide sugars (Bailey and Hassid, 1966; Ray et al., 1969, 1976; Smith and Stone, 1973; Ray, 1980; Hayashi and Matsuda, 1981a; Gordon and Maclachlan, 1989; Gibeaut and Carpita, 1993).When micromolar concentrations of substrates were used, only small chains of the glycan products were typically made in vitro. For example, xyloglucan oligomers with the characteristic α-d-Xyl-(1→6)-d-glucosyl unit, isoprimeverose, were synthesized with isolated microsomal membranes and low concentrations of UDP-Glc and UDP-Xyl (Ray et al., 1976; Hayashi and Matsuda, 1981b). When concentrations of each nucleotide sugar were increased to millimolar concentrations, then polysaccharides of about 250 kD were synthesized containing the characteristic XXXG heptasaccharide unit structure (Gordon and Maclachlan, 1989). Immunocytochemical evidence with antibodies directed against the terminal nonreducing xylosyl and fucosyl residues confirm that synthesis of the xyloglucan backbone begins in the cis-Golgi membrane and culminates with fucosylation in the trans-Golgi membrane and trans-Golgi network (Moore et al., 1991; Lynch and Staehelin, 1992; Zhang and Staehelin, 1992). The fucosyl transferase responsible for xyloglucan side chain decoration was also shown to be a Golgi-resident protein by in vitro synthesis of xyloglucan polymers (Camirand and Maclachlan, 1986).In Poales species, including all grasses, the mixed-linkage (1→3),(1→4)-β-d-glucan is a major cross-linking glycan that appears transiently during cell elongation in growing tissues and accumulates to large amounts in the cell walls of the endosperm of certain grains (Stone and Clarke, 1992; Trethewey et al., 2005). Bailey and Hassid (1966) demonstrated the synthesis in vitro of noncellulosic glucans with microsomal membranes from grasses. Henry and Stone (1982) used the Bacillus subtilis endoglucanase, an enzyme that generates diagnostic cellodextrin-(1→3)-β-Glc units from (1→3),(1→4)-β-d-glucan to show that the mixed-linkage β-glucan was made specifically with UDP-Glc and microsomal membranes. We used flotation centrifugation to obtain highly enriched Golgi membranes from which (1→3),(1→4)-β-d-glucans of an average of about 250 kD were synthesized (Gibeaut and Carpita, 1993).The BG1 monoclonal antibody recognizes the (1→3),(1→4)-β-d-glucan with high specificity (Meikle et al., 1994). This monoclonal antibody has been used to show dramatic changes in epitope abundance of (1→3),(1→4)-β-d-glucan in the cell walls of developing tissues (Meikle et al., 1994; Trethewey et al., 2005; McCann et al., 2007) and its appearance in the cell walls of Arabidopsis (Arabidopsis thaliana) following heterologous expression of genes thought to encode its synthases (Burton et al., 2006; Doblin et al., 2009). The failure to detect (1→3),(1→4)-β-d-glucan in Golgi membranes and only in the cell wall prompted Fincher (2009) to conclude that cellodextrin oligomers of the (1→3),(1→4)-β-d-glucan may be initiated in the Golgi membrane, but the actual polymerization of the polysaccharide occurs at the plasma membrane.While there is little question that synthesis of full-length polymers is possible in vitro with isolated Golgi membranes and UDP-Glc (Gibeaut and Carpita, 1993; Buckeridge et al., 1999, 2001; Urbanowicz et al., 2004), Fincher (2009) asserts correctly that there exists no experimental evidence that the polymer is made in vivo within the Golgi membrane in intact tissues. In fact, earlier work showing the paucity of immunolabeling of (1→3),(1→4)-β-d-glucan in Golgi membranes of developing wheat (Triticum aestivum) endosperm at a time of active deposition called to question the site of synthesis in vivo (Philippe et al., 2006). There is precedence for the synthesis of chitin in vitro with precociously activated chitisomes (Bracker et al., 1976), a vesicular package of chitin synthase that in vivo is quiescent until reaching the plasma membrane. No activity of chitin synthase from isolated plasma membranes could be demonstrated. In a similar way, the Golgi synthase activity of (1→3),(1→4)-β-d-glucan could be a precocious activation in vitro of a plasma membrane activity.As in vitro synthesis studies clearly show synthesis of full-length (1→3),(1→4)-β-d-glucan only at the Golgi, we reexamined the puzzling finding of its absence from Golgi bodies to determine the true site of synthesis in vivo. In contrast to Fincher (2009), our own immunocytochemistry shows (1→3),(1→4)-β-d-glucan is indeed in the Golgi membrane in 2-d-old coleoptiles, when rapid growth is just beginning. However, we are unable to detect the β-glucan in Golgi after the peak rate of elongation. We pulse labeled maize (Zea mays) seedlings with radiolabeled CO₂ and followed the fate of label captured by photosynthesis and translocated to elongating cells at the base of the seedling. We found by flotation centrifugation that Golgi membranes contain (1→3),(1→4)-β-d-glucan of at least 250 kD, similar to that of the product of in vitro synthesis at optimal UDP-Glc concentrations and commercial preparations of barley (Hordeum vulgare) endosperm (1→3),(1→4)-β-d-glucan (Gibeaut and Carpita, 1993; Buckeridge et al., 1999, 2001; Urbanowicz et al., 2004). When polysaccharides are extracted sequentially from the cell walls by hot ammonium oxalate, and increasing concentrations of NaOH to 4 m, the (1→3),(1→4)-β-d-glucans are found mostly in the higher concentrations of alkali fractions. While 250 kD polymers are observed, most of the (1→3),(1→4)-β-d-glucans eluted in fractions containing glucuronoarabinoxylans (GAXs), which are much larger, indicating either that an aggregation with GAXs increase the apparent size or that trans-glucosylation events increase the degree of polymerization of the (1→3),(1→4)-β-d-glucans.     

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-β-(1→ 3),(1→ 4)-glucanase useful for the preparation of oligosaccharides from barley β-glucan

An endo--(13),(14)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo--(13),(14)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis, transformed with the recombinant plasmid pBLC771, produced an extracellular endo--(13),(14)-glucanase that was 130 times (7176 mU ml^–1) more active than that of the gene donor cells (55 mU ml^–1), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml^–1) more active than that of the gene donor cells. M _r of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble -glucan hydrolyzed by over-produced endo--(13),(14)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.     

Synthesis and stability assay of 4-methylumbelliferyl (1→3)-β-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 3 are described. The (1 → 3)-β-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 → 3)-β-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 → 3)-β-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 → 3)-β-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with β-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

DFTr studies of five- and six-residue cyclic-β(1→4) cellulosic molecules

Density functional (DFT) conformational in vacuo studies of cellobiose have shown that ?_H‐anti conformations are low in energy relative to the syn forms, while the ψ_H‐anti forms are higher in energy. Further, as the cellulosic fragments became larger than a disaccharide and new hydrogen bonding interactions between multiple residues become available, stable low energy ?_H‐anti, and ψ_H‐anti cellulosic structures became possible. To test the stability of cyclic anti‐conformations, a number of β‐linked five‐ and six‐residue molecules were created and then energy optimized in solvent (water, n‐heptane) using the implicit solvation method COSMO at the B3LYP level of theory. The created symmetric cyclic structures were without distortion. Upon optimization some cyclic conformations were found to be of low energy when compared with linear five‐ and six‐residue chains, after correcting the energy for the exclusion of a water molecule upon cyclization. It was also obvious from the hydrogen bonding network formed above and below the plane of the cyclic structure that these structures could exhibit strong synergistic tendencies. The conformational energy preferences for clockwise “c” and counter‐clockwise “r” hydroxyl groups and preference for the hydroxymethyl rotamers is described. Because these structures contain energetically unfavorable flipped conformations in water, that is, dihedral angles of ～180°/0° or ～0°/180° in ?_H/ψ_H, it is clear that the synthesis of these compounds will be challenging. © 2012 Wiley Periodicals, Inc. Biopolymers 97:568–576, 2012.     

Studies of two low-molecular-weight endo-1(1→4)-β-d-xylanases constitutively synthesised by the cellulolytic fungus Trichoderma koningii

Two endo-(1→4)-β-d-xylanases (xylanases 1 and 2), which were constitutively synthesised by the fungus Trichoderma koningii, were purified to homogeneity on gel-filtration media and by isoelectric focusing. They had molecular weights of 29,000 (xylanase 1) and 18,000 (xylanase 2), and isoelectric pHs of 7.24 (xylanase 1) and 7.3 (xylanase 2); neither enzyme was associated with carbohydrate. Xylanase 1 had an optimum at the remarkably high temperature of 60–65°. Each enzyme liberated a different range of oligosaccharides from oat-straw arabinoxylan, but only xylanase 1 released l-arabinose and d-xylose. Both xylanases were free from cellulase activity.     

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (13)--glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (13)--glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (13, 14)--glucan-BSA conjugate. Binding was inhibited by (13)--oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (13)--linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 10⁴M^–1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (13)--glucan in the inner wall layer of thin sections of the N. alata pollen tubes.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline - ELISA enzyme linked immunosorbent assay - DP degree of polymerization - PVC polyvinyl chloride P.J.M. is an Australian Postdoctoral Research Fellow. We wish to thank Joan Hoogenraad for her technical assistance with the tissue culture, and Althea Wright for her assistance in the preparation of this paper.