Purification of endo-(1→3)-β-D-glucanases lysing yeast cell walls from Rhizoctonia solani

Three forms of $\text{endo}\text{-(1→3)-β-}\text{g}\text{-glucanases}$ lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of $\text{endo}\text{-(1→3)-β-}\text{D}\text{-glucanases}$ lysing yeast cell walls.     

A hemicellulosic β-glucan from the hypocotyls of Phaseolus aureus

A glucan of $\text{DP}$_nca 80 has been isolated from the hypocotyls of mung bean plants (Phaseolus aureus). Methylation analysis and periodate oxidation studies showed that the glucan has (1 → 3) and (1 → 4) linked d-glucopyranosyl residues in the molar ratio 1·0:1·7. Oligosaccharides containing both β(1 → 3) and β(1 → 4) linked residues were isolated from partial hydrolysates.     

Steric factors involved in the action of glycosidases and galactose oxidase

α-(1→2)-$\text{L}\text{=}$-Fucosidase, β-$\text{D}\text{=}$-galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β-$\text{D}\text{=}$-Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor $\text{N}$-acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by $\text{α-}\text{N}\text{-acetylgalactosaminidase}$. 3) α-(1→2)-$\text{L}\text{=}$-Fucosidase action is inhibited by $\text{α-(1→3)-}\text{N}\text{-acetylgalactosaminyl}$ or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action.

     

Urinary excretion of a novel hexasaccharide and glycopeptide analogue in fucosidosis.

Repeated Biogel P6 chromatography of the urine from a patient with fucosidosis yielded several fractions containing fucosyloligosaccharides and glycopeptides. Two of these were shown by ¹H nuclear magnetic resonance (¹H-n.m.r.) spectroscopy and permethylation analysis to have the following structures respectively: (I) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) glcNAc and (II) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) βglcNAc (1→4) [αfuc ($\text{1→}\text{3}\text{6}$)] βglcNAc-Asn.     

Structure of the ceramide octadekahexoside isolated from gastric mucosa

A fucose-containingceramide octadekahexoside exhibiting blood-group (A+H) activity has been isolated from hog gastric mucosa. Based on the results of partial acid hydrolysis, sequential degradation with specific glycosidases, oxidation with periodate and chromium trioxide, and permethylation analysis, we propose that the carbohydrate chain of this fucolipid contains four branches. Two of the branches are terminated by βGall→4βGlcNAc, one by $\text{αFucl→2βGall→}\text{3}\text{4}\text{βGlcNAc}$ and one by $\text{αGalNAcl→3(αFucl→2)βGall→}\text{3}\text{4}\text{βGlcNAc}$.     

Changes in composition of harvested mushrooms (Agaricus bisporus)

Post-harvest changes in the biochemical composition of the mushroom were studied. Non-structural polysaccharide was found at levels greater than 10% dry wt in the fresh mushroom. After 4 days storage, the level had decreased to below 5% dry weight. The polysaccharide appeared to contain only glucose residues joined by α-1,4 and α-1,6 linkages. The chitin content of cell walls increased by ca 50% during 4 days storage, while cell wall glucan decreased. There was a large increase in urea content.     

Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall

Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with β(1-3)- or β(1-6)glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to β(1-6)glucan. With this technique, crh1Δ crh2Δ mutants still appeared to contain a substantial percentage of chitin linked to β(1-3)glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a β(1-3)glucan, for β(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1Δ crh2Δ strains is free. Reexamination of the previously used procedure revealed that the β(1-3)glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both β(1-3)- and β(1-6)glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.The fungal cell wall protects the cell against internal turgor pressure and external mechanical injury. To fulfill these functions, it must be endowed with a resilient structure. Presumably, the cell wall strength is largely due to the cross-links that bind together its components, mainly polysaccharides, giving rise to a tightly knit mesh (6, 11-13). Interestingly, the cross-links must be created outside the plasma membrane, because most of the polysaccharides are extruded as they are synthesized at the membrane; therefore, they do not exist inside the cell. This posits a thermodynamic problem, because there are no obvious sources of energy in the periplasmic space. About 20 years ago we proposed that the free energy may come from existing bonds in the polysaccharide chains and that the new cross-links may be originated by transglycosylation, thus creating a new linkage for each one that is broken (5).Ascertaining the mechanism of cross-link formation seemed a worthwhile endeavor, both because of the theoretical implications and because the cell wall is a proven target for antifungal compounds; therefore, more knowledge about its synthesis can be of practical interest. For this type of investigation to proceed, it was necessary to devise some method for the quantitative analysis of cell wall cross-links. We developed such a procedure for the evaluation of the proportion of cell wall chitin that is free or bound to β(1-3)- or β(1-6)glucan (4). In this methodology, chitin was specifically labeled in vivo with [¹⁴C]glucosamine; cell walls were isolated, and their proteins were eliminated by alkali treatment. The insoluble residue was solubilized by carboxymethylation and analyzed by size fractionation chromatography. By treating the cell walls with different glucanases before carboxymethylation and comparing the chromatographic profiles, we were able to determine the amount of chitin bound to the different glucans, as well as the fraction that was free (4). Armed with this procedure, we could now analyze the cell wall of different mutants that appeared to be candidates for cross-links defects. In this way we found that the two putative transglycosylases Crh1p and Crh2p were redundantly required for the formation of the chitin-β(1-6)glucan linkage. A double mutant crh1Δ crh2Δ had no chitin attached to β(1-6)glucan, although it still contained apparently normal amounts of chitin-β(1-3)glucan complex (7). Further work supported the notion that Crh1p and Crh2p function as transglycosylases, transferring portions of chitin chains to glucan (8). This confirmed our earlier hypothesis.With the initial intention of finding easier and faster methods, I devised two novel procedures for cell wall analysis. One is based on the affinity between β(1-3)glucan chains, the other on the conversion of chitin in situ into its deacetylated product, chitosan, followed by extraction of the chitosan with acetic acid before or after treatment with specific glucanases. With a wild-type strain, both procedures gave similar results to those of the carboxymethylation-chromatography technique. However, in the double mutant crh1Δ crh2Δ all of the chitin appeared to be free with both new methods. Further investigation showed that the older procedure led to erroneous results for the double mutant, because of the presence of a small amount of chitinase in the β(1-3)glucanase preparation used. After reconciling the results, I conclude that Crh1p and Crh2p are necessary for the formation of cross-links between chitin and either β(1-6) or β(1-3)glucan.     

An uncommon fucosyl linkage in surface membranes of human neuroblastoma cells

The surface membranes of human neuroblastoma cells contain a fucosyl linkage, defined by using an α-L-fucosidase from almond emulsin specific for the cleavage of Fucα1→3G1cNAc and Fucα1→4G1cNAc. These linkages are not found in significant amounts on the surface of mouse neuroblastoma cells, or human or hamster fibroblasts. The enzyme released fucose from glycoproteins as well as glycopeptides, making it particularly useful for $\text{in}$$\text{vivo}$ studies.     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Cell Walls of Germinating Uredospores: II. Carbohydrate Polymers

Polymeric carbohydrates in ¹⁴C-labeled germ tube and uredospore walls of Uromyces phaseoli var. typica were studied by permethylation and by enzymatic hydrolysis. The native structure of the uredospore wall limited the effectiveness of both techniques with this wall, but evidence for two distinct polysaccharides was obtained. A linear (1→3) glucan, containing minor quantities of (1→6) linkages, may account for most of the glucose in the uredospore wall. A second uredospore polymer was a glucomannan similar to one reported for other rust fungi in that it consisted of approximately equal numbers of β(1→3) and β(1→4) mannosidic linkages with glucose as a minor component at the nonreducing end. Branching, most likely by (1→6) mannose links, was low. In contrast to uredospore wall, considerably more germ tube polysaccharide was accessible to enzymes and to methylation. Methylation studies indicate that (1→3) glucose and mannose bonds occur predominantly. Evidence from hydrolysis with exo- (β)-(1→3) glucanase suggests distinct wall regions of β(1→3) glycan, highly branched by (1→6) bonds, as well as wall regions of a glucomannan with alternating (1→3) glucose and (1→3) mannose residues. Polymer heterogeneity was indicated by differences in the proportions of mannose, glucose, and galactose as reducing end groups in different solubility fractions. In germ tube walls, but not in uredospore walls, glucosamine apparently existed as part of chitin polymer as evidenced by the isolation of N,N-diacetylchitobiose from chitinase digestion.     

Bis(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)amine obtained by a fusion reaction

A unique, alkali-soluble polysaccharide has been isolated from the cell walls of the basidiomycete Coprinus macrorhizus microsporus. The polysaccharide, which is primarily a glucan, contains a large proportion of α-(1→4)-linked d-glucose residues and a smaller amount of β-(1→3) and (1→6) linkages, as suggested by methylation, partial acid hydrolysis, periodate oxidation, and enzymic studies. Hydrolysis of the methylated polysaccharide gave equimolar amounts of 2,4-di- and 2,3-di-O-methyl-d-glucose; no 2,6-di-O-methyl-d-glucose was identified, indicating the absence of branch points joined through O-1, O-3, and O-4. The isolation and identification of 2-O-α- glucopyranosylerythritol from the periodate-oxidized polysaccharide suggests that segments of the a-(1→4)-linked d-glucose residues are joined by single (1→3)-linkages. An extracellular enzyme-preparation from Sporotrichum dimorphosporum (QM 806) containing both β-(1→3)- and α-(1→4)-d-glucanohydrolase activity released 76% of the reducing groups from the polysaccharide. The polysaccharide also contains minor proportions of xylose, mannose, 2-amino-2-deoxyglucose, and amino acids.     

Biosynthesis of glycerol teichoic acid in Bacillus cereus: formation of linkage unit disaccharide on a lipid intermediate

A tunicamycin-like antibiotic 24010 at a concentration of 1 μg/ml selectively inhibited the $\text{in vivo}$ synthesis of glycerol teichoic acid of cell walls in $\text{Bacillus cereus}$ AHU 1030. Incubation of membranes of this strain with $\text{N}$-acetylglucosaminyl pyrophosphorylundecaprenol and UDP-$\text{N}$-acetylmannosamine led to formation of a glycolipid having a saccharide moiety identical with the cell wall teichoic acid linkage unit, $\text{N}$-acetylmannosaminylβ(1→4)-$\text{N}$-acetylglucosamine. The membranes also catalyzed transfer of glycerol phosphate units from CDP-glycerol to this disaccharide-linked lipid. Thus the biosynthesis of the cell wall glycerol teichoic acid in this strain seems to involve the disaccharide-linked lipid as an intermediate.     

Structure of the water-insoluble α-d-glucan of streptococcus salivarius HHT

Water-insoluble, non-adherent α-d-glucans have been obtained from Streptococcus salivarius HHT under two sets of conditions: from a growing culture, or synthesized enzymically by using a glucosyltransferase. In the former case, the glucan ([α]_d + 197°) was shown by methylation analysis to have a slightly branched structure containing a relatively high proportion (80 %) of (1→3)-α-d-glucosidic linkages, together with small proportions of (1→6)- and (1→4)-α-d-glucosidic linkages. The enzymically synthesized glucan had a much less-branched structure, containing 88 % of (1→3)-α-d-glucosidic linkages. Both glucans, on Smith degradation (sequential periodate oxidation, borohydride reduction, and mild acid hydrolysis), gave linear, (1→3)-α-d-glucosidic polysaccharides (yields, 82-90%) that constitute the backbone chains. The presence of small proportions of glycerol, erythritol, 1-O-α-d-glucosyl-d-glycerol, and also 2-O-α-d-glucosyl-d-erythritol in the products of Smith degradation suggests that the short side-chains are attached to the backbone chain by (1→4)-, (1→6)-, and (1→3)-α-d-glucosidic linkages     

Heterozygosity for the IVS-I-5(G→C) mutation with a G→A change at codon 18 (Val→Met; Hb Baden) in cis and a T→G mutation at codon 126 (Val→Gly; Hb Djonburi) in trans resulting in a thalassemia intermedia

We have analyzed the hemoglobins of a young German patient with β-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the β-globin through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the $\text{G}\text{T}\text{G}\text{→}\text{G}\text{G}\text{G}$ mutation at codon 126 leading to the synthesis of Hb Dhoburi or α₂β₂126(H₄)Val→Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G→C) mutation that leads to a rather severe β⁺-thalassemia and the $\text{G}\text{TG}\text{→}\text{A}\text{TG}$ mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered β-chain variant, named Hb Baden, was present for only 2–3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G→C) thalassemic mutation. The chromosome with the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) and the IVS-I-5 (G→C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G→C) mutation living in different countries failed to detect the codon 18 ($\text{G}\text{TG}\text{→}\text{A}\text{TG}$) change.     

Molecular and crystal structure of the regenerated form of (1→3)-α-d-glucan

The crystal structure of a regenerated form of (1→3)-α-d-glucan, obtained by solid state deacetylation of the triacetate derivative, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. The structure crystallizes in an orthorhombic unit cell with parameters $\text{a = 16.46}\text{A}\text{?}\text{, b = 9.55}\text{A}\text{?}$ and c (fibre repeat)=8.44 Å, and space group P2₁2₁2₁. The chain conformation is nearly completely extended and is very close to a 2/1 helix, even though the dimer residue is the crystallographic repeat unit. An intramolecular O(2)  O(4)′ hydrogen bond stabilizes the conformation and extensive intermolecular hydrogen-bonding abilizes the packing. The resulting structure is sheet-like, with an alternating polarity of chain directions within the sheet. In its sheet-like character, extensive hydrogen-bonding, and insolubility in water, this polymorph of (1→3)-α-d-glucan resembles regenerated cellulose. The reliability of the structure analysis is indicated by the X-ray residual R=0.206.     

Total hemicelluloses from Hordeum vulgare plants at different stages of maturity

The changes in the composition of the total hemicelluloses of leaf and stem tissues of field-grown barley plants have been examined at different stages of maturation. In each plant the proportion of xylose residues in the total hemicellulose increases with tissue maturity, that of galactose varies little, and the proportions of arabinose, glucose and uronic acid residues decrease. The ratio of β(1 → 3) to β(1 → 4) linkages in the β-glucans decreases with tissue maturity and there is a decrease in the $\text{DP}$_n of these β-glucans.     

Isolation and partial characterization of a novel disulfoglycosphingolipid from rat kidney

A novel sulfoglycosphingolipid containing two sulfate ester groups was isolated from the lipid extract of rat kidney by a procedure involving mild alkaline methanolysis and column chromatographies on DEAE-Sephacel and silicic acid. The component carbohydrates were galactose, glucose and $\text{N}$-acetylgalactosamine in equimolar amounts. Infrared spectroscopy, permethylation study, periodate oxidation and solvolysis suggested that the sulfoglycolipid was GalNAc1-4Gal1-4GlcCer sulfated at the C3 hydroxyls of both galactose and $\text{N}$-acetylgalactosamine. The yield of this sulfoglycolipid was 11.2 nmol/g tissue.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma capsulatum

Chemical and ultrastructural studies of the cell walls of the yeastlike (Y) and mycelial (M) forms ofHistoplasma capsulatum G-184B revealed that the Y form contained about 46.5% ofα-glucan, 31.0% ofβ-glucan, 7.7% of galactomannan and 11.5% of chitin, whereas the M form cell wall contained about 18.8% ofβ-glucan, 24.7% of galactomannan, 25.8% of chitin, and essentially noα-glucan. Theα-glucan of the Y form contained mainly anα-(1 → 3)-linkage. Theβ-glucans of both forms may have mainly aβ-(1 → 3)-linkage. Chitin microfibrils were located mainly in the inner portion of the cell walls of the Y and M forms, whereas theα-glucan fibers were observed only in the outer portion of the Y form cell wall.     

A hemicellulosic β-D-glucan from the endosperm of sorghum grain

A hemicellulosic β-D-glucan of $\text{d.p.}$ ≈26 has been isolated from the endosperm of sorghum grain. Methylation analysis, partial hydrolysis with acid, and periodateoxidation studies showed that the glucan is linear and has both (1 → 3)- and (1 → 4)-linked D-glucopyranose residues in the ratio of 3:2. The low, positive, specific rotation and chromium trioxide oxidation studies indicated that the D-glucose residues are β-linked.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [¹⁴C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is $\text{Gal}\text{β1 → 3([}{}^{14}\text{C}\text{]}\text{Gal}\text{β1 → 4}\text{GlcNac}\text{β1 → 6)}\text{GalNAcol}$. Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.