Mannan Backbone Research Articles

Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase.

The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ((14)C)Gal from UDP-((14)C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1-->6)-alpha, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.

Application of a mannan-specific antibody for the detection of galactomannans in foods

An immunoassay is described to detect galactomannans in foods. The assay uses a monoclonal antibody that specifically binds the (1→4)-β-linked mannan backbone of galactomannans. Carob and guar galactomannan additives in commercial food samples can be detected at concentrations as low as 5 ng/ml without prior sample preparation other than dilution.

Relation between the molecular structure of some polysaccharides and original properties in sol and gel states

This paper concerns the properties of a few natural polysaccharides investigated in our laboratory. We also present the different techniques used in our group for a long time and their advantages to characterize polysaccharides. In a first part, the behavior of galactomannans with different chemical structures is discussed; the distribution of galactose units along the mannan backbone is very important. Then, xanthan is developed as one of the best thickening polymers due to its high intrinsic stiffness related to the helical conformation stabilized in the presence of excess salt. Gellan under native or deacylated structure is described to demonstrate the role of substituents on the physical properties in solution and especially on the sol–gel transition. The last part concerns the cooperative interaction between xanthan and galactomannan giving in specific conditions two different gels with a gel–sol melting transition around 25°C and 50–60°C, respectively, depending on the galactomannan microstructure.

Structural studies of the antigen III cell wall polysaccharide of Trichosporon domesticum

Cell wall and soluble polysaccharides that reacted with Trichosporon domesticum factor III serum were isolated from the type strain of T. domesticum. The fractions contained O-acetyl groups, which contributed to the serological reactivity. The antigenic structure was characterized by chromatographic and spectroscopic methods. The polysaccharide has an α-(1→3)- d-mannan backbone with hetero-oligosaccharide side chains consisting of a 2- O-substituted β- d-glucuronic acid residue bound to O-2 of the mannose residue, β- d-xylopyranosyl residues located in the middle of the side chain, and a nonreducing terminal α- l-arabinopyranosyl residue bound to O-4 of xylose. The mannan backbone is O-acetylated at O-6 of the mannose residues.

In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with a α-galactosidase gene cloned from senna

Galactomannans are composed of a β 1to4 mannan backbone with varying degrees of α 1to6 galactose substitutions and are found in the cell walls of legume endosperm. Like other cell wall polysaccharides, many factors controlling the metabolism of galactomannans remain to be elucidated. In the endospermous legume senna (Senna occidentalis) increased α-galactosidase activity has previously been observed to coincide temporarily with a decrease of the galactose content of the galactomannan. To evaluate the potential role of α-galactosidase for the control of the final galactose content, a α-galactosidase gene expressed in immature senna seeds was cloned and transformed into the related high-yielding species guar (Cyamopsis tetragonoloba). The isolated cDNA encoded a 406 amino acid protein with a calculated molecular mass of 44 313 Da. The amino acid sequence was 75% identical to the galactomannan hydrolysing α-galactosidases from germinating guar and coffee bean. The senna α-galactosidase gene was inserted behind a wheat high-molecular-weight glutenin promoter in the vector employed for transformation of guar by Agrobacterium tumefaciens-mediated gene transfer. About 30% of the guar transformants produced endosperm with galactomannans where the galactose content was significantly reduced. After self-fertilization of primary transformants displaying the highest galactose reduction of the galactomannan, endosperms of R1 plants were analysed demonstrating that this property was inherited stably to the progeny and that it was 100% coupled to the presence of the senna α-galactosidase gene. This suggests that α-galactosidases can be involved in the determination of the final galactose content of endosperm galactomannans, demonstrating that cell wall polysaccharide biosynthesis can be modified in vivo.

N-Glycosylation of yeast, with emphasis onCandida albicans

Fungal cell wall N-linked glycans have been studied most extensively in Saccharomyces cerevisiae and in Candida albicans. The glycans are located on the fungal cell surface in the form of phosphomannoprotein complexes and the amount of glycosylation is influenced both by genetics and environmental factors. The glycans, which are comprised mostly of mannan, are important in fungal-host interactions, as they make first contact with the immune system. Initial N-linked glycosylation events take place in the endoplasmic reticulum and are conserved throughout all eukaryotes, but yeasts are capable of additional glycosylation that may result in a glycan comprised of more than 200 mannose units. In C. albicans, the glycan can be delineated into an inner mannan core, which is similar to mammalian glycoproteins, an alpha-linked mannan backbone with alpha-oligomannosyl side chains, and beta(1,2)-oligomannosides which are phosphodiester linked to the alpha-mannan. Both the beta-oligomannosides, which make up the acid-labile part of the phosphomannan complex, and alpha-oligomannosides, which make up the acid-stable part of the complex, serve as adhesins in the attachment of C. albicans yeast cells to host splenic and lymph node macrophages. The beta-oligomannosides can induce release of tumour necrosis factor (TNF)-alpha, and antibodies specific to certain beta-oligomannosides enhance host resistance to various forms of candidiasis. The importance of the N-linked glycans in fungal-host interactions provides rationale for further studies, which may well lead to effective immunotherapeutic strategies for prevention and, possibly, treatment of disease.

Extracellular Tremella polysaccharides : structure, properties and applications

Tremella sp. is a yeast-like fungus from which polysaccharides can be isolated from either the yeast or the fungus phase, depending on the species. All of the polymers synthesized by these organisms consist of a mannan backbone to which small xylose side chains and glucuronic acid are attached. The main interest in these glucuronoxylomannans is their application in medicine to enhance the immune system.

Diffusion of Macromolecules in Polymer Solutions and Gels: A Laser Scanning Confocal Microscopy Study

Laser scanning confocal microscopy combined with fluorescence recovery after photobleaching is an effective tool to measure the diffusion coefficients of macromolecules in cross-linked hydrogels and polymer solutions. In this study, the effects of enzyme treatment on the diffusion of macromolecules (FITC-dextran) in guar solutions and titanium-guar hydrogels are examined. Enzyme treatment with β-mannanase, a polymer backbone cleaving enzyme, quickly increases the diffusion coefficient of the probe molecules in both solutions and hydrogels to that in water. Enzyme treatment of guar solutions and hydrogels with α-galactosidase, a side chain cleaving enzyme, displays a unique behavior due to changes in the fine structure of guar. The removal of galactose branches from the mannan backbone of guar creates additional hyperentanglements (i.e., cross-links), which reduce the water holding capacity of guar and induce syneresis. If the depth at which the diffusion coefficient is measured remains constant, a minimum is observed in the diffusion coefficient as α-galactosidase enzyme treatment time increases. At the site of measurement, the sample changes from a homogeneous guar system to a phase-separated polymer-rich hydrogel and finally to a dilute polymer phase as the polymer-rich hydrogel phase precipitates below the site of measurement. The diffusion coefficient in the dilute polymer phase increases to that in water, while the diffusion coefficient in the hydrogel phase continues to decrease to a value of approximately 6 × 10-8 cm2/s.

Pathogenesis and treatment of hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a granulomatous interstitial lung disease resulting from an immunologic reaction to the repeated inhalation of organic dusts and active chemicals. There are 50 or more groups of HP, but the prevalence varies from country to country, and even within a country, depending on a variety of occupational or environmental inhalants. In Western coutries farmer's lung, bird fancier's disease, humidifier lung, and air-conditioner disease are common, but in Japan summer-type HP is the most prevalent group. Summer-type HP is a house-related illnes induced by Trichosporon asahii and Trichosporon mucoides which contaminate the patients' home environments in hot and humid conditions. The polysaccharide antigen contains mannan backbone attached with short side chains consi-sting of mannose, xylose, and glucuronic acid residues. Both immune complex-mediated and T cell-mediated immune responses to the yeast are involved in the induction and development of the disease. Host factors such as HLA-DQw3 and cigarette smoking also play an important role in the develop-ment or suppression of the disease. An assay for serum anti-Trichosporon antibody by a Triko Kit is very useful for the serodiagnosis, and sanitization by cleaning, disinfecting, and removing from the colonizing location of Trichosporon prevents recurrence of the disease. Summer-type HP induced by Trichosporon is a new type of HP. It can be found in other countries including most Western countries, because Trichosporon asahii and Trichosporon mucoides distribute in the temperate and subtropical areas of the world.

The aman6 gene encoding a yeast mannan backbone degrading 1,6-alpha-D-mannanase in Bacillus circulans: cloning, sequence analysis, and expression.

A gene (aman6) encoding endo-1,6-alpha-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1,767 base pairs long and encoded a mature 1,6-alpha-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-alpha-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-alpha-D-mannanase from Bacillus circulans TN-31.

Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis.

Galactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP-mannose dependent (1-->4)-beta-D-mannan synthase and a UDP-galactose dependent (1-->6)-alpha-D-galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta-(1-->4)-linked D-manno-oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose-substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan alpha-galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane-spanning helix, was expressed.

The Saccharomyces cerevisiae Protein Mnn10p/Bed1p Is a Subunit of a Golgi Mannosyltransferase Complex

In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex.

Interactions between locust bean gum and cellulose characterized by 13C n.m.r. spectroscopy

Molecular interactions between locust bean gum (LBG) and cellulose crystallite surfaces appear to involve most mannosyl residues of the mannan backbone, not just the small proportion contained in long segments which lack galactosyl residues. This conclusion is based on: (1) relative strengths of 13C n.m.r. signals at 102.2 ppm in the cross-polarization (CP) spectrum and 101.3 ppm in the single-pulse excitation (SPE) spectrum, assigned to mannosyl C-1 in bound and non-bound segments of LBG, respectively; (2) displacement of a 13C n.m.r. signal assigned to mannosyl C-4 to 81 ppm, indicating a change of conformation in the mannan backbone relative to a gel phase; (3) similarities between proton spin relaxation time constants for cellulose and LBG, indicating proton spin diffusion between polymers in close contact; (4) broadening of CP n.m.r. signals at 83.9 and 85.0 ppm, assigned to C-4 of cellulose chains exposed on crystallite surfaces. The galactosyl C-1 signal appears at 99.9 ppm in the SPE n.m.r. spectrum of the LBG-cellulose complex but shows a poor response to CP n.m.r., indicating that galactosyl residues are not fixed in rigid conformations relative to the mannan backbones.

Morphology of galactomannans: an X-ray structure analysis of guaran

The three-dimensional structure of a galactomannan has been determined by careful analysis of the X-ray data obtained from polycrystalline and well oriented fibers of guaran. The polymer forms a flat 2-fold helix of pitch 10.38 Å. The galactosyl side-chain hydrogen bonds to the mannan backbone intramolecularly and provides structural stability. Four helices are packed in an orthorhombic unit cell of dimensions a=9.26, b=30.84 and c (fiber axis)=10.38 Å according to space group symmetry I222. Each chemical repeat consisting of 1.0 mannosyl and 0.6 galactosyl units is associated with two structured water molecules which are responsible for the formation and stability of sheets of guaran helices in the crystalline lattice. The crystallographic R-value is 0.185 for the best model. The guaran molecular structure and packing arrangement presented here are readily adoptable to the entire galactomannan family.

NMR assignment of the galactomannan of Candida lipolytica

The chemical structure of the cell wall galactomannan of Candida lipolytica was analyzed using two-dimensional NMR techniques without chemical fragmentation. The H-1-H-2-correlated cross-peaks of the galactomannan indicated that it consists of an α-1,6-linked mannan backbone moiety with side chains. A sequential NMR assignment of the side chains through nuclear Overhauser effect (NOE) cross-peaks indicated that the triose side chain contains an α-1,2-linked galactopyranose unit at the non-reducing terminal. The structure was significantly different from the galactomannan of Trichophyton. The molar ratio of the side chains calculated from the H-1 signal dimensions indicated that ca. 45% of the backbone α-1,6-linked mannose units are not substituted with side chains and are responsible for the reactivity of the galactomannan with factor 9 serum.

Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida

We investigated the antigenic determinants of factors 8, 9, and 34 of the genus Candida among pathogenic yeasts by enzyme-linked immunosorbent assay (ELISA) using mannans of Saccharomyces cerevisiae wild type and mutant types, mnn 1- mnn 4 and mnn 2. Results of ELISA including antisera against the antigenic factors of genus Candida (Candida Check, Iatron; FAbs) indicated that these three types of mannan distinctly react with FAbs 34, 8 and 9, respectively. To identify the recognition sites of these FAbs, we compared the ability of various oligosaccharides to inhibit the binding of the mannans to FAbs. The results indicated that FAb 34 preferentially recognizes linear side chains containing a non-reducing terminal α-1,3-linked mannose residue, Man α1 → 3Man α1 → (2Man α1 →) n 2Man ( n ≥ 0), and that one of the recognition sites of FAb 9 is linear α-1,6-linked oligomannosyl series, Man α1 → (6Man α1 →) n 6-Man ( n ≥ 2). On the other hand, the recognition site of FAb 8 apparently consisted of two α-1,2-linked oligomannosyl side chains and an α-1,6-linked mannose residue that originated from the mannan backbone, Man α1 → 2Man α1 → 2(Man α1 → 2Man α1 → 6)Man.

Characterisation of acidic heteroglycans from Tremella fuciformis Berk with cytokine stimulating activity

Four kinds of acidic heteroglycan, T3a-T3d, were isolated from the body of the fungus Tremella fuciformis Berk. The molecular weights of T3a-T3d were 550, 420, 55, and 48 kDa. respectively. Glycosidic linkage analysis showed that they had a mannan backbone consisting of 3-linked Man p, and side chains containing glucosyl, mannosyl, fucosyl, xylosyl, and glucuronic acid residues attached through O-2, O-4, or O-6 of about half of the backbone mannosyl residues. A partial acidic hydrolysate of T3a could be divided into a low-branching region (T3a-1, 2, 3, 4) mainly branched at the 2-position of 3-linked Man p in the mannan backbone, and a high-branching region (T3a-5A) branched at the 2,4- or 2,4,6-positions of 3-linked Man p in the backbone. The low-branching region, which is predominant in the backbone, was substituted with non-reducing terminal Glc pA, Fuc p and short side chains consisting of (1 → 6)-linked Glc p and (1 → 2)-linked Man p linked through C-2 of the mannan backbone. The high-branching region, which is a minor component of the backbone, was linked with long side chains of (1 → 6)-linked Glc p and (1 → 4)-linked Glc pA in their branching points. T3a-T3d were able to induce human monocytes to produce interleukin-1, interleukin-6, and tumor necrosis factor in vitro. The different fragments of the acidic hydrolysate of T3a (T3a-1, 2, 3, 4, 5A) also induced monocytes to secret interleukin-6 with high potency, indicating that the activity may be caused by a common structure (1 → 3)-mannan in the four heteroglycans and their fragments. The change of molecular weight had no obvious influence on the activity of the heteroglycans.

Structural Analysis of the Mannan Region of Lipoarabinomannan from Mycobacterium bovis BCG.: HETEROGENEITY IN PHOSPHORYLATION STATE

Lipoarabinomannan (LAM) is a major antigen of mycobacterial cell walls, involved in host-Mycobacterium interactions. In a previous work, LAM from the vaccine strain, Mycobacterium bovis BCG, was found to exhibit mannooligosaccharides at its arabinan nonreducing ends (ManLAM). The present report concerns the mannan core structure of this ManLAM. After partial hydrolysis of ManLAM, two populations of mannans (Ma1 and Ma2) were obtained by gel filtration chromatography. Their structural features were defined by means of two-dimensional homo- and heteronuclear (1H-13C) NMR sequences and methylation analysis. They were both found to be composed of an alpha-(1-->6)-linked mannan backbone with alpha-(1-->2)-Manp-linked side chains. They are highly branched, and Ma2 presents a higher frequency of branching than Ma1. Moreover, chemical analysis indicates that only Ma1 is phosphorylated. By a two-dimensional heteronuclear 1H-31P total correlation experiment, the phosphate was found to be involved in a phosphodiester bond between inositol C-1 and glycerol C-3. Then, the molecular mass of mannan was established by mass spectrometry, which revealed a molecular mass of 3517 Da for the major molecular species of Ma1. Likewise, analysis of unfractionated mannans showed the occurrence of other, quantitatively minor molecular species, endowed with two phosphates. This study clearly indicates that the mannan region of M. bovis BCG ManLAM exists as a heterogeneous population of molecules whose structures differ in their degree of glycosylation, level of branching, and phosphorylation state. The hypothesis that the relative abundance of these different molecules modulates the biological functions of LAM is discussed.

Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.

Purification and characterization of the serotype-specific polysaccharide antigen of Trichosporon cutaneum serotype II: a disease-related antigen of Japanese summer-type hypersensitivity pneumonitis.

Summer-type hypersensitivity pneumonitis (SHP) is a unique type of hypersensitivity pneumonitis and the most prevalent in Japan. Our previous study clarified that the causative agent of the disease is Trichosporon cutaneum, and that the patients with SHP have high titres of antibodies against the serotype-specific antigen of polysaccharide nature which exist in the high molecular weight fraction of the culture supernatant of the yeast. In this study, we purified the serotype-specific antigen of serotype II T. cutaneum by gel filtration and affinity chromatography using a monoclonal antibody, D-8, specific for a high molecular weight antigen of serotype II T. cutaneum, and elucidated the structure of the antigen. This affinity-purified antigen was shown to be an essentially acidic polysaccharide comprising mannose, xylose, and glucuronic acid (6:44:4.7). Chemical analysis showed that this polysaccharide antigen contains a (1-3)-linked mannan backbone attached with short side chains of (1-4)-linked mannose and a small proportion of (1-2)-linked xylose residues by substituting the 2- or 4-positions of the (1-3)-linked mannose residues of the main chain. Approximately one-fifth of the side chains were terminated with glucuronic acid residues. The antigenic epitope of the serotype-specific antigen was shown to involve the terminal glucoronic acid residues as revealed by immunodiffusion test and sandwich enzyme-linked immunosorbent assay using monoclonal antibody D-8.