Mannose-containing Polysaccharides Research Articles

Dectin-2-mediated initiation of immune responses caused by influenza virus hemagglutinin.

Antigen-presenting cells express pattern recognition receptors (PRRs), which sense pathogen-associated molecular patterns from microorganisms and lead to the induction of inflammatory responses. C-type lectin receptors (CLRs), the representative PRRs, bind to microbial polysaccharides, among which Dectin-2 and Mincle recognize mannose-containing polysaccharides. Because influenza virus (IFV) hemagglutinin (HA) is rich in mannose polysaccharides, Dectin-2 or Mincle may contribute to the recognition of HA. In this study, we addressed the possible involvement of Dectin-2 and Mincle in the viral recognition and the initiation of cytokine production. Interleukin (IL)-12p40 and IL-6 production by bone marrow-derived dendritic cells (BM-DCs) upon stimulation with HA was significantly reduced in Dectin-2 knockout (KO) mice compared to wild-type (WT) mice whereas there was no difference between WT mice and Mincle KO mice. BM-DCs that were treated with Syk inhibitor resulted in a significant reduction of cytokine production upon stimulation with HA. The treatment of BM-DCs with methyl-α-D-mannopyranoside (ManP) also led to a significant reduction in cytokine production by BM-DCs that were stimulated with HA, except for the A/H1N1pdm09 subtype. IL-12p40 and IL-6 synthesis by BM-DCs was completely diminished upon stimulation with HA treated with concanavalin A (ConA)-bound sepharose beads. Finally, GFP expression was detected in reporter cells that were transfected with the Dectin-2 gene, but not with the Mincle gene, when stimulated with HA derived from the A/H3N2 subtype. These data suggested that Dectin-2 may be a key molecule as the sensor for IFV to initiate the immune response and regulate the pathogenesis of IFV infection.

Optimization of medium composition for production of chitin-glucan complex and mannose-containing polysaccharides by the yeast Komagataella pastoris

Komagataella pastoris was recently proposed as a source of valuable polysaccharides, namely, the co-polymer chitin-glucan complex (CGC) and mannose-containing polymers (mannans), that are extracted from its cell-wall. In this study, a novel cultivation medium, Medium K, was developed envisaging the simultaneous production of both types of cell-wall polysaccharides. The use of Medium K for the cultivation of K. pastoris resulted in high contents of CGC (19 wt%) and mannans (21 wt%) in the biomass, corresponding to significantly higher products’ volumetric productivities (17.5 and 19.2 g/L day, respectively) compared to previous studies. The produced CGC had a chitin:β-glucan molar ratio of 12:88, similarly to previously reported values for K. pastoris CGC (11:89–19:81), while the mannans were mainly composed of mannose units, with a protein content of 10 wt%. These results demonstrated that the developed optimized medium was suitable for cultivation of K. pastoris and the production of both CGC and mannans. It comprised fewer components with considerably reduced salts content, thus representing a significant simplification of the bioprocess with no precipitation problems, without impacting on the polymers’ composition.

Mixed alcoholic fermentation of Schizosaccharomyces pombe and Lachancea thermotolerans and its influence on mannose-containing polysaccharides wine Composition

This study researched the winemaking performance of new biotechnology involving the cooperation of Lachancea and Schizosaccharomyces genera in the production of wine. In all fermentations where Lachancea thermotolerans was involved, higher lactic acid concentrations appeared, while all fermentations where Schizosaccharomyces pombe was involved, lower levels in malic acid concentration took place. The sensorial properties of the final wines varied accordingly. Differences in mouthfeel properties and acidity occurred in the different fermentation trials. Fermentations with the highest concentration of hydrolyzed mannose showed the highest mouthfeel properties, but the lack of acidity reduced their overall impression. Wines made from a combination of L. thermotolerans and S. pombe showed the highest overall impression and were preferred by the tasters due to the balance between mouthfeel properties and acidity.

The GDP-mannose transporter gene (DoGMT) from Dendrobium officinale is critical for mannan biosynthesis in plant growth and development

Dendrobium officinale is a precious traditional Chinese medicinal herb because it is abundant in mannose-containing polysaccharides (MCPs). GDP-mannose transporter (GMT), which translocates GDP-mannose into the Golgi lumen, is indispensable for the biosynthesis of MCPs. In this study, we found that the dominant polysaccharides in D. officinale were MCPs in a range of varieties and different physiological phases. After a positive correlation between the accumulation of mannose and the transcript levels of candidate GMT genes was found, three GMT genes (DoGMT1-3) were identified in D. officinale. DoGMT1, DoGMT2 and DoGMT3 exhibited the highest transcript level in stem that an organ for MCPs storage. All three DoGMT proteins were targeted to Golgi apparatus, and had a GDP binding domain (GXL/VNK) that was homologous to a specially characterized GMT protein GONST1 in Arabidopsis thaliana. Moreover, DoGMT1, DoGMT2 and DoGMT3 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), meanwhile they also demonstrated a higher GDP-mannose uptake activity. Therefore, we conclude that DoGMT1, DoGMT2 and DoGMT3 are able to transport GDP-mannose while the expression patterns of these genes correspond to the accumulation of MCPs in D. officinale. These findings support the importance of GMT genes from D. officinale in the biosynthesis of MCPs.

DoGMP1 from Dendrobium officinale contributes to mannose content of water-soluble polysaccharides and plays a role in salt stress response

GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth.

Loops Adjacent to Catalytic Region and Molecular Stability of Man1312.

Hemicelluloses are the second major polysaccharides in nature and can be converted to ethanol product by a variety of enzymes including mannanases. Mannanase is an important enzyme that hydrolyses mannose-containing polysaccharides which are abundant in plants. An optimized mannanase could help to improve conversion process and make the technology efficiently and competitively. In this work, the effects of loops adjacent to active region on enzymic properties of Man1312 were investigated. Loop 6 and 10 are two loops neighboring to Man1312 catalytic region, and deletion mutagenesis and residue substitution were performed on both loops. Deletion on sites S145, Q148, N244, and S255 and substitution on sites N146, S147, S156, and T157 gave significant increased stability to enzyme. The quadruplet mutant ManD4I4 combined all the mutations and had higher optimal temperature and T m value by 5 and 4°C than Man1312, respectively. From our data, we are able to conclude the loops of enzymes are important to design mutagenesis and obtain improved properties, especially the loops neighboring to catalytic region from tertiary structure. In our experiment, residue deletion and substitution on loops neighboring to catalytic region made significant improvement on enzyme properties.

Insights into the structure and function of fungal β-mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme.

Hemicellulose is an important part of the plant cell wall biomass, and is relevant to cellulosic ethanol technologies. β-Mannosidases are enzymes capable of cleaving nonreducing residues of β-d-mannose from β-d-mannosides and hemicellulose mannose-containing polysaccharides, such as mannans and galactomannans. β-Mannosidases are distributed between glycoside hydrolase (GH) families 1, 2, and 5, and only a handful of the enzymes have been structurally characterized to date. The only published X-ray structure of a GH family 2 mannosidase is that of the bacterial Bacteroides thetaiotaomicron enzyme. No structures of eukaryotic mannosidases of this family are currently available. To fill this gap, we set out to solve the structure of Trichoderma harzianum GH family 2 β-mannosidase and to refine it to 1.9-Å resolution. Structural comparisons of the T. harzianum GH2 β-mannosidase highlight similarities in its structural architecture with other members of GH family 2, reveal the molecular mechanism of β-mannoside binding and recognition, and shed light on its putative galactomannan-binding site. Coordinates and observed structure factor amplitudes have been deposited with the Protein Data Bank (4CVU and 4UOJ). The T. harzianum β-mannosidase 2A nucleotide sequence has GenBank accession number BankIt1712036 GeneMark.hmm KJ624918.

Three sulphated polysaccharides isolated from the mucilage of mud snail, Bullacta exarata philippi: Characterization and antitumour activity

Three sulphated polysaccharides, coded as BEMPA, BEMPB1, BEMPB2, were extracted from the mucilage of mud snail of Bullacta exarata and purified by DEAE-cellulose ion-exchange and size-exclusion chromatography. Structural analysis of purified polysaccharides by chemical and biochemical methods revealed BEMPA was a high (1→3,4)-linked mannose-containing polysaccharide with molecular weight of 22,977Da. BEMPB1, with molecular weight of 64,117Da, was a high (1→3)-linked arabinose-containing polysaccharide. BEMPB2 was mainly composed of (1→3,4)-linked mannose with molecular weight of 47,507Da. The comparison between sulphated polysaccharides and their desulphated products showed that sulphate substitutions of BEMPB1 were deduced to be at the C-3 of (1→4)-linked mannose, while sulphate substitutions of BEMPA and BEMPB2 were at C-4 of (1→3)-linked mannose. Furthermore, BEMPA exhibited highest inhibitory effects on growth of B-16 melanoma cells, and IC50 were 31.1μg/mL.

Role of Dectin-2 in the host defense against Streptococcus pneumoniae infection (45.8)

Abstract Streptococcus pneumoniae possesses a thick polysaccharide capsule. Host defense to this bacterium is mediated by neutrophilic inflammatory responses, which is critically regulated by innate immune mechanism. S. pneumoniae has been reported to be sensed by pattern-recognition receptors such as TLR2, TLR4 and SIGN-R1. In this study, we examined the role of Dectin-2 in the host defense to S. pneumoniae infection. When bone marrow-derived dendritic cells from wild-type (WT) and Dectin-2KO mice were stimulated with culture supernatants (Sup) of a strain of S. pneumoniae (URF918) or its ConA-bound fraction, IL-12p40 production was abrogated in Dectin-2KO mice. In a NFAT-GFP reporter assay, Sup provided a positive signal in Dectin-2 gene-transfected reporter cells. These results suggest that Dectin-2 senses mannose-containing polysaccharides in S. pneumoniae capsule. Dectin-2KO mice infected intratracheally with S. pneumoniae showed shorter survival and larger bacterial burden and lower IFN-γ production in lungs than WT mice. Although the neutrophilic infiltration in lungs was equivalent in both mice, the ratio of engulfed/un-engulfed neutrophils was lower in KO mice than in WT mice. The levels of anti-pneumococcal polysaccharide Ab in bronchoalveolar lavage fluid and serum were lower in KO mice than WT mice. These results suggest that Dectin-2 plays critical roles in the host defense to S. pneumoniae infection through promoting the Ab-mediated phagocytosis by neutrophils.

Comparison of Cell Wall Polysaccharide Hydrolysis by a Dilute Acid/Enzymatic Saccharification Process and Rumen Microorganisms

Evaluation of biomass crops for breeding or pricing purposes requires an assay that predicts performance in the bioenergy conversion process. Cell wall polysaccharide hydrolysis was compared for a dilute sulfuric acid pretreatment at 121°C followed with cellulase hydrolysis for 72 h conversion assay (CONV) with in vitro rumen microflora incubation for 72 h (RUMEN) for a set of maize (Zea mays L.) stover samples with a wide range in cell wall composition. Residual polysaccharides from the assays were analyzed for sugar components and extent of hydrolysis calculated. Cell wall polysaccharide hydrolysis was different for all sugar components between the CONV and RUMEN assays. The CONV assay hydrolyzed xylose-, arabinose-, galactose-, and uronic acid-containing polysaccharides to a greater degree than did the RUMEN assay, whereas the RUMEN assay was more effective at hydrolyzing glucose- and mannose-containing polysaccharides. Greater hydrolysis of hemicelluloses and pectins by CONV can be attributed to the acid hydrolysis mechanism of the CONV assay for noncellulosic polysaccharides, whereas the RUMEN assay was dependent on enzymatic hydrolysis. While CONV and RUMEN hydrolysis were correlated for most polysaccharide components, the greatest correlation was only r = 0.70 for glucose-containing polysaccharides. Linear correlations and multiple regressions indicated that polysaccharide hydrolysis by the RUMEN assay was negatively associated with lignin concentration and ferulate ether cross linking as expected. Corresponding correlations and regressions for CONV were less consistent and occasionally positive. Use of rumen microbial hydrolysis to characterize biomass performance in a conversion process may have some limited usefulness for genetic evaluations, but such assays would be unreliable for biomass pricing.

Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.

Polysaccharides from seeds of higher plants. water-soluble polysaccharides from plant seeds of the family Apiaceae

We studied previously plants of the families Liliaceae [1, 2], Amaryllidacea [3], Malvaceae [4], and Caryophyllaceae [5]. In continuation of work in this area, we studied water-soluble polysaccharides (WSPS) from seeds of higher plants of the family Apiaceae (Umbelliferae). The composition and content and physicochemical characteristics of seeds from representatives of this family have not been reported. Furthermore, many Umbelliferae plants have great economic value as food, drug, feed, and technical plants [6]. Therefore, our goal was to isolate and determine qualitatively and quantitatively WSPS from seeds of 15 plant species of the family Apiaceae. The common determination method for WSPS from all seeds included grinding air-dried seeds (10 g), treatment with EtOH:CHCl3 (1:2, v/v) to remove lipids and pigments, and extraction (3 ) of WSPS with water at room temperature (1:10 ratio). The extracts were evaporated. Polysaccharides were precipitated by alcohol. The precipitate was washed with acetone and dried. The monosaccharide composition of WSPS was established after total acid hydrolysis (H2SO4, 2 N, 100°C, 5 h) and subsequent analysis. GC of aldononitrile acetates [7] was performed on a Chrom-5 instrument with a flame-ionization detector, glass column (250 0.20 mm), 5% Silicone XE-60 on inerton NAW (0.200–0.250 m), 205°C, N2 carrier gas, 30 mL/min. Samples of WSPS were white powders with a cream tint. They did not have reducing power and did not contain starch according to a negative test with iodine. Fehling solution was added to aqueous solutions of the polysaccharides using the principle of selective precipitation of mannose-containing polysaccharide [8]. A precipitate formed for seeds of Angelica ternata, Coriandrum sativum, Heraclim lehmannianum, Ferula tenuisecta, F. diversivittata, Foeniculum vulgare, and Mediasia macrophylla. Polysaccharides of the mannan type were probably present in the isolated WSPS. Table 1 shows that the studied polysaccharides were obtained in different yields from the seeds. The WSPS content was 0.38-5.96% and was greater in seeds of Anethum graveolens, Daucus carota L., Ferula tschimganica (>4%); and in Foeniculum vulgare (5.6%) and Mediasia macrophylla (5.96%). The hydrolysis products of the WSPS contained from two to five monosaccharides. Rhamnose was present in all studied polysaccharides from plant seeds. The principal components were rhamnose, arabinose, glucose, and galactose. This indicated that the polysaccharide fractions contained rhamnoarabinogalactan, rhamnoxylogalactan, glucan, and rhamnoglucan. Thus, screening of seeds from 15 plant species of the family Apiaceae found that the isolated WSPS differed in quantitative content of WSPS and monosaccharide composition. Some WSPS (A. ternata, C. sativum, H. lehmannianum, F. tsehimganica, F. diversivittata, F. vulgare, M. macrophylla) contained mannan-type polysaccharides. As stated above, polysaccharides affect the physiological activity of many food additives [9]. In this respect, screening of WSPS from seeds of Anethum graveolens, C. sativum, Ferula, Foeniculum vulgare, and M. macrophylla are interesting for further medical and biological testing.

Sequential solvent extraction and structural characterization of polysaccharides from the endosperm cell walls of barley grown in different environments

The objective of this study was to examine the composition and molecular structure of the endosperm cell walls (CW) derived from barley grain grown in three environments in Canada, and differing in grain hardness, protein, and total β-glucan contents. The endosperm CW were isolated from barley, cv. Metcalfe, grown in Davidson, SK (Sample A), Hythe, AB (sample B), and Hamiota, MB (sample C). The CW were sequentially extracted with water at 65oC, saturated Ba(OH)2, again with water at 25oC, and 1M NaOH, resulting in fractions designated WE65, BaE, Ba/WE, and NaE, respectively. The monosaccharide analysis indicated the presence of β-glucans, arabinoxylans, and small amounts of arabinogalactans, glucomannans, and xyloglucans. Cellulose was detected in the CW remnants. The CW of sample A, exhibiting a lower grain hardness than sample B, contained the lowest amount of β-glucans, but the highest amount of arabinoxylans and the mannose-containing polysaccharides. The CW of sample C, characterized by very high protein content in the grain, contained the highest amount of β-glucans and the lowest amount of other polysaccharides. Polysaccharides in the CW of sample B, exhibiting the highest grain hardness, were characterized by the highest weight average molecular weights (Mw). β-Glucans in the CW of Sample B showed the highest ratio of DP3/DP4 and the longest cellulosic fragments in the polymeric chains. Of the three barley samples, arabinoxylans in the endosperm CW of sample A exhibited the lowest degree of branching, the highest amount of unsubstituted Xyl residues, and the highest ratio of singly to doubly substituted Xylp. The highest water solubility of the CW of sample C was associated with the highest concentration of β-glucans, the lowest DP3/DP4 ratio, and the lowest Mw of the polymeric constituents. Arabinoxylans with the lowest amount of doubly substituted but the highest amount of unsubstituted xylose residues and long sequences of unsubstituted xylan regions were found in the NaE fractions. The NaE fractions showed a high ratio of →4)-Glcp-(1→ to →3)-Glcp-(1→ linkages and some →4)-Manp-(1→ linkages, indicating a high level of long cellulosic regions in β-glucan chains and the presence of glucomannans.

Effect of lysozyme on the lectin-mediated phagocytosis of Bacillus cereus by haemocytes of the cockroach, Blaberus discoidalis

Lysozyme-like activity has been demonstrated in both cell-free haemolymph and, more abundantly, in haemocyte–lysate supernatants of Blaberus discoidalis. This activity was non-inducible, but heat-stable, with a maximum activity at pH 6.2. When B. cereus was pre-incubated in a concentration of chicken egg-white lysozyme equivalent to the concentration of lysozyme-like activity in cell-free haemolymph, the phagocytosis of B. cereus opsonized with GlcNAc-specific lectins, i.e. BDL2, WGA and HPA, was significantly reduced by up to 50%, while phagocytosis of B. cereus opsonized with mannose-specific lectins, such as BDL1 and Con A, was significantly increased. Pre-incubation of B. cereus in a higher concentration of lysozyme resulted in a smaller, shorter lived increase in the phagocytic rate of bacteria opsonized with these mannose-specific lectins. The action of lysozyme on the peptidoglycan in the cell wall of B. cereus probably resulted in a reduction in the number of binding sites for the GlcNAc-specific lectins, and, therefore, reduced the phagocytic rate of BDL2, HPA and WGA-opsonized B. cereus. Concomitantly, the breakdown of peptidoglycan probably exposed mannose-containing polysaccharides, previously embedded in the peptidoglycan layer, resulting in an increase in the phagocytic rate of the BDL1- and Con A-opsonized B. cereus. These results are discussed in relation to the immune-potential of B. discoidalis.

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.

Molecular cloning of a cDNA encoding a (1-->4)-beta-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum).

Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1-->4)-beta-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1-->4)-beta-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5'-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated M(r) of 38,950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28-30% sequence identity with fungal (1-->4)-beta-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1-->4)-beta-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.

STRUCTURAL STUDIES ON THE GALACTOMANNANS OF LICHENS OF THE GENUSCLADONIA

Galactomannans were isolated fromCladonia signata,C. furcata,C. imperialis, andC. clathratavia successive alkaline extraction and precipitation with Fehling solution and Cetavlon. They were investigated using13C-NMR spectroscopy, methylation analysis, and Smith degradation, and their specific rotations and monosaccharide compositions determined. As with galactomannans of otherCladoniaspecies, they contained (1→6)-linked main chains of α-mannopyranose, which were non-substituted (structure4in Fig. 2), mono-substituted at O-2 with α-mannopyranose (structure6) or α-galactopyranose (structure1), O-4 with β-galactopyranose (structure2), and disubstituted at O-2 and O-4 with α-mannopyranosyl and β-galactopyranosyl units, respectively (structure5). Disubstitution was present to a greater extent in the galactomannans ofC. clathrataandC. imperialisthan in those ofC. signataandC. furcata. In the case of the galactomannans ofC. furcata,C. clathrata, andC. imperialis, substitution also occurred at O-2 withO-β-galactofuranosyl-(1→6)-O-α-mannopyranosyl units (structure7). As observed in previous investigations, the C-1 portion of the13C-NMR of mannose-containing polysaccharides is typical of the lichen species. However, those of galactomannans ofC. imperialisandC. clathrataare almost identical and, although other chemical data showed many structures in common, some differences were evident.

Structural Studies on the Galactomannans of Lichens of the Genus Cladonia

Galactomannans were isolated from Cladonia signata, C. furcata, C. imperialis, and C. clathrata via successive alkaline extraction and precipitation with Fehling solution and Cetavlon. They were investigated using ⊃13C-NMR spectroscopy, methylation analysis, and Smith degradation, and their specific rotations and monosaccharide compositions determined. As with galactomannans of other Cladonia species, they contained (l →6)-linked main chains of α-mannopyranose, which were non-substituted (structure 4 in Fig. 2), monosubstituted at O-2 with α-mannopyranose (structure 6) or α-galactopyranose (structure 1), O-4 with β-galactopyranose (structure 2), and disubstituted at O-2 and O-4 with α-mannopyranosyl and β-galactopyranosyl units, respectively (structure 5). Disubstitution was present to a greater extent in the galactomannans of C. clathrata and C. imperialis than in those of C. signata and C. furcata. In the case of the galactomannans of C. furcata, C. clathrata, and C. imperialis, substitution also occurred at O-2 with O-β-galactofuranosyl-(l→6)-O-α-mannopyranosyl units (structure 7). As observed in previous investigations, the C-l portion of the ⊃13C-NMR of mannose-containing polysaccharides is typical of the lichen species. However, those of galactomannans of C. imperialis and C. clathrata are almost identical and, although other chemical data showed many structures in common, some differences were evident.

Chemotypes of mannose-containing polysaccharides of lichen mycobionts: a possible aid in classification and identification

Chemotypes of mannose-containing polysaccharides of lichen mycobionts: a possible aid in classification and identification

The alpha-mannosyl-binding lectin from leaves of the orchid twayblade (Listera ovata). Application to separation of alpha-D-mannans from alpha-D-glucans.

The carbohydrate-binding specificity of an alpha-D-mannose-specific lectin isolated from leaves of the orchid twayblade (Listera ovata) was elucidated by quantitative precipitation of mannose-containing polysaccharides and glycoproteins, hapten inhibition, and affinity chromatography on the immobilized lectin. L. ovata agglutinin (LOA) interacted with various alpha-mannans and galactomannans of yeasts, fungi and bacteria, but not with alpha-glucans, e.g., dextran and glycogen, as do mannose/glucose-binding lectins. This lectin, LOA, appears to be highly specific for alpha 1-3 mannosidic linkages. It reacted with a linear alpha 1-3-mannan (D. P. 15) and, surprisingly, even with a linear alpha 1-3-mannoheptasaccharide. The LOA/C. tropicalis mannan precipitation reaction was inhibited by alpha-linked mannooligosaccharides, in the order, alpha 1-3 > alpha 1-6 > alpha 1-2 linkages; alpha 1-3 [Man]4 and [Man]5 were the best inhibitors among various mannooligosaccharides tested, having 7-times greater potency than alpha 1-3 [Man]2, and 18-times that of methyl, alpha-mannoside. LOA/mannan interaction was also inhibited by periodate-oxidized and reduced alpha 1-3 [Man]5 which had an inhibitory potency similar to that of alpha 1-3 [Man]3, confirming that LOA also recognizes the internal alpha 1-3-mannosidic linkages of carbohydrate chains. Complete resolution of mannan and glycogen from yeast cells, by affinity chromatography on an immobilized LOA column, and retention of several high-mannose-glycoproteins suggest this lectin to be a useful tool for purification and structural investigation of alpha-mannosyl-containing polysaccharides and glycoconjugates.