Mannan Biosynthesis Research Articles

ROLE AND REGULATION OF GLYCOSYLATION PATHWAYS IN LEISHMANIA PARASITES

Leishmania are insect-borne protozoan parasites that cause a number of important diseases in humans. These parasites alternate between two major developmental stages that proliferate within the digestive tract of the sandfly vector and the phagolysosome compartment of macrophages in the mammalian host. The insect stages that initiate infection in the mammalian host are coated with a complex glycocalyx composed of GPI-anchored glycoproteins, phosphoglycans and proteophosphoglycans. Most of these molecules are shed following differentiation to the obligate intracellular stage, leaving a minimal surface coat composed of parasite and host-acquired glycolipids. In contrast, intracellular parasites accumulate an unusual class of oligosaccharides composed of β 1,2-linked mannose, that constitute the primary reserve material of these parasites. We have defined the novel pathways of mannan biosynthesis and turnover, and investigated how the pathways of cell surface and intracellular glycan biosynthesis are reciprocally regulated. We also show that the biosynthesis of these molecules in mammalian stages is dependent on gluconeogenesis and scavenging of amino acids from the macrophage phagolysosome. These analyses provide new insights into the role and regulation of different Leishmania glycosylation pathways in mammalian stages and suggest that Leishmania spp have had to adapt to life in the lysosome in order to satisfy their hexose requirements.

Analysis of Organ-specific, Expressed Genes in Oncidium Orchid by Subtractive Expressed Sequence Tags Library

The pseudobulb of Oncidium orchid plays a key role in water, carbohydrate, and other nutrition support during floral development, yet a large scale of gene expression analysis involved in the metabolisms have not been evaluated. By subtracting RsaI-digested cDNAs of leaf from those of psuedobulb, an efficient subtractive cDNA library was developed. In total, 1080 subtractive expressed sequence tags (ESTs) were obtained. Analysis revealed approximately 636 unique gene parts, 120 clusters and 516 singles. Of these sequences, 74.8% were annotated on the database of NCBI GenBank. Peroxidase, sodium/dicarboxylate cotransporter, and mannose-binding lectin were highly expressed. Some gene profiles were identified as related to carbohydrate metabolism involved in mannan, pectin, starch and sucrose biosynthesis. A large fraction of the ESTs (35%) were classified into transportation, stress-related, cell cycle, or regulatory functions. Most genes that were differentially expressed are important in early flowering development, carbohydrate metabolism and stress-response physiology. This efficient organ-specific EST library represented an explicit transcriptome profile of Oncidium pseudobulb.

Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae.

The antifungal agent caspofungin (CAS) specifically interferes with glucan synthesis and cell wall formation. To further study the cellular processes affected by CAS, we analyzed a Saccharomyces cerevisiae mutant collection (4,787 individual knockout mutations) to identify new genes affecting susceptibility to the drug. This collection was screened for increased CAS sensitivity (CAS-IS) or increased CAS resistance (CAS-IR). MICs were determined by the broth microdilution method. Disruption of 20 genes led to CAS-IS (four- to eightfold reductions in the MIC). Eleven of the 20 genes are involved in cell wall and membrane function, notably in the protein kinase C (PKC) integrity pathway (MID2, FKS1, SMI1, and BCK1), chitin and mannan biosynthesis (CHS3, CHS4, CHS7, and MNN10), and ergosterol biosynthesis (ERG5 and ERG6). Four of the 20 genes (TPO1, VPS65, VPS25, and CHC1) are involved in vacuole and transport functions, 3 of the 20 genes (CCR4, POP2, and NPL3) are involved in the control of transcription, and 2 of the 20 genes are of unknown function. Disruption of nine additional genes led to CAS-IR (a fourfold increase of MIC). Five of these nine genes (SLG1, ERG3, VRP1, CSG2, and CKA2) are involved in cell wall function and signal transduction, and two of the nine genes (VPS67 and SAC2) are involved in vacuole function. To assess the specificity of susceptibility to CAS, the MICs of amphotericin B, fluconazole, flucytosine, and calcofluor for the strains were tested. Seven of 20 CAS-IS strains (with disruption of FKS1, SMI1, BCK1, CHS4, ERG5, TPO1, and ILM1) and 1 of 9 CAS-IR strains (with disruption of SLG1) demonstrated selective susceptibility to CAS. To further explore the importance of PKC in CAS susceptibility, the activity of the PKC inhibitor staurosporine in combination with CAS was tested against eight Aspergillus clinical isolates by the microdilution assay. Synergistic or synergistic-to-additive activities were found against all eight isolates by use of both MIC and minimum effective concentration endpoints.

The histidine kinases of Candida albicans: regulation of cell wall mannan biosynthesis.

Previously, we have used both biochemical and immunological approaches to determine that the two-component, histidine kinase Chk1p regulates cell wall biosynthesis in Candida albicans. These data were obtained by comparing wild-type cells to a strain of C. albicans deleted in CHK1. The dysregulation of cell wall biosynthesis in the mutant reduces its adherence to human esophageal tissue and results in avirulence. In the current study, we used transmission immune electron microscopy (IEM) to visualize the cell surface of both wild-type (CAF2) and the chk1 mutant (CHK21). IEM was performed using two IgM monoclonal antibodies to either an acid-stable mannan epitope (Mab B6) or to an acid-labile mannan epitope (Mab B6.1). We observed that the cell surface of the CHK21 mutant was more reactive than wild-type cells with Mab B6, while the reactivity of Mab B6.1 was similar for both CAF2 and CHK21. These observations correlate with previous data on the Western blotting of mutant and wild-type cells using the same monoclonal antibodies, i.e., greater activity with Mab B6 than with Mab B6.1. In addition to CHK1, two other histidine kinases (SLN1 and NIK1) have been described in C. albicans. Mutants in both sln1Delta and nik1Delta were compared by Western blotting using Mab B6 and Mab B6.1. Reactivity of each mutant to Mab B6 was similar to that observed with the chk1 mutant; on the other hand, the mannoprotein profiles obtained with Mab B6.1 in all mutants were similar to wild-type cells. We also compared the expression of 29 genes involved in mannan synthesis by reverse transcription-polymerase chain reaction (RT-PCR) and found that expression of a subset of six genes (ALG2, ALG6, ALG8, MNT3, PMT6, KRT2) was upregulated in all histidine kinase mutants, while increased expression of ALG7 was only observed in the sln1 and nik1 mutants, MNN1 was upregulated in the chk1 and nik1 mutants, and MNN4 was upregulated in the nik1Delta. Our data indicate that each of the C. albicans HK proteins may regulate similar functions in cell wall biosynthesis. This activity could be achieved in either a common or parallel, redundant signal transduction pathway(s).

The role of the histidine kinase [) gene in the regulation of cell wall mannan and glucan biosynthesis

The publisher regrets that in the above-mentioned article the title and authors were printed wrong. The correct version is reprinted here. “The role of the Candida albicans histidine kinase ( CHK1 ) gene in the regulation of cell wall mannan …

Crystalline structure and packing of mannan I

AbstractThe x‐ray fiber diffraction pattern of the mannan I polymorph has been reexamined using the linked atom least squares (LALS) model‐building program. The chain conformation and packing were refined using atomic coordinates derived from the x‐ray crystallographic structure of mannotriose. The study confirmed the conclusions of previous investigations and showed that, in mannan I, the chains are antiparallel. Mannan polymorphism is compared with other related polysaccharides and the biological significance of this result in relation to the biosynthesis of mannan is discussed.

In vitro‐Einbau von 14C‐Hexose‐6‐phosphat in Mannan, β‐Glucan und Glycogen bei Candida spec. H und ihren Mutanten

AbstractMannose‐6‐p is an activator of the 14C‐mannose incorporation from GDP‐14C‐mannose in the mono‐ and oligosaccharides and in the mannopolymers of the cell wall proteophosphomannan produced by the food protein yeast Candida spec. H.Moreover, mannose‐6‐p is a precursor of proteophosphomannan: 14C‐Mannose‐6‐p has been incorporated in absense of GTP. Corresponding behaviour shows glucose‐6‐p by synthesis of β‐glucan and glycogen. Mutants of Candida spec. H with different efficiency in the biosynthesis of mannan, β‐glucan and glycogen incorporate hexose‐6‐p in a different extent.

Biosynthesis of yeast mannan. Properties of a mannosylphosphate transferase in Saccharomyces cerevisiae.

A homogenate of mechanically broken, freshly grown Saccharomyces cerevisiae X2180 cells catalyzes the transfer of mannosylphosphate units from guanosine diphosphate mannose to reduced alpha1 leads to 2-[3H]mannotetraose to yield reduced mannosylphosphoryl [3H]-mannotetraose. The product is analogous in structure to the phosphorylated mannan side chains, which suggests that the enzymic activity is involved in mannoprotein biosynthesis in the intact cell. The mannosylphosphate transferase activity, localized in a membrane fraction obtained by differential centrifugation at 100,000 x g, was solubilized by Triton X-155 and purified 250-fold by ammonium sulfate precipitation and by ion exchange and gell filtration chromatographies. The enzyme requires MN2+ OR Co2+ ions for activity and is stimulated by various detergents. The mnn2 and mnn3 mannan mutants of S. cerevisiae possess normal levels of mannosylphosphate transferase activity, whereas the mnn4 mutant cells contain very low, if any, activity. This is consistent with a previous conclusion that the mnn4 mutation affects the mannosylphosphate transferase activity, whereas the mnn2 and mnn3 strains possess phosphate-deficient mannans because they are unable to synthesize the appropriate side chain precursors. A new mannan mutant class with the mnn4 chemotype was isolated, but the mutation proved to be recessive and nonallelic with the mnn4 locus. This new locus is designated mnn6.

Biosynthesis of mannan and mannolipids from GDP-Man by membrane fractions of sycamore cell cultures

Membrane fractions have been prepared from sycamore (Acer pseudoplatanus) cell suspensions grown in liquid medium. These fractions catalyzed the transfer of mannoxyl-units from GDP-(14C) Man into a polymannoside, a mannolipid and oligosaccharide-lipids. The polymannoside was partially solubilized by proteolytic digestion or maceration in sodium dodecyl sulfate-urea mixtures. However no evidence is available of a covalent linkage between the biosynthesized glycan and peptides. The structural analysis of the (14C)mannan showed that the polysaccharide was a homopolymer of beta-(1 leads to 4) linked mannose with a few branches. During the incubation of the membranes with the substrate, the polymer chains elongated with a large number of sugar units and contained 25 to 40 hexose residues per non-reducing end monomer. GDP-Glc was a competitive inhibitor of the GDP-Man: beta-mannan mannosyl-transferase, whereas GDP-Man activated a GDP-Gle: beta-glucan glucosyl-transferase present in the same membrane preparation. Two kinds of glycolipids were synthesized in the presence of GDP-(14C)Man. The first (I) contained a polar moiety characterized as a mannose-phosphate and was very similar to polyprenyl-phosphate-mannose identified in plants by Alam and Hemming. The other mannolipid (II) was hydrolyzed by mild acid into labeled oligosaccharides of high molecular weight. This material was separated into two oligosaccharide fractions, the first (II A) of MW over 5000, the second (II B) of MW around 1700. II B contained at most two labeled mannose residues per chain, linked to the non-reducing end of unlabeled units which probably contained neutral sugars and N-acetyl-osamine(s) near the reducing end. Oligosaccharide II A seemed to contain one or several II B chains.

Biosynthesis of yeast mannan: Diversity of mannosyltransferases in the mannan-synthesizing enzyme system from yeast

1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an α-linked mannosyl unit at the nonreducing position of their moleculae were effective as receptors. Monodeoxyanalogues of mannose as well as α-mannose phosphates did not serve as receptors in the above reaction. 3. The structure of the product formed with mannose as receptor was determined to be O- α-D-mannosyl-(1→2)-mannose; with αMan(1→Man(1→6)mannose as the acceptor, the product was αMan(1→6)αMan(1→6)mannose and with αMan-(1→2)mannose the product was tentatively characterized as a mixture of αMan-(1→3)αMan(1→2)mannose and αMan(1→2)αMan(1→2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysacchride to a minor extent under the same conditions.

Structure and Biosynthesis of the Mannan Component of the Yeast Cell Envelope

Publisher Summary The chapter focuses on the new developments concerning yeast mannan structure and biosynthesis as they relate to the organization of the cell wall and the roles played by these glycoproteins. The mechanism of mannan biosynthesis is still in a rudimentary state, partly because of the insufficient knowledge concerning mannan structure and partly because of the inherent difficulties in working with such a complex macromolecule. Detailed information of Saccharomyces cerevisiae mannan can now be sketched and facilitates the planning of experiments on, and the interpretation of results from, biosynthetic studies. Moreover, the availability of mannan mutants with known polysaccharide structural alterations should prove advantageous. Studies of mannan biosynthesis have taken three general tacks. One approach has been directed toward an elucidation of the overall process of mannan formation and translocation within the cell using “pulsechase” techniques and autoradiography. The second approach has dealt with the formation and secretion of intact mannan molecules by yeast protoplasts and the inhibition of this process by compounds that prevent protein or polysaccharide synthesis. The third approach has investigations of the enzymic processes involved in mannan synthesis, using cell-free extracts and following incorporation of radioactive mannose into endogenous and exogenous acceptors. The chapter discusses the detailed structures of specific yeast mannans.

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [ 14C]mannose from GDP-[ 14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [ 14C]mannosyl-l-phosphorylundecaprenol. In contrast, this is the major manno-lipid synthesized from GDP-[ 14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [ 14C]mannose from purified [ 14C]mannosyl-l-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [ 14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.

Genetic control of yeast mannan structure: mapping genes mnn2 and mnn4 in Saccharomyces cerevisiae.

Two mutations concerned with mannan biosynthesis in the yeast Saccharomyces cerevisiae have been mapping. The mnn2 mutation, which affects the addition to the polysaccharide backbone of the first side-chain D-mannose unit in alpha1-leads to2 linkage, was located on chromosome II linked to the centromere and the gall locus. The mn4 locus, which regulates the synthesis of mannosylphosphate groups on the mannan side chains, was placed on chromosome XI near trp3 and ural and a locus previously reported to regulate the ability of a S. diastaticus strain to bind alcian blue (Friis and Ottolenghi, 1970). The mnn4 mutant also fails to bind alcain blue, but the gene responsible for alcian blud binding in this strain segregates independently from the dye-binding locus of S. diastaticus, and therefore must be a different gene. A diploid heterozygous for mnn4 fails to bind dye, indicating dominance of this mutant genotype. The alcian blue dye binding locus dbll, reported to Friis and Ottolenghi (1970), is also dominant. Thus, there are at least two independent genes that control the formation of the mannosylphosphate units in the mannan side chains, and both have the property of dominance in the mutant form.

Biosynthesis of yeast mannan. Isolation of Kluyveromyces lactis mannan mutants and a study of the incorporation of N-acetyl-D-glucosamine into the polysaccharide side chains.

One side chain in the cell wall mannan of the yeast Kluyveromyces lactis has the structure (see article). (Raschke, W. C., and Ballou, C. E. (1972) Biochemistry 11, 3807). This (Man)4GNAc unit (the N-acetyl-D-glucosamine derivative of mannotetroase) and the (Man)4 side chain, aMan(1 yields 3)aMan(1 yields 2)aMan(1 yields 2)Man, are the principle immunochemical determinants on the cell surface. Two classes of mutants were obtained which lack the N-acetyl-D-glucosamine-containing determinant. The mannan of one class, designated mmnl, lacks both the (Man)4GNAc and (Man)4 side chains. Apparently, it has a defective alpha-1 yields 3-mannosyltransferase and the (Man)4 unit must be formed to serve as the acceptor before the alpha-1 yields 2-N-acetyl-glucosamine transferase can act. The other mutant class, mnn2, lacks only the (Man)4GNAc determinant and must be defective in adding N-acetylglucosamine to the mannotetrasose side chains. Two members of this class were obtained, one which still showed a wild type N-acetylglucosamine transferase activity in cell-free extracts and the other lacking it. They are allelic or tightly linked, and were designated mnn2-1 mnn2-2. Protoplast particles from the wild type cells catalyzed a Mn2+-dependent transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the mannotetraose side chain of endogenous acceptors. Exogenous mannotetraose also served as an acceptor in a Mn2+-dependent reaction and yielded (Man)4GNAc. Related oligosaccharides with terminal alpha (1 yields 3)mannosyl units were also good acceptors. The product from the reaction with alphaMan(1 yields 3)Man had the N-acetylglucosamine attached to the mannose unit at the reducing end, which supports the conclusion that the cell-free glycosyltransferase activity is identical with that involved in mannan synthesis. The reaction was inhibited by uridine diphosphate. Protoplast particles from the mmnl mutants showed wild type N-acetylglucosamine transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains. Particles from the mnn2-1 mutant failed to catalyze N-acetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent alpha-1 yields 2-N-acetylglucosamine transferase with kinetic properties similar to those of the wild type enzyme.

Konjak mannan biosynthesis. II. Composition of soluble nucleotides in growing corms of Amorphophallus konjac C. Koch.

The nucleotides and the activities of both sucrose synthetase and granular starch synthetase in the konjak corm (Amorphophallus konjac C. Koch) have been investigated as a preliminary experiment on konjak mannan biosynthesis. On chromatographic separation on anion exchange resin and paper of compounds present in the acid ethanol extract from the corms, ascorbic acid, AMP, ADP, ATP, ADP-glucose, UTP, UDP-glucose, GTP, and GDP-mannose were isolated and tentatively identified. An unidentified nucleotide was also isolated.Of the three nucleotide sugars, UDP-glucose was the most plentiful, while ADP-glucose was the least. The sucrose synthetase in konjak corms was as active as that in other plants. These observations suggest that the mechanism involved in sucrose cleavage in konjak corms is the same as that in other plants, such as sweet potato roots. Starch synthetase bound to starch granules in konjak corms was also found to be active when ADP-glucose was used as glucose donor. But UDP-glucose could not be s...

Metabolism of 2-deoxy- d-glucose by baker's yeast. VI. A study on cell wall mannan

The incorporation of 2-deoxy- d-glucose into cell wall mannan of growing Saccharomyces cerevisiae proceeded continuously during culture growth and followed the cell multiplication. About 10% of mannan labelled with deoxyglucose was concurrently released into the medium. The distribution of deoxyglucose between the side-chains and the main chain of mannan has been established. Approximately 90% of deoxyglucose present in the polysaccharide was bound in the side-chains and only 10% was located in the (1 å 6)-linked main chain. This result suggested that deoxyglucose metabolites serving as glycosyl donors in mannan biosynthesis were much worse substrates for the enzyme(s) responsible for the formation of the main chain of the polysaccharide than for the mannosyl transferases involved in the formation of the mannan side-chains. Degradation of deoxyglucose-containing mannan by α-mannosidase of Arthrobacter GJM-1 stopped at the deoxyglucosyl residues.

Site of mannan synthesis in yeast. An autoradiographic study.

The combination of high-resolution autoradiography and biochemical methods has been used to ascertain the site of mannan synthesis in the yeastSaccharomyces cerevisiae. High-resolution autoradiography has been performed under conditions when addedd-mannose-3H was incorporated exclusively into mannan. Application of “pulse-chase” labelling technique revealed that the radio-active mannose is fixed primarily in the cytoplasmic space from where it is transported into the cell wall. Additional experiments with separated membrane fractions from the same yeast strongly support the hypothesis that the plasmalemma is not directly involved in the biosynthesis of yeast mannan and that the membranes of the endoplasmic reticulum are the sites where the polymerization of mannosyl units takes place.

Identification of the lipid intermediate in yeast mannan biosynthesis.

The unsaponifiable lipid fraction of Saccharomyces cerevisiae contains a phosphorylated component which accepts a mannosyl residue from GDP‐mannose and transfers it to growing mannan chains. Both reactions are catalyzed by membrane‐bound enzymes. The lipid component has been purified more than 1000‐fold (based on lipid‐bound phosphate). After dephosphorylation with an orthophosphoric monoester phosphohydrolase this acceptor was shown by mass spectroscopy to be identical with authentic yeast dolichol. The “lipid” intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol. The “lipid” intermediate intermediate in yeast cell wall biosynthesis, therefore, is mannosylated dolichol monophosphate.Yeast dolichol is a mixture of five homologous polyprenols with saturated α‐isoprene units, as shown by Dunphy et al. in 1967. All these homologues seem also to be present in the cells as monophosphates. As compared to free dolichols the phosphorylated forms only amount to about 10–20%.

Genetic Control of Yeast Mannan Structure: MAPPING THE FIRST GENE CONCERNED WITH MANNAN BIOSYNTHESIS

Abstract Two wild type strains of the yeast Saccharomyces cerevisiae, which differ in the structure of their cell wall mannans, have been hybridized and the genetic control of the mannan structure expressed in the diploid has been investigated. The mannan of one strain (X2180) is characterized by the presence of a mannotetraose side chain, while the other (strain 4484-24D) possesses a mannosylphosphorylmannotriose side chain in place of the mannotetraose unit. The diploid had the X2180 mannan chemotype rather than an average mixture of the two. Sporulation and tetrad analysis of the diploid hybrids demonstrated that the two mannan chemotypes segregated 2:2 as though the difference were under the control of a single gene, and from a detailed genetic analysis we have mapped the dominant gene to chromosome V, tightly linked to ura3 and the centromere. It is the first gene concerned with mannan biosynthesis to be placed on the yeast genetic map and it has been designated mnn1. From the observed differences between the structures of the mannans of strains X2180 and 4484-24D, we conclude that the gene is involved in the synthesis of an α-1→3-mannosyltransferase which adds the terminal α-1→3-linked mannose unit to α-1→2-linked mannotriose side chains in the mannan. The mechanism which prevents the expression of the mannosylphosphate group on the surface of the hybrid diploid cell is unknown, but it must be related to the processes which control the organization of the mannan in the cell wall.

Biosynthesis of mannan in Saccharomyces cerevisiae. Isolation of a lipid intermediate and its identification as a mannosyl-1-phosphoryl polyprenol

Biosynthesis of mannan in Saccharomyces cerevisiae. Isolation of a lipid intermediate and its identification as a mannosyl-1-phosphoryl polyprenol