Mannan Structure Research Articles

The mannan formed by the yeastBullera tsugae

The structure of an extracellular mannan formed by the yeastBullera tsugae has been studied. It has been shown that the repeating unit of this polysaccharide contains β-(1→3)- and β-(1→4)-bound D-mannopyranose residues in equimolar ratio. A comparison has been made of the polymer under investigation with the extracellular mannan from a yeast of the genusRhodotorula.

Structure of Cell Wall and Extracellular Mannans from Saccharomyces rouxii and Their Relationship to a High Concentration of NaCl in the Growth Medium

Cells of Saccharomyces rouxii (a salt-tolerant yeast) were grown in the presence of two levels of NaCl, 0 and 15%. Mannans obtained from both the cell walls and culture filtrates (extracellular) were examined. Yields based on the dry weight of cells demonstrated that the levels of both cell wall and extracellular mannans were lower when cells were grown in the presence of 15% NaCl. However, the carbohydrate and protein contents of the mannan preparations were not altered. The cell wall mannans obtained from the two growth conditions had similar molecular weights, whereas the extracellular mannans had different molecular weight distributions. Structural analyses showed that the cell wall and extracellular mannans had similar structures. Both had an alpha1-6-linked backbone to which single mannose and mannobiose units were connected as side chains, predominantly by alpha1-2 linkages. The mannans also contained mannosyloligosaccharides, mannotriose, mannobiose, and mannose attached to protein through an O-glycosidic bond. The molecular structure of the cell wall mannans remained unchanged at both levels of NaCl. However, in the presence of 15% NaCl, the side chains consisting of a mannobiose unit were slightly reduced.

Synthetic approach to glycan chains of a glycoprotein and a proteoglycan

Abstract

Cell wall composition of yeast- and rod-forms of Candida krusei.

Cell walls were obtained from yeast (Y)- and rod (R)-forms of Candida krusei. These Y- and R-form cells were grown in synthetic media containing lysine and alanine as nitrogen sources, respectively. Comparative analysis of their walls showed that the Y-form wall had greater quantities of alkali-soluble glucan, alkali-insoluble glucan and mannan than the R-form wall had.The glucan from Y-form walls had a larger amount of β-1,3-glycosidic linkage with a few branches at the C-6 position and a smaller amount of linear β-1,6-glycosidic linkage than the glucan from R-form walls had. There was, however, no significant difference in the chemical structure of mannan between the walls of these two forms.The results are discussed in relation to the chemical composition and structure of polysaccharides of walls in the yeast-rod dimorphism.

A Cell Wall Proteo-Heteroglycan from Piricularia oryzae: Further Studies of the Structure

The carbohydrate part of a proteo-heteroglycan from Piricularia oryzae was further studied by chemical and immunological methods. Acetolysis studies of the proteo-heteroglycan and exo-alpha-D-mannanase resistant core showed a (1 leads to 6) mannan back-bone structure with side chains composed of one to four mannose units, and some of which are terminated by D-glucose or D-galactofuranose. The mode of attachment of the terminal glucose was characterized to be alphaGlc(1 leads to 6)Man by inhibition reaction with oligosaccharides. Rabbit anti-serum formed against P. oryzae cells had three specificities, the first one for alphaGlc(1 leads to 6)alphamannosyl, the second one for alphaMan(1 leads to 3)alphamannosyl, and the last one for alphaGal-f(1 leads to 2)alphamannosyl residues. The most immunodominant side chain structure of the P. oryzae heteroglycan was shown to be alphaGlc(1 leads to 6)alphaMan(1 leads to 2)alphaMan(1 leads to 2)Man.

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.

Structure of cell wall and exocellular mannans from the yeast Hansenula holstii: II. Mannans produced in phosphate-limited medium

1. 1. Cell wall mannan and exocellular phosphomannan from Hansenula holstii NCYC 560 grown under phosphate-limited conditions were analyzed to determine their structures. 2. 2. Both preparations were homogeneous with respect to molecular weight (1. 10 6), but heterogeneous toward ion-exchange resins. 3. 3. Mannans studied were reduced in their phosphate content with O-acetyl groups replacing most of the phosphates as constituents of these polymers. 4. 4. Both mannans had a branched structure similar to some extent to that described for Saccharomyces cerevisiae cell wall mannan, the main difference being the incorporation of O-acetyl groups within the molecules. 5. 5. A role as ion-exchanger in the outer regions of the cell wall is proposed for these mannans.

Structure of cell wall and exocellular mannans from the yeast Hansenula holstii: I. Mannans produced in phosphate-containing medium

1. 1. Cell wall mannan and exocellular phosphomanna from Hansenula holstii NCYC 560 were analysed to determine their structures. 2. 2. Both preparations were composed of a mixture of several polysaccharides (molecular weight = 1. 10 6) differing in degree of phosphorylation and in structural arrangement. 3. 3. The cell wall mannan was a mixture of two branched polysaccharides containing 1,2- and 1,3-linkages, one of the fractions having β-linkages in addition to the α-linkages usually found in these polymers. 4. 4. The exocellular phosphomannan was composed of three polymers, all of them with an α-1,6-linked backbone to which α-1,2- and α-1,3-linked side chains of various lengths were attached. 5. 5. The cell wall mannan contained phosphodiester linkages as reported for mannans from other species. However, the highly phosphorylated exocellular mannan was a novel kind of polysaccharide having its phosphates in the monoester form. 6. 6. It was concluded that the exocellular phosphomannan was not a product of autolysis of the cell wall material but rather an independently synthesized material.

Some aspects of the structure, immunochemistry, and genetic control of yeast mannans.

Some aspects of the structure, immunochemistry, and genetic control of yeast mannans.

Genetic Control of Yeast Mannan Structure: ISOLATION AND CHARACTERIZATION OF MANNAN MUTANTS

Abstract The yeast Saccharomyces cerevisiae X2180, the strain most commonly used for genetic studies on yeasts, was treated with the mutagen ethyl methanesulfonate, and mutants defective in the synthesis of cell wall mannan were isolated. The initial selection of mutants was made on the basis of their failure to agglutinate with antiserum that was specific for the mannotetraose side chain of the mannan, the immuno-dominant group of this yeast strain, which has the structure αMan(1→3)αMan(1→2)αMan(1→2)Man. Nonagglutinating cells were assumed to have modified mannan structures, and this was confirmed by chemical analysis of the isolated mutant mannans. Two classes of mutants were obtained. One was apparently defective in the formation of the α-1→3-mannosyl-transferase (mnn1) which is presumed to be involved in the addition of the terminal α-1→3-linked mannose unit to form the mannotetraose side chain. The other class was defective in one of two α-1→2-mannosyltransferases that must be involved in adding the two α-1→2-linked mannose units, one of them (mnn2) directly to the α-1→6-linked mannose units in the backbone of the polysaccharide and the other (mnn3) to this first side chain mannose unit. No mutant was obtained that was defective in forming the backbone. Since such a mutation would yield a cell completely lacking mannan, it might be lethal. Complementation studies on diploid crosses of representatives of the different mutant classes gave results that were consistent with the interpretations based on chemical analysis of the mutant mannans. Thus, diploids obtained from crosses between mnn1 x mnn2, mnn2 x mnn3, or mnn1 x mnn3 mutants all made mannan of the X2180 chemotype. We conclude that all of the mutations involve structural genes for the various mannosyltransferases that are required for mannan synthesis and not regulatory genes that control expression of a particular mannan chemotype.

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.

The Crystalline Structure of Poly-β,D(1→4′)mannose: Mannan I

The crystallographic structure of mannan I has been examined by X-ray fiber diffraction, polarized i.r. spectroscopy, and computer chain-packing methods. The combination of X-ray intensity data and the chain packing analyses have shown that the overall number of permissible structures is very limited so that van der Waals' forces play a major role in packing. It was concluded that the chain senses are anti-parallel. The interpretation of the polarized i.r. spectra in the OH stretching region is in line with the presence of an intra-molecular hydrogen-bond between O(3)… O(5′). The awkward "knobby" shape of the mannan molecule, largely a result of the axial disposition of O(2)—H, lends itself to a close packing mainly determined by van der Waals' forces. The considerable similarity between X-ray and i.r. data from mannans and glucomannans would indicate that structural conclusions about the former might relate to the latter.

Method for fingerprinting yeast cell wall mannans.

Controlled acetolysis of yeast mannans yields mixtures of oligosaccharides with (1-->2) and (1-->3) linkages between the mannose units, whereas the less stable (1-->6) linkages of the polysaccharide backbone are cleaved. The "fingerprints," obtained by gel filtration of the oligosaccharide mixtures, can be used to distinguish between the different yeast mannans. The general method may be useful for determining the taxonomy of yeasts and for making correlations between immunochemical reactivity and mannan structure.

The structure of a glycopeptide isolated from the yeast cell wall.

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.

Non-cellulosic structural polysaccharides in algal cell walls - III. Mannan in siphoneous green algae

The cell walls of a number of green seaweeds, all members of the Codiaceae and the Dasy-cladaceae and includingCodiumandAcetabularia, are shown to containβ-1.4-linked mannan as the sole crystalline polysaccharide in the complete absence of cellulose. The X-ray diagram of the native mannan (almost identical with that of the mannan of ivory nut and of other palm-seed endosperms) has been indexed to an orthorhombic unit cella= 7.21 Å,b(fibre axis) = 10.27 Å,c= 8.82 Å. After treatment with alkali solutions the mannan recrystallizes in a different lattice; by analogy with cellulose we propose to name this form mannan II and the native mannan, mannan I. The lamellated walls of the central siphon of some of these algae (includingDasycladus,BatophoraandCymopolia) may be separated into two layers. X-ray diffraction analysis and polarization microscopy show that the mannan crystallites of the outer layer tend to lie transversely to the siphon axis, with some dispersion, while those in the inner layer lie longitudinally. The inner layers therefore yield good X-ray fibre diagrams from which a provisional structure of mannan I has been derived. It has proved impossible to reveal in the electron microscope, by the techniques used, the presence of true microfibrils in these plants even when the mannan is well oriented. Electron microscope images of carbon replicas reveal at most the appearance of short rodlets some 100 Å wide. The outer and inner layers resemble respectively the primary and secondary wall layers of higher plants. Some peculiar growth habits of members of the Dasycladaceae are discussed in terms of wall architecture.

On the structure of cell walls and cell wall mannans from ivory nuts and from dates

The submicroscopic structure of date and ivory-nut-endosperm cell walls has been studied with polarisation and electron microscopes. The structure of the two main components of these cell walls, mannan A and mannan B, has been investigated. Mannan A was found to be a granular material that was X-ray crystalline both in the native and in the isolated state. Native mannan B was found to be microfibrillar and to show birefringence; it was X-ray amorphous both before and after isolation and purification. The degree of polymerisation ( DP n ) of mannans A and B was determined osmometrically. This gave values between 17 and 21 for mannan A and about 80 for mannan B. The actual values, however, might be somewhat lower. Carbohydrate analyses of the isolated and purified mannan A showed only traces of galactose and glucose; mannan B after purification contained 6.6% of sugars other than mannose.