Mannan Moiety Research Articles

An exopolysaccharide from Lactobacillus pentosus YY-112: structure and effect on the human intestinal microbiota.

The development of novel prebiotics, which could regulate the intestinal microbiota, may help prevent and treat intestinal diseases. Here, we studied a homogeneous polysaccharide, LPE-2, produced by Lactobacillus pentosus YY-112 during fermentation. Methylation and gas chromatography-mass spectrometry analysis, combined with nuclear magnetic resonance results, suggested that the structural unit of LPE-2 comprises a branched mannan moiety and a linear glucan moiety. In vitro simulated intestinal fermentation showed that LPE-2 reduced harmful intestinal gas production and promoted short-chain fatty acid production (especially propionic acid). Moreover, it reduced the relative abundance of Escherichia-Shigella, increased that of Bifidobacterium and Lactobacillus, and had a stronger regulatory effect on intestinal flora in women than in men. The potential sex-specific prebiotic effects of LPE-2 on human intestinal health, were possibly related to its mannan branch with (1→2) and (1→3) linkages and backbones with flexible α configurations, which are sheared and degraded/utilized easier by Bifidobacterium and Lactobacillus.

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M. smegmatis.

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.

Analysis of the 3H8 antigen of Candida albicans reveals new aspects of the organization of fungal cell wall proteins.

The walls of both, yeast and mycelial cells of Candida albicans possess a species-specific antigen that is recognized by a monoclonal antibody (MAb 3H8). This antigen can be extracted in the form of a very high Mr complex, close or over 106 Da, by treatment, with β-1,3-glucanase, β mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall β-1,3 glucans, and to proteins by disulfide bonds, but not to β-1,6 glucans. Through its sensitivity to trypsin and different deglycosylation procedures, it was concluded that the epitope is associated to a glycoprotein containing N-glycosidic, but not O-glycosidic mannan moieties. By means of electrophoresis in polycrylamide gradient gels, followed by mass spectrometric analysis, the epitope was pinpointed to a very high MW complex containing Agglutinin-Like Sequence (ALS) family proteins, and other cytoplasmic, membrane and secreted proteins. The components of this complex are bound by unknown covalent bonds. The material extracted with β mercaptoethanol or dilute alkali appeared under the electron microscope as large aggregates in the form of spheroidal and mostly web-like structures of large sizes. These, and additional data, suggest that this protein complex may constitute an important part of the basic glycoprotein structure of C. albicans. The possibility that similar complexes exist in the wall of other fungi is an attractive, although yet untested possibility.

Synthetic arabinomannan glycolipids impede mycobacterial growth, sliding motility and biofilm structure.

Mycobacterium has evolved distinct cell wall and strategies such as biofilm formation, which helps it to survive in hostile conditions. We have reported previously that arabinofuranoside containing glycolipids exhibit inhibition activities against the above functions of the mycobacterial species M. smegmatis. In search for activities mediated by oligosaccharide glycolipids, we report herein the inhibitory activities of a linear and a branched pentasaccharides having arabinan and mannan moieties. In the presence of the pentasaccharide glycolipids, a significant reduction in mycobacterial growth is observed, concomitant with reductions in sliding motility and colonization through biofilm formation, at the optimal glycolipid concentrations of 50-100μg mL(-1). Especially the biofilm coat is ruptured by ~80-85% in the presence of glycolipids. Pentasaccharides alone without the lipidic chain show only a weak effect. The glycolipids are non-toxic, as evaluated through their effect on RBCs. Analysis of the mycolic acid profile of glycolipid treated biofilm shows that α- and epoxy mycolic acids are downregulated significantly, in comparison to glycolipid untreated biofilms. Lipidomics profile analysis through mass spectrometry further reveals profound downregulation of phosphatidylinositol mannosides, acylatedphosphoglycerols and mycolic acid family, namely, keto-, alpha- and methoxymycolic acids.

Vasculitis and anaphylactoid shock in mice induced by the polysaccharide fraction secreted into culture supernatants by the fungus Candida metapsilosis

The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a completely synthesized medium, were examined to determine whether CMWS induces vasculitis similar to that seen in Kawasaki disease, and anaphylactoid shock, in mice. It was found that intraperitoneal injection of CMWS induces coronary arteritis and i.v. injection induces acute anaphylactoid shock in mice, similar to Candida albicans water-soluble fraction (CAWS)-induced arteritis and anaphylactoid shock. The mannan structure of the polysaccharide fraction was then analyzed by performing antiserum reactivity tests and nuclear magnetic resonance spectroscopy. The mannan structure was investigated because the present authors have recently found that the mannan moiety within the polysaccharide fraction might be responsible for these pathogenic activities. The structural analysis showed that the mannan structure within CMWS expresses α-mannan residues, but not β-mannan. In addition, the mannan structure of CMWS is quite similar to that of CAWS. The present findings indicate that the polysaccharide fraction from C. metapsilosis, which is mainly composed of mannan, contributes to coronary arteritis and acute shock, and that the mannan structure could be responsible for this pathogenicity.

ACandida albicansMannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells and Induces T-Cell Activation without Stimulating Proinflammatory Cytokine Production

Mannoproteins are cell wall components of pathogenic fungi and play major virulence and immunogenic roles with both their mannan and protein moieties. The 65-kDa mannoprotein (MP65) of Candida albicans is a beta-glucanase adhesin recognized as a major target of the human immune response against this fungus, and its recombinant product (rMP65; devoid of the mannan moiety) is presently under consideration as a vaccine candidate. Here we investigated cellular and molecular aspects of the interaction of rMP65 with human antigen-presenting cells. We also assessed the ability of rMP65 to initiate a T-cell response. Both the native mannosylated MP65 (nMP65) and the recombinant product were efficiently bound and taken up by macrophages and dendritic cells. However, contrarily to nMP65, rMP65 did not induce tumor necrosis factor alpha and interleukin-6 release from these cells. On the other hand, rMP65 was rapidly endocytosed by both macrophages and dendritic cells, in a process involving both clathrin-dependent and clathrin-independent mechanisms. Moreover, the RGD sequence inhibited rMP65 uptake to some extent. After internalization, rMP65 partially colocalized with lysosomal membrane-associated glycoproteins 1 and 2. This possibly resulted in efficient protein degradation and presentation to CD4(+) T cells, which proliferated and produced gamma interferon. Collectively, these results demonstrate that the absence of the mannan moiety does not deprive MP65 of the capacity to initiate the pattern of cellular and molecular events leading to antigen presentation and T-cell activation, which are essential features for further consideration of MP65 as a potential vaccine candidate.

Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A.

Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups. Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa. The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates. Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6. The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance. Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates. Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan). In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.

Antibody directed against mannan of the Mycobacterium tuberculosis cell envelope provokes blood–brain barrier breakdown

Previous studies demonstrated that blood–brain barrier (BBB) breakdown observed at the cerebral level during experimental allergic encephalomyelitis (EAE) arises from the presence of Mycobacterium tuberculosis in Freund's adjuvant and not only from the encephalitogenic antigens. The main objective of this study was to check if the mannan moiety of lipoarabinomannan present in the mycobacterial cell envelope is responsible for an immune response provoking a BBB breakdown. The results showed that: firstly, the complete Freund's adjuvant (CFA) contains a high release of polysaccharides; secondly, the rats immunized with the CFA present important serum concentration of anti-mannan antibody; and finally, a single 2-mg dose of anti-mannan antibody injected intravenously in naive rats provokes an immediate and reversible BBB breakdown. These results suggest that mannan arising from the solubilization of the mycobacterial cell wall in Freund's adjuvant induces a high production of anti-mannan antibody, which, in turn, provokes a BBB breakdown and possibly facilitates the induction of EAE.

NMR study of the galactomannans of Trichophyton mentagrophytes and Trichophyton rubrum.

Around 90% of chronic dermatophyte infections are caused by the fungi Trichophyton mentagrophytes and Trichophyton rubrum. One of the causes of the chronic infection resides in the immunosuppressive effects of the cell-wall components of these organisms. Therefore we have attempted to identify the chemical structure of galactomannan, one of the major cell-wall components. The cell-wall polysaccharides secreted by T. mentagrophytes and T. rubrum were isolated from the culture medium and fractionated into three subfractions by DEAE-Sephadex chromatography. Analysis of each subfraction by NMR indicated that there are two kinds of polysaccharides present, i.e. mannan and galactomannan. The mannan has a linear backbone consisting of alpha1,6-linked mannose units, with alpha1,2-linked mannose units as side chains. The core mannan moiety of the galactomannan was analysed by a sequential NMR assignment method after removing the galactofuranose units by acid treatment. The result indicates that the mannan moiety has a linear repeating structure of alpha1,2-linked mannotetraose units connected by an alpha1,6 linkage. The H-1 signals of the two intermediary alpha1, 2-linked mannoses of the tetraose unit showed a significant upfield shift (Deltadelta=0.05-0.08 p.p.m.), due to the steric effect of an alpha1,6-linked mannose unit. The attachment point of the galactofuranose units was determined at C-3 of the core mannan by the assignment of the downfield-shifted 13C signals of the galactomannan compared with those of the acid-modified product. In these galactomannans there were no polygalactofuranosyl chains which have been found in Penicillium charlesii and Aspergillus fumigatus.

Molecular bases of adhesion of Candida albicans.

The purpose of this review is to focus on the location and the adhesion activity of the protein (peptide) and the mannan moieties of the mannoprotein in the outer surface of the Candida albicans cell wall. A macromolecule of the mannoprotein located on the outermost surface is undoubtedly a strong adhesin comprising several adhesion molecules including protein and mannan. Mannoproteins can be divided into two classes, higher molecular weight peptidomannans (260 kDa) and lower molecular weight mannoproteins (50-66 kDa), both of which consist of similar mannans and disparate proteins or peptides which have distinct adhesion specificities. The protein moiety of mannoprotein can be divided functionally into two groups, lectin-like proteins and proteins recognizing arginine-glycine-aspartic acid (RGD) ligands. The latter proteins are further subdivided into two groups, CR2/CR3-like proteins and proteins binding extracellular matrix (ECM) proteins. Hydrophobicity of the cell surface of C. albicans influences adhesion of the organisms to epithelial cells. Degree of glycosylation of cell surface mannoproteins that affect yeast cell surface hydrophobicity affects adhesion of C. albicans to epithelial cells. The hydrophobic proteins may have low levels of glycosylation, and changes in glycosylation may determine exposure of hydrophobic protein regions at the cell surface. The serotype A-specific oligosaccharide of antigen 6 (pentaose or hexaose of mannan moiety) has been shown to exhibit marked adhesion ability for epithelial cells, and mannotetraose related to antigenic factor 5 which is present in both serotypes A and B showed adhesive activity for tissue macrophages. Proteinoceous adhesins of C. albicans are expressed preferably on the mycelial form. It is suggested that several of the adhesion molecules of C. albicans described above appear to complementarily utilize multiple adhesion mechanisms.

Immunogenic and immunomodulatory properties of mannoproteins from Candida albicans

Candida albicans is a human commensal and opportunistic fungal organism that expresses on its surface and releases into the external milieu a variety of mannoprotein molecules that are relevant in many aspects of host–Candida relationship. We have attempted to relate mannoprotein constituents to the microbial function and (or) host response. For instance, we have recently found that a protein moiety of a 65-kDa mannoprotein is a major target of cell-mediated immune response. The pattern of cytokines produced by mannoprotein-stimulated human mononuclear cell cultures and mannoprotein-specific T cell lines demonstrated abundant interferon-γ and interleukin-2 production with very low or no production of interleukin-4 and interleukin-10, which suggests that MP-65 is recognized by CD4+ cells of T helper 1 subset. A similar cytokine pattern was seen in splenocyte cultures of mice chronically infected with a low-virulence Candida strain (CA-2) and then stimulated in vitro with MP-65-containing mannoprotein fractions, or also in mice immunized with these fractions. On the other hand, the mannan moieties of mannoproteins bear major B cell epitopes that are responsible for serological specificity, and the ordinary antibody response mounted during normal host colonization. Indirect evidence for the importance of this response for the host–Candida relationship is the dramatic modulation of mannan epitopes on the cell surface during growth and morphological development in vivo. One such epitope is a phosphorylated β-1,2-oligomannoside recognized by the monoclonal antibody AF1, which is present on the surface of infecting yeast cells but then lost during an experimental vaginal infection. Finally, mannan moieties also exert powerful activation of the antimicrobial activity of polymorphonuclear cells and release of a variety of pro-inflammatory and immunomodulatory cytokines, in particular interleukin-1β and tumor necrosis factor-α. Overall, these studies emphasize the need for further definition of individual mannoprotein constituents to dissect the multiple biological actions of these highly complex, multifunctional molecular within C. albicans. Key words: mannoproteins, Candida albicans, immunogenicity, immunomodulation.

Mannan glycopeptide elicits p-coumaroylamino acids in Ephedra distachya cultures

A mannan glycopeptide (Con A-II) which elicits the accumulation of p-coumaroylamino acids in Ephedra distachya cells was purified ca 20-fold from commercial yeast extract by ethanol precipitation, DEAE-cellulose, CM cellulose, gel filtration and Con A-Sepharose chromatography, consecutively. There was no significant elicitor activity in the glucan fraction. The elicitor active fraction (Con A-II) was partially hydrolysed with 2 M TFA and the hydrolysate chromatographed on Sephadex G-25 followed by ethanol precipitation. The activity was found in the 60% ethanol soluble but 80% ethanol insoluble fraction (MGP) whose M r on gel filtration HPLC was nearly homogeneous ( ca 4600). The GC-mass spectral analysis of the partially O-methylated alditol acetate and 13C NMR spectroscopy revealed that MGP contained terminal-(25.4%), α-1,2-(33.6%), α-1,6-(20.6%) and α-1,2,6-(20.4%) mannan glycosyl linkages. MGP elicitor also contained 17.4% (by weight) of peptide which was composed of 10 kinds of amino acid. Ser and Thr comprised 45 molar% of the total amino acid content. Hydrazinolysis of MGP revealed that the mannan moiety was possibly linked to Ser and Thr of the peptide through O-glycosidic bonds. Enzymatic degradation of MGP using α-mannosidase and proteinase K suggested that both mannan and peptide moieties were essential for elicitor activity.

Structure of cell wall mannan of Candida kefyr IFO 0586

We conducted a structural analysis of the antigenic cell wall mannoprotein (mannan) isolated from Candida kefyr (formerly Candida pseudotropicalis) IFO 0586. The result of two-dimensional homonuclear Hartmann-Hahn analysis of this mannan indicates that the molecule is constructed from alpha-1,2- and alpha-1,6-linked mannopyranose residues. Upon alkali treatment (beta-elimination reaction), this mannan released two alpha-1,2-linked mannooligosaccharides, biose and triose. The structure of the alkali-stable mannan (outer chain) moiety was investigated by acetolysis. The structures of the resultant oligosaccharides, biose and triose, from the outer chain moiety were found to be the same as those of the alkali-released ones. Further, the treatment of the parent mannan with an Arthrobacter GJM-1 exo-alpha-mannosidase gave a linear mannan consisting solely of alpha-1,6-linked mannopyranose residues. These results indicate that the mannan forms the long backbone of the alpha-1,6 linkage, with a large number of short alpha-1,2-linked oligomannosyl side chains forming a comblike structure. Moreover, we investigated the serological properties of this mannan by performing an inhibition assay of a slide agglutination reaction with mannooligosaccharides and polyclonal factor sera (Candida Check; Iatron). The result indicates that the factor 1 serum preferentially recognizes the alpha-1,2-linked oligomannosyl side chains in this mannan. On the other hand, the fact that the mannan does not contain an antigenic determinant(s) corresponding to factor 8 suggests that the epitope(s) of this factor resides in other molecules on the cell surface of this strain.

Role of specific determinants in mannan of Candida albicans serotype A in adherence to human buccal epithelial cells.

Candida albicans serotype A (C. albicans A) possesses a specific antigen, designated antigen 6, which resides in mannans on the cell surface. To determine the role of the mannan moiety of the C. albicans cell wall in adherence to buccal epithelial cells, we used antigen 6-deficient mutants which had been isolated by screening with an agglutinating monoclonal antibody against antigen 6 (MAb-6). 1H nuclear magnetic resonance spectral analysis of the purified mannans from the mutants showed a loss of the signals related to that beta-linkage of the side chains. Moreover, acetolyzed fragments of the mutant mannans showed a decreased amount of mannohexaose and mannopentaose. The mutant yeast cells exhibited significantly reduced ability to adhere both to exfoliated buccal epithelial cells and to a human buccal cell line. A number of strains of C. albicans A, C. tropicalis, and C. glabrata, all of which bear antigen 6, showed significantly higher adherence to the cell line than did those of C. albicans serotype B, which lack antigen 6. The whole mannan from the C. albicans A parent inhibited the adherence of C. albicans A to epithelial cells dose dependently, whereas mannan from a mutant strains did not. Moreover, C. albicans A treated with MAb-6 or polyclonal factor 6 serum showed reduced adherence. A close correlation was found between adhesive ability and agglutinability with MAb-6 in the C. albicans A parent, the antigenic mutants, and their spontaneous revertants. These results suggest that so far as mannan adhesion is concerned, serotype A-specific determinants are largely involved in the mechanisms of adherence of C. albicans A to human buccal epithelial cells.

Structural study of a cell wall mannan-protein complex of the pathogenic yeast Candida glabrata IFO 0622 strain

We conducted a structural analysis of the cell wall mannan-protein complex (mannan) isolated from a pathogenic yeast, Candida glabrata IFO 0622 strain. The chemical structure of mannobiose released from this mannan by treatment with 10 m m HCl at 100 °C for 1 h was identified as Man pβ1–2Man. The treatment of this mannan with 100 m m NaOH at 25 °C for 18 h gave a mixture of α-1,2- and α-1,3-linked oligosaccharides, from tetraose to biose, and mannose. The acid- and alkalistable mannan moiety was subjected to mild acetolysis with a 100:100:1 (v/v) mixture of (CH 3CO) 2O, CH 3COOH, and H 2SO 4 at 40 °C for 36 h. The resultant three novel oligosaccharides, tetraose, hexaose, and heptaose, were identified as Man pβ1–2Man pα1–2Man pα1–2Man, Man pα1–2Man pα1–2 Man pα1-6Man pα1- 2Man pα1–2Man, and Man pα1-3Man pα1–2Manpal-2Man pα-16Man pα1–2Man pα1–2Man, respectively, in addition to the three known oligosaccharides, Man pα1–2Man, Man pα1–2Man pα1–2Man, and Man pα1-3Man pα1–2Man pα1–2Man. A sequential analytical procedure involving partial acid hydrolysis with hot 0.3 m H 2SO 4, methylation, fast atom bombardment mass, and 1H NMR analyses was quite effective in the structural determination of the novel oligosaccharides. The results indicate that this mannan possesses a structure closely resembling that of Saccharomyces cerevisiae X2180-1A wild type strain, with the presence of small amounts of oligomannosyl residue, Man pβ1–2Man pα1-X, corresponding to one of the epitopes dominating serotype-A specificity of Candida spp., in addition to branches corresponding to hexaose and heptaose each containing one intermediary α-1,6 linkage.

Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 m m HCl at 100 °C for 60 min gave a mixture of β-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100: 1 ( v v ) mixture of (CH 3CO) 2O, CH 3COOH, and H 2SO 4, at 40 °C for 36 h in order to avoid cleavage of the β-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 α-mannosidase in order to cleave the enzymesusceptible α-1,2 and α-1,3 linkages, leaving manno-oligosaccharides containing the β-1, 2 linkage at their nonreducing terminal sites, Man pβ1 → 2Man pα1 → 2Man pα1 → 2Man pα1 → 2Man, Man pβ1 → 2Man pβ1 → 2Man pα1 → 2Man pα1 → 2Man pα1 → 2Man, and Man pβ1 → 2Man pβ1 → 2Man pβ1 → 2Man pα1 → 2Man pα1 → 2Man pα1 → 2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 ( v v ) mixture of (CH 3CO) 3O, CH 3COOH, and H 2SO 4 at 40 °C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.

Antitumor effect of a baker's yeast mannan—mitomycin C conjugate against mouse hepatoma, MH134, in vivo and in vitro

A conjugate of mitomycin C and the mannan of baker's yeast ( Saccharomyces cerevisiae wild type strain) was synthesized. Assay of its growth inhibitory effect on MH134 hepatoma solid tumor implanted in C3H/He slc mice showed that this conjugate exhibited a higher growth — inhibition ratio than those of free mitomycin C and the same bakers' yeast mannan in the corresponding effective doses. This conjugate was also found to kill the same hepatoma cells in vitro. Because a dextran — mitomycin C conjugate did not manifest any antitumor effect against this tumor, it was postulated that interaction of the mannan moiety in the mannan — mitomycin C conjugate with the mannose receptor located on the cell surface of immunocytes and/or with hepatoma cells participated in the common initiating reaction of in vitro and in vivo antitumor effects.

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties.

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.

Characterization of phosphomannan-protein complexes isolated from viable cells of yeast and mycelial forms of Candida albicans NIH B-792 strain by the action of Zymolyase-100T

The isolation of phosphomannan-protein complexes from the viable cells of yeast (Y) and mycelial (M) forms of Candida albicans NIH B-792 strain was conducted by treatment with Zymolyase-100T followed by fractional precipitation with cetyltrimethylammonium bromide. The M-form complex was found to contain smaller amount of phosphate (1.3%) than that of the Y-form complex (1.6%). Proton magnetic resonance (PMR) spectra of these complexes indicated that the content of β-1,2-linked oligomannosyl and nonreducing terminal α-1,3-linked mannopyranosyl residues in the M-form complex was lower than that of the Y-form complex. With hot 10 m m HCl, the Y-form complex released a mixture of oligosaccharides ranging from mannose to mannoheptaose, while the M-form complex produced lower oligosaccharides, from mannose to mannotetraose. Upon acetolysis, the acid-modified complex of the M form gave mainly mannotetraose, while that of the Y form produced mainly mannopentaose and mannohexaose in addition to mannotetraose. The average length of branching moieties of the mannan of Y-form cells was therefore longer than that of M-form cells. These results indicate that the Y to M transformation of this C. albicans strain accompanies the suppression of enzyme activity concerning the biosynthesis of mannan such as β-1,2- and α-1,3-mannosyltransferases to synthesize the phosphomannan-protein complex containing mannan moiety with incomplete structure.

Isolation of mannan-protein complexes from viable cells of Saccharomyces cerevisiae X2180-1A wild type and Saccharomyces cerevisiae X2180-1 A-5 mutant strains by the action of Zymolyase-60,000

The viable whole cells of Saccharomyces cerevisiae X2180-1A wild type and its mannan mutant strain S. cerevisiae X2180-1A-5, were treated with an Arthrobacter sp. beta-1,3-glucanase in the presence of a serine protease inhibitor, phenyl-methylsulfonyl fluoride. Fractionation of the solubilized materials of each strain with Cetavlon (cetyltrimethylammonium bromide) yielded one mannan-protein complex. Molecular weights of these complexes were almost the same as that of the mannoprotein of the mutant strain prepared by Nakajima and Ballou, which had a molecular weight of 133,000 and were approximately three times larger than those of the mannans isolated from the same cells by hot-water extraction. Each mannan-protein complex contained up to 2% glucose residue, which was not removed by specific precipitation with anti-mannan sera or by affinity chromatography on a column of concanavalin A-Sepharose. Treatment of these complexes with alkaline NaBH4 produced peptide-free mannan containing small amounts of glucose nearly identical to those of the parent complexes. The above findings provide evidence that the glucose residues exist in a covalently linked form to the mannan moiety. Fractionation of the mannan-protein complex of the S. cerevisiae wild-type strain by DEAE-Sephadex chromatography yielded five subfractions of different phosphate content, indicating that these highly intact mannan-protein complexes were of heterogeneous material consisting of many molecular species of different phosphate content.