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At least 19 recordsLinked to original sources
PubMed1984

Influence of yeast mannan on release of myeloperoxidase by human neutrophils: determination of structural features of mannan required for formation of myeloperoxidase-mannan-neutrophil complexes.

Structural features of mannan which participate in the formation of myeloperoxidase-mannan-neutrophil complexes have been studied by using a battery of structurally modified mannans. Mannan was isolated from Saccharomyces cerevisiae X2180 wild type and modified by strong alkaline degradation, selective (mild) alkaline degradation, and selective acetolysis. Mannose oligosaccharides bound to the peptide portion of mannan appeared to be required for binding of mannan to the neutrophil. The interaction of mannan with myeloperoxidase appeared to occur through phosphate groups of the mannan outer chain. The myeloperoxidase-mannan interaction was determined to be ionic in nature. The mannan-neutrophil interaction may involve cell membrane receptors for mannose.

Carbohydrate Conformation↗
PubMed2006

Antioxidant and antimutagenic activity of mannan neoglycoconjugates: mannan-human serum albumin and mannan-penicillin G acylase.

The antioxidant and antimutagenic activity of the yeast cell-wall mannan and mannan conjugates--in particular the mannan of Saccharomyces cerevisiae (M-S.c.) and conjugates of mannan S. cerevisiae with human serum albumin (M-HSA1, M-HSA2) and the microbial enzyme penicillin G acylase (M-PGA)--were evaluated in vitro in the unicellular flagellate Euglena gracilis exposed to the genotoxic agents ofloxacin and acridine orange (AO). M-S.c., M-HSA1, M-HSA2 and M-PGA show a statistically significant, concentration-dependent protective antigenotoxic activity against both compounds. M-PGA was the most efficient inhibitor of ofloxacin- and AO-induced chloroplast DNA damage, whereas M-HSA2 and M-HSA1 were less effective and M-S.c. had the lowest antigenotoxic activity. It is suggested that different mechanisms may be involved in their protective effect--antioxidant activity in the case of ofloxacin-induced DNA damage and direct adsorption of AO on mannan conjugates as possible mechanisms of protection, based on spectrophotometric measurements. The important characteristics of yeast cell-wall mannans and mannan conjugates, such as their high water solubility, their broad spectrum of biological activity, low toxicity, stability and their antimutagenic effects via different modes of action, appear to be promising features for their practical application as antioxidants and antimutagenic agents.

Acridine Orange↗
PubMed1976

Biosynthesis of yeast mannan. Characterization of mannan-synthesizing enzyme systems from mutants defective in mannan structure.

The yeast Saccharomyces cerevisiae X2180-1A (wild) and its mutants X2180-1A-4 (mnn 1) and X2180-1A-5 (mnn 2) defective in mannan biosynthesis were used as enzyme sources to catalyze in vitro mannosyl transfer from GDP-[14C-U]-mannose to endogenous glycoproteins as well as to exogenous, low-molecular weight acceptors. While the enzyme preparation from the wild strain exhibited all mannosyl transferase activities involved in mannan biosynthesis by catalyzing the synthesis of characteristic mannoprotein, the enzyme from mnn 1 mutant failed to catalyze the synthesis of alpha(1 leads to 3) mannoside linkages both with endogenous as well as with exogenous acceptors. The enzyme preparation from the mnn 2 mutant catalyzed the formation of mannoprotein very similar to that obtained with the enzyme from the wild strain. The most important difference was the formation of a higher number of unsubstituted mannosyl units in the alpha(1 leads to 6) linked mannan backbone. The observed results support the hypothesis that in the mnn 1 the mutation has altered the structural gene involved in biosynthesis of an alpha(1 leads to 3) mannosyl transferase catalyzing the addition of alpha(1 leads to 3) linked mannosyl units to alpha(1 leads to 2) linked mannotrioses in the polysaccharide side chains and in the oligosaccharides attached to serine and/or threonine in the protein part of mannan molecule. The mnn 2 mutant represents most probably a kind of regulatory mutation where the activity of an alpha(1 leads to 2) mannosyl transferase adding the mannosyl units directly to alpha(1 leads to 6) linked backbone in the outer region of polysaccharide part of yeast mannan is repressed in vivo but becomes significant in vitro.

Electron Transport↗
PubMed1981

Comparison of the mannan structure from the cell-wall mutant Candida sp. M-7002 and its wild type. I. Characterization of the proteo-mannan from the mutant and the wild-type cells.

The cell-wall mutant of a hydrocarbon-assimilating yeast, Candida sp. M-7002 and its wild type have shown a significant difference in mannose content. Each mannan was isolated from the mutant and the wild-type cells by fractionation with Cetavlon and copper reagent. Both mannans contain D-mannose, D-glucose and phosphate. The mutant mannan has a relatively high content of protein (18% in weight bases) whereas the wild-type mannan has a low protein content (5.1%) with a high amount of carbohydrate (greater than 90%). Structural analyses by enzymatic and chemical methods showed that both mannans had a mannosidic (alpha 1--6)-linked back bone substituted at O-2 by side chains of varying length. The side chains of the mutant mannan were shown to consist of single mannose units and disaccharide units whose linkages were predominantly alpha 1--2, while the wild-type mannan had two additional side chains of disaccharides. These additional side chains had alpha 1--3 linkages which were scarcely found in the mutant mannan. beta-Elimination reaction demonstrated that the mannans also contain mannosyl oligosaccharides linked to protein through O-glycosidic linkage. The chemical properties of the mannan of the Candida mutant indicates that the mutation might occur not only in the side chain structure but also in the (alpha 1--6)-linked mannan back bone.

Candida↗
PubMed1983

Identification of the mannan-binding protein from rat livers as a hepatocyte protein distinct from the mannan receptor on sinusoidal cells.

Cellular distribution in rat livers of the mannan-binding protein, a liver lectin specific for mannose and N-acetylglucosamine was investigated. Estimation of mannan binding activity of isolated cells demonstrated that more than 99% of the mannan binding activity recovered from whole livers was derived from hepatocytes. Intracellular localization of the mannan-binding protein disproved its function as a receptor of endocytosis. A small quantity of mannan binding activity detectable in sinusoidal cells seems to represent a receptor for mannan endocytosis. The receptor resembled the mannan-binding protein in its high affinity for mannan, requirement of Ca2+ for binding, and specificity for mannose and N-acetylglucosamine. However, the receptor was clearly distinct from the mannan-binding protein as indicated by the lack of response to antimannan-binding protein antiserum, by the potent inhibition by L-fucose and by the differences of specificity for various glycoproteins. The results in this study confirm the recent report of Maynard and Baenziger (Maynard, Y., and Baenziger, J. (1982) J. Biol. Chem. 257, 3788-3794) with quantitative data using different methodology and ligands, and provide additional evidence indicating the receptor on sinusoidal cells to be distinct from the hepatic binding protein. A possible role of the mannan-binding protein in the intracellular transport of intermediates of glycoprotein biosynthesis is also discussed.

Animals↗
PubMed1996

Structure and antigenicity of the mannans of Candida famata and Candida saitoana: comparative study with the mannan of Candida guilliermondii.

The chemical structure of the mannans of antigenic factor 9-expressing yeast, Candida famata and Candida saitoana, was analyzed by acetolysis and NMR. The structural study of the oligosaccharides and mannans using one- and two-dimensional NMR indicated that the mannan of C. saitoana contains a third type of beta-1,2-linked mannose unit. On the other hand, the mannan of C. famata does not contain any beta-1,2-linked mannose units. The mannan of C. saitoana gave two groups of beta-1,2 linkage-containing oligosaccharides by acetolysis. One contains one beta-1,2-linked mannose unit and the other contains two consecutive beta-1,2-linked mannose units at the nonreducing terminal. The inhibition of the reactivity of factor 9 serum on an enzyme-linked immunosorbent assay (ELISA) with these oligosaccharides indicated that the inhibition activity of the former oligosaccharide is 1/20 of that of the latter ones. The ELISA of the mannans of Candida guilliermondii, C. saitoana, and Saccharomyces kluyveri, all of which contain the third type of beta-1,2-linked mannose unit, indicated that Man(beta)1--> 2Man(beta)1-->2Man(alpha)1-->3Man(alpha)1-->works as the antigenic factor 9 but Man(beta)1-->2Man(alpha)1-->3Man(alpha)1--> weakly behaves as both antigenic factors 6 and 9. The epitope structures of the side chain oligosaccharides agreed well with that proposed from the 2D-HOHAHA spectra of the mannans. This result demonstrates the usefulness of the H-1 - H-2-correlated cross-peak pattern, which was reported in a preceding paper (Shibata, N., Akagi, R., Hosoya, T., Kawahara, K., Suzuki, A., Ikuta, K., Kobayashi, H., Hisamichi, K., Okawa, Y., and Suzuki, S. (1996) J. Biol. Chem. 271, 9259-9266) for the determination of the epitope structure of Candida mannans without any chemical fragmentation.

Acetic Acid↗
PubMed2004

Genetic variants of the mannan-binding lectin are associated with immune reactivity to mannans in Crohn's disease.

BACKGROUND & AIMS: Some patients with Crohn's disease (CD) develop antibodies against mannan, a component of the yeast Saccharomyces cerevisiae cell wall. Mannan-binding lectin (MBL), a component of the innate immune system, can bind to S. cerevisiae . MBL concentration depends on genetic polymorphisms. The aim of this study was to evaluate whether low MBL contributes to anti-S. cerevisiae antibody (ASCA) production. METHODS: ASCA and MBL concentrations in sera from patients with CD (n = 74), ulcerative colitis (UC) (n = 22), and healthy controls (n = 32) were measured by an enzyme-linked immunosorbent assay (ELISA). Genetic MBL variants were determined from 58 CD patients, 18 UC patients, and 47 controls by DNA sequencing. Lymphocytes were tested for proliferative response after stimulation with mannan. RESULTS: ASCA were found in 47% of the patients with CD and in 0% of the controls. More ASCA-positive patients (52%) had low serum MBL concentrations compared with ASCA-negative patients (4%) (P < 0.0001). T-cell proliferation in response to mannan stimulation was observed in ASCA-positive patients and could be inhibited by the addition of MBL. These patients had significantly lower MBL serum concentrations than patients whose lymphocytes did not proliferate on mannan stimulation (P < 0.0001). Homozygous or compound heterozygous MBL mutations in the exon 1 and promoter occurred in 12 patients with cellular or humoral immune reactivity to mannan as compared with only 1 nonreactive patient (P < 0.0001). CONCLUSIONS: A subgroup of CD patients is characterized by ASCA positivity, T-cell proliferation on mannan stimulation, and mutations in the MBL gene that result in MBL deficiency. Thus, we propose that enhanced mannan exposure stimulates specific immune responses in a subgroup of CD patients with genetically determined low MBL concentrations. This enhanced exposure contributes to the generation of ASCA.

Antibodies, Fungal↗
PubMed1990

Different effects of native Candida albicans mannan and mannan-derived oligosaccharides on antigen-stimulated lymphoproliferation in vitro.

Yeast cell wall mannan polysaccharide has been proposed to contribute to immune dysfunction associated with chronic infections involving Candida albicans. This influence of mannan has been suggested based partially upon studies of the in vitro immunoinhibitory effects of mannans prepared from Saccharomyces cerevisiae or C. albicans by precipitation with Fehling's reagent, which provides a structurally modified product contaminated with copper. We have therefore evaluated the immunoinhibitory influence of a more native C. albicans mannan prepared by complexation with cetyltrimethylammonium bromide (CTAB). CTAB mannan was a potent stimulator of lymphoproliferation when added to human PMBC from donors responsive to Candida; it has no inhibitory influence on lympho-proliferation induced by Candida or other Ag. In contrast, members of a family of mannose oligosaccharides (disaccharide through hexasaccharide) derived from the CTAB mannan by weak alkaline degradation did not stimulate lymphoproliferation, but were potent inhibitors of lymphoproliferation stimulated by Candida and other Ag. Fifty percent inhibitory doses were 260 to 34 microM, respectively. Compositional analyses of these immunoinhibitory oligomers showed them to be composed of mannose and free of contaminating protein. We propose that mannan-derived oligosaccharides produced by catabolism of mannan in vivo are immunoinhibitory and contribute to the deficit in cell-mediated immune function associated with chronic candidiasis.

Antigens, Fungal↗
PubMed1990

Structure of the D-mannan of Candida stellatoidea IFO 1397 strain. Comparison with that of the phospho-D-mannan of Candida albicans NIH B-792 strain.

The structure of the D-mannan of Candida stellatoidea IFO 1397 strain, which has properties identical to those of the phospho-D-mannan of C. albicans serotype B strain, does not contain phosphate groups, and its 1H- and 13C-n.m.r. spectra are quite similar to those of the phospho-D-mannan of C. albicans NIH B-792 strain. However, the 1H-n.m.r. and 1H-13C-correlation n.m.r. spectra of the products obtained by digestion with alpha-D-mannosidase of C. stellatoidea D-mannan considerably differed from those of the corresponding digestion products of the C. albicans phospho-D-mannan. Additionally, the enzyme-linked immunosorbent assay, by means of a monoclonal antibody corresponding to (1----2)-linked beta-D-oligomannosyl residues, of the phospho-D-mannan of the same C. albicans strain indicated that the C. stellatoidea D-mannan does not contain any (1----2)-linked beta-D-oligomannosyl residues. The absence of these residues may be used as one of the criteria of chemotaxonomical identification of C. stellatoidea spp.

Candida↗
PubMed1988

Detection of Candida sp. mannan antigen by indirect ELISA-inhibition. In vitro crossed reactivity among mannans obtained from C. albicans A, C. albicans B, and C. tropicalis.

We have obtained mannans from four Candida species: C. albicans A, C. albicans B and C. tropicalis; anti-mannan sera against C. albicans A, C. albicans B and C. tropicalis were obtained by immunizing rabbits subcutaneously with the respective yeast extract. The efficacy of these sera in reacting with mannans obtained from three Candida sp. has been proven by indirect ELISA-inhibition. Any of three immune sera can be used to detect mannan antigen from the three Candida sp. tested. This confirms the existence of crossed reactivity and the possibility of detecting mannan antigen in serum from patients infected by different Candida sp., although we had only one immune serum and one Candida mannan.

Animals↗
PubMed1993

Synthesis of antimony complexes of yeast mannan and mannan derivatives and their effect on Leishmania-infected macrophages.

Antimony(Sb)-yeast mannan complexes were synthesized as a strategy to introduce Sb into macrophages infected with Leishmania amastigotes. The complexes were taken up by endocytosis after specific recognition by alpha-D-mannosyl receptors on the macrophage membrane. About 90% of the intracellular parasites were destroyed by Sb-mannan in vitro, whereas the corresponding Sb concentration used as the pentavalent antimonial drug glucantime destroyed about 60% of the amastigotes. None of the Sb complexes prepared with mannan acid or basic derivatives was as effective as the simple Sb-mannan complex in clearing macrophage infection by Leishmania (L) amazonensis. The leishmanicidal effect of Sb-mannan was also demonstrated in vivo with infected hamsters. The alternative use of Sb-mannan complex in the treatment of human leishmaniasis is envisaged on the basis of parasite-killing efficiency and the use of a low antimony dose.

Animals↗
PubMed1981

Comparison of the mannan structure from the cell-wall mutant Candida sp. M-7002 and its wild type. II. Immunochemical properties of the mannans.

The structure of the proteomannans from the cell-wall mutant Candida sp. and its parent strain was further studied by immunochemical methods. Alkaline titration showed that the phosphate is present in the form of diester and mild acid treatment released mannose from both of the mannans. In the wild-type mannan, the phosphorylated side-chain oligosaccharides gave 85% inhibition and a mannopentaose side chain having alpha 1--3 linkage in the nonreducing terminal also gave 40% inhibition in the homologous precipitin reaction. This indicates that the immunodominant side chain of the Candida sp. wild type mannan is the phosphorylated mannopentaose which has an (alpha 1--3)-linked nonreducing terminal. In the mutant system, a significant reactivity in the cross reaction between the mutant antiserum and Kloeckera brevis mannan indicates that the immunodeterminant of the mutant mannan may be a structural analogue of the side-chain oligosaccharide from K. brevis, which has phosphorylated (alpha 1--3)-linked mannotriose. However, no definite results were obtained in the inhibition studies. On the basis of the results from chemical, enzymatical and immunological experiments, the side-chain structure and macromolecular models of Candida sp. wild-type and mutant mannans are proposed.

Antigen-Antibody Complex↗
PubMed2006

Hydrolysis of (1,4)-beta-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-beta-D-mannan endohydrolase and beta-D-mannosidase.

A family GH5 (family 5 glycoside hydrolase) (1,4)-beta-D-mannan endohydrolase or beta-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K(m) constants of 0.16 mg x ml(-1) and 5.3 mM and kcat constants of 12.9 and 3.9 s(-1) respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-beta-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-beta-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-beta-D-manno-oligosaccharides to Man is a family GH1 beta-D-mannosidase, now designated HvbetaMANNOS1, but previously identified as a beta-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) beta-D-mannoside three times faster than 4NP beta-D-glucoside, and has an action pattern typical of a (1,4)-beta-D-mannan exohydrolase.

Amino Acid Sequence↗
PubMed1990

The level of mannan-binding protein regulates the binding of complement-derived opsonins to mannan and zymosan at low serum concentrations.

When sera diluted to 5% in a buffer containing calcium and magnesium were incubated with mannan-coated ELISA plates, C4 fragments, properdin and factor B were bound to the plates as well as the expected opsonic C3 fragments, C3b and C3bi. The calcium-dependent lectin mannan-binding protein, which is structurally similar to C1q, was also shown to bind in this assay and analysis of sera from 179 healthy blood donors revealed that the binding levels of all these proteins were highly significantly correlated. Results obtained with a previously described C3b opsonic assay using zymosan also correlated with the mannan-binding levels. When the sera were diluted to 5% in the presence of Mg-EGTA there was no detectable binding of complement proteins to the mannan surface, confirming that no alternative pathway activation occurred at this serum concentration. When sera were diluted to 5% in a buffer containing EDTA in order to study immunoglobulin binding in the absence of complement activation, the levels of bound IgG1, IgG2, IgG3, IgA and IgM antibodies were found to be completely unrelated to the C3bi binding levels previously observed. The results suggest that in this experimental system using low concentrations of serum, mannan-binding protein initiates an antibody-independent mechanism of cleavage of the classical pathway component C4, which subsequently regulates the degree of cleavage of C3 and recruitment of alternative pathway proteins.

Carrier Proteins↗
PubMed1987

Mannan-specific and mannan-induced T-cell suppressive activity in patients with chronic mucocutaneous candidiasis.

We have studied T- and B-cell responses to antigens of Candida albicans in 18 patients suffering from chronic mucocutaneous candidiasis. We have shown that in vitro production of antibody to one of these antigens, mannan, was absent during the active phase of the disease and that this absence was consequent to the activation of specific CD8(+) and CD8(-) suppressor T lymphocytes. Such activation was also observed when control T lymphocytes were incubated in the presence of monocytes and a high concentration of mannan. This suppressive effect was specific to antigens of Candida albicans, was radiosensitive, and was not consequent to the secretion of prostaglandin E2. It appeared as well that the induction of these suppressor T cells was HLA-DQ restricted. The suppressor T-cell activity induced by antigens of Candida albicans in vitro is thus comparable to the suppressor T-cell activity observed in vivo in patients affected with chronic mucocutaneous candidiasis. Defective handling of mannan by monocytes could result in the accumulation of mannan, resulting in the activation of specific T suppressor cells and in the consequent cellular immunodeficiency specific to Candida albicans. Successful treatment of the candidiasis resulted in complete correction of the immune abnormalities.

Adolescent↗
PubMed2000

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Affinity Labels↗
PubMed1995

Existence of branched side chains in the cell wall mannan of pathogenic yeast, Candida albicans. Structure-antigenicity relationship between the cell wall mannans of Candida albicans and Candida parapsilosis.

Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by 1H and 13C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans mannan. [sequence: see text] It was observed that the H-1 proton chemical shifts of the second and the third mannose units from the reducing terminus in each oligosaccharide are shifted upfield by substitution with an alpha-linked mannose unit at position 6 of the 3-O-substituted mannose unit. An agglutination inhibition assay between factor 4 serum and cells of Candida stellatoidea IFO 1397 lacking the beta-1,2-linked mannose unit, with oligosaccharides obtained from these mannans, indicated that only the branched oligosaccharides were active. This finding suggests that the branched oligosaccharides correspond to the epitope of antigenic factor 4. The presence of the branched structure in other mannans was detected by the characteristic H-1-H-2-correlated cross-peak of the alpha-1,2-linked mannose unit connected with the 3,6-di-O-substituted one by two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Acids↗
PubMed1975

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.

Acetylglucosamine↗