Terminal Mannosyl Residues Research Articles

Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT11.

Under general aqueous conditions, xanthan appears in an ordered conformation, which makes its backbone largely resistant to degradation by known cellulases. Therefore, the xanthan degradation mechanism is still unclear because of the lack of an efficient hydrolase. Here, we report the catalytic properties of MiXen, a xanthan-degrading enzyme identified from the genus Microbacterium MiXen is a 952-amino-acid protein that is unique to strain XT11. Both the sequence and structural features suggested that MiXen belongs to a new branch of the GH9 family and has a multimodular structure in which a catalytic (α/α)6 barrel is flanked by an N-terminal Ig-like domain and by a C-terminal domain that has very few homologues in sequence databases and functions as a carbohydrate-binding module (CBM). Based on circular dichroism, shear-dependent viscosity, and reducing sugar and gel permeation chromatography analysis, we demonstrated that recombinant MiXen efficiently and randomly cleaved glucosidic bonds within the highly ordered xanthan substrate. A MiXen mutant free of the C-terminal CBM domain partially lost its xanthan-hydrolyzing ability because of decreased affinity toward xanthan, indicating the CBM domain assisted MiXen in hydrolyzing highly ordered xanthan via recognizing and binding to the substrate. Furthermore, side chain substituents and the terminal mannosyl residue significantly influenced the activity of MiXen via the formation of barriers to enzymolysis. Overall, the results of this study provide insight into the hydrolysis mechanism and enzymatic properties of a novel endotype xanthanase that will benefit future applications.IMPORTANCE This work characterized a novel endotype xanthanase, MiXen, and elucidated that the C-terminal carbohydrate-binding module of MiXen could drastically enhance the hydrolysis activity of the enzyme toward highly ordered xanthan. Both the sequence and structural analysis demonstrated that the catalytic domain and carbohydrate-binding module of MiXen belong to the novel branch of the GH9 family and CBMs, respectively. This xanthan cleaver can help further reveal the enzymolysis mechanism of xanthan and provide an efficient tool for the production of molecular modified xanthan with new physicochemical and physiological functions.

Identification of a Novel Mycobacterial Arabinosyltransferase Activity Which Adds an Arabinosyl Residue to α-d-Mannosyl Residues.

The arabinosyltransferases responsible for the biosynthesis of the arabinan domains of two abundant heteropolysaccharides of the cell envelope of all mycobacterial species, lipoarabinomannan and arabinogalactan, are validated drug targets. Using a cell envelope preparation from Mycobacterium smegmatis as the enzyme source and di- and trimannoside synthetic acceptors, we uncovered a previously undetected arabinosyltransferase activity. Thin layer chromatography, GC/MS, and LC/MS/MS analyses of the major enzymatic product are consistent with the transfer of an arabinose residue to the 6 position of the terminal mannosyl residue at the nonreducing end of the acceptors. The newly identified enzymatic activity is resistant to ethambutol and could correspond to the priming arabinosyl transfer reaction that occurs during lipoarabinomannan biosynthesis.

Novel template-assembled oligosaccharide clusters as epitope mimics for HIV-neutralizing antibody 2G12. Design, synthesis, and antibody binding study

The synthesis of a new class of template-assembled oligomannose clusters as the mimics of the epitope of the HIV-neutralizing antibody 2G12 is described. The novel oligomannose clusters were successfully assembled on a cyclic decapeptide template using the Cu(I)-catalyzed 1,3-dipolar cycloaddition of azides to alkynes by introducing four units of a synthetic D1 arm tetrasaccharide (Manalpha1,2Manalpha1,2Manalpha1,3Manalpha-) of high-mannose N-glycan on one face of the template and two T-helper epitope peptides on the other face of the template. Their binding to human antibody 2G12 was studied using surface plasmon resonance (SPR) technology. It was found that while the synthetic monomeric D1 arm oligosaccharide and its fluorinated derivative interacted with 2G12 only weakly, the corresponding template-assembled oligosaccharide clusters showed high affinity to antibody 2G12, indicating a clear clustering effect in 2G12 recognition. Interestingly, the fluorinated D1 arm cluster, in which the 6-OH of the terminal mannosyl residue was replaced with a fluorine atom, showed a distinct kinetic model in 2G12 binding as compared with the cluster of the natural D1 arm oligosaccharides. The oligosaccharide clusters with varied length of spacer demonstrated different affinity to 2G12, suggesting that an appropriate spatial orientation of the sugar chains in the cluster was crucial for high affinity binding to the antibody 2G12. It was also found that the introduction of two T-helper epitopes onto the template did not affect the structural integrity of the oligomannose cluster. The novel synthetic glycoconjugates represent a new type of immunogen that may be able to raise carbohydrate-specific neutralizing antibodies against HIV-1.

Rapid, iterative assembly of octyl α-1,6-oligomannosides and their 6-deoxy equivalents

Mycobacterium tuberculosis is the cause of the deadly human disease tuberculosis. In studies over the last 40 years it has been revealed that this organism possesses a complex cell wall including glycophospholipids such as the phosphatidylinositiol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM). These glycolipids all contain a common alpha-1,6-linked mannoside core, and the higher PIMs and LAM possess alpha-1,2-linked mannosyl residues. It has been shown that simple alpha-1,6-linked oligomannosides can act as substrates for alpha-1,6-mannosyltransferases in mycobacteria. Here we report a simple iterative synthesis of a series of hydrophobic octyl alpha-1,6-linked oligomannosides from mono- through to tetrasaccharides. We have utilized a single thioglycoside donor and alcohol acceptor. Further, we have developed conditions for the conversion of each of these compounds to the 6-deoxy congeners. Deoxygenation of the 6-position of the terminal mannosyl residue should prevent these compounds acting as substrates for the abundant alpha-1,6-mannosyltransferases in mycobacteria and should permit detection of the elusive alpha-1,2-mannosyltransferase activity responsible for elaboration of LM to mature LAM and the biosynthesis of the higher PIMs.

Evaluation of fluorescently labeled lectins for noninvasive localization of extracellular polymeric substances in Sphingomonas biofilms.

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.

Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.

Cytometric detection of mycobacterial surface antigens: exposure of mannosyl epitopes and of the arabinan segment of arabinomannans

The physical arrangement of cell envelope components leads to the exposure of selected structural motifs which in turn may influence host-parasite interactions. To gain insight into the exposed epitopes, the present study describes a flow cytometric method designed to probe defined molecules on dispersed mycobacteria. The hydrophobic fluorophore N-hexadecanoyl aminofluorescein inserted in the mycobacterial cell envelope permitted focusing of fluorescence-activated cell sorter analysis on cells that were further labeled with defined monoclonal antibodies and fluorochrome-coupled streptavidin. The use of antibodies directed against the lipooligosaccharide of Mycobacterium tuberculosis demonstrated the specific detection of the antigen on the cell surface of a Canetti-like strain of M. tuberculosis, and not on those of mycobacterial strains that were devoid of the glycolipid. Thus, the method was applied to investigate the relative amounts of surface-exposed mannosylated compounds and D-arabinan-containing substances of different strains of the tubercle bacillus and a strain of the rapidly growing nonpathogenic species Mycobacterium smegmatis. Both M. tuberculosis and M. smegmatis are endowed with mannosyl and arabinan epitopes on their surfaces, although there are many differences in terms of exposed mannosyl epitopes between the various strains of the tubercle bacillus examined. These differences are correlated with the amounts of terminal mannosyl residues that cap the surface-exposed arabinomannans (A. Ortalo-Magné, A. B. Andersen, and M. Daffé, Microbiology 142:927-935, 1996) but not with the degrees of virulence of the strains. This novel approach could provide new insights into the distribution of defined surface-exposed antigens and thereby into the architecture of the cell envelopes.

Acid hydrolysis and high-performance liquid chromatography of xanthan

A method for the routine determination of the hexose and organic acid content of xanthan preparations based on acid hydrolysis of the exopolysaccharide followed by high-performance liquid chromatography (HPLC) of the hydrolysate on two cation-exchange columns was investigated. A kinetic model was fitted to the recoveries of these components over a range of hydrolysis conditions using non-linear regression, affording values for the rate constants and the initial concentration of each compotent in the unhydrolysed polysaccharide. The ratio of glucose to mannose was greater than the assumed 1:1 ratio in unhydrolysed xanthan, suggesting that the polysaccharide may contain fewer terminal mannosyl residues than previously proposed. Quantification of glucuronic acid was complicated by its lactonisation and consequent elution as two peaks and by interference from its degradation products. The possibility of determining this component as its aldobiouronic acid [β- d-GlcA p-(1→2)- d-Man p] precursor was investigated. Acetic acid recovery was not significantly affected by the hydrolysis conditions over the range studied. The difference between the maximum recoveries and the fitted initial concentrations of each component indicate that compositional analyses of xanthan samples based on single time and acid strength conditions should be treated with caution as they are unlikely to yield quantitatively accurate values.

The structure and physiological activity of a glycoprotein secreted from conidia of Mycosphaerella pinodes. II.

The chemical structure of a glycoprotein secreted from conidia of Mycosphaerella pinodes into the medium during germination was investigated, and its elicitor activity to induce pisatin synthesis in peas was examined. The glycoprotein showed a higher elicitor activity than the intra-conidial polysaccharide. The glycoprotein, whose molecular weight is about 130×104, has ap partial structure in which a reducing terminal mannosyl residue of a trisaccharide 1Man6-α←1Man6β←1Glu is Ο-glycosidically attached to serine in the protein portion.

POLYSACCHARIDES FROM THE ENVELOPES OF HETEROCYSTS AND SPORES OF THE BLUE‐GREEN ALGAE ANABAENA VARIABILIS AND CYLINDROSPERMUM LICHENIFORME1

ABSTRACTThe polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3‐linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose‐containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4‐linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.

Structural analysis of mannans from Candida albicans serotypes A and B and from Torulopsis glabrata by methylation gas chromatography mass spectrometry and exo-alpha-mannanase.

Mannans of Candida albicans serotypes A and B and of Torulopsis glabrata were subjected to methylation analysis and the resulting monosaccharides were converted to the peracetylaldononitrile derivatives and subsequently analyzed by gas chromatography mass spectrometry. Serotype A differed from B in having more 1 leads to 2 linked mannosyl residues (46.2 vs 37.1 mol%) indicating longer oligomannosyl sidechains; mannosyl branch points linked through C-1, C-3 and C-6 were detected for the first time in C. albicans mannans and were more abundant in serotype B (9.2 vs 5.3 mol%). T. glabrata mannan differed from that of C. albicans in having virtually no (1.6 mol%) unsubstituted sugars in the linear backbone and less 1 leads to 2 linked mannosyl residues (33.1 mol%) interpreted as shorter oligomannosyl sidechains. Model building of a repeating unit for the outer chain region of these mannans was prevented by the large amount of nonreducing terminal mannosyl residues, 23.0 mol% for C. albicans A, that probably arise from the inner core region. These mannans were exposed to exo-alpha-mannanase and the percentage of digestion, measured as reducing sugar released, was 33.5% for C. albicans B; 27.4% for T. glabrata, and C. albicans A was refractory (5.4% digested).

Isolation and properties of alpha-D-mannose:beta-1,2-N-acetylglucosaminyltransferase from trachea mucosa.

A beta-1,2-N-acetylglucosaminyltransferase which transfers N-acetylglucosamine from UDP-N-acetylglucosamine to the branched terminal mannosyl residues found in the complex type oligosaccharide units of N-linked glycoproteins was isolated from swine trachea mucosa. The enzyme was purified from microsomes after solubilization with 1% Triton X-100 and 1% Nonidet P-40, and it was also isolated from a soluble extract of this tissue. The enzyme was purified by chromatography on DEAE-cellulose, cellulose phosphate, and Sephacryl S300 columns and by affinity chromatography on a Sepharose 4B column containing covalently bound ovomucoid. The purified enzyme forms beta-1,2 bonds between N-acetylglucosamine and terminal-branched mannosyl residues of glycoproteins and glycopeptides. The enzyme has an absolute specificity for terminal branched mannosyl residues, and solute specificity for terminal branched mannosyl residues, and no activity is observed with mannose, p-nitrophenyl alpha-D-mannoside, p-nitrophenyl beta-O-mannoside, and glycopeptides which contain unbranched oligosaccharide chains terminating in free mannose. Evidence obtained by kinetic and structural analysis shows that the purified enzyme catalyzes the reactions shown in Scheme I.

The 5-O-β-d-Galactofuranosyl-containing Exocellular Glycopeptide of Penicillium charlesii: OCCURRENCE OF ETHANOLAMINE AND PARTIAL CHARACTERIZATION OF THE PEPTIDE PORTION AND THE CARBOHYDRATE-PEPTIDE LINKAGE

Abstract The major exocellular glycopeptide (peptidophosphogalactomannan) produced by Penicillium charlesii was shown to contain 30 to 32 amino acid residues, and serine and threonine accounted for approximately 15 residues. In addition, the glycopeptide was found to contain approximately 1 ethanolamine residue per molecule. Treatment of the glycopeptide with alkali resulted in an increase in absorbance at 241 nm with the concomitant release of peptide from monosaccharides, oligosaccharides, and phosphogalactomannan. The number of seryl and threonyl residues decreased markedly following alkaline borohydride-PdCl2 treatment of the glycopeptide. This treatment also resulted in an increase in alanine and the appearance of α-aminobutyric acid after hydrolyzing the reaction mixture in 6 n HCl. In addition, treatment of the glycopeptide with alkaline NaB3H4 followed by hydrolyzing the phosphogalactomannan in 2 n HCl resulted in the formation of [3H]mannitol. We conclude that the phosphogalactomannan is attached to the peptide through O-glycosidic linkage of the terminal mannosyl residue to the hydroxyl group of serine or threonine.