Oligomannosyl Residues Research Articles

A novel lectin (morniga M) from mulberry (Morus nigra) bark recognizes oligomannosyl residues in N-Glycans

Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine alpha1-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Man alpha1-->6(Man alpha1-->3) Man], Penta-Man oligomer [Man alpha1-->6(Man alpha1-->3)Man alpha1-->6(Man alpha1-->3) Man] > or = Man alpha1-->2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.

Lectin structure-activity: the story is never over.

Advances in plant lectin biochemistry have made great strides during the past decade. Technical advances in biophysical techniques and molecular biology, the availability of synthetic oligosaccharides, and characterization of lectins with unique carbohydrate-binding properties are responsible for these advances. Studies in this laboratory support the view that interesting new discoveries are yet to be made. A new lectin was recently isolated from a fungus (Polyporous squamosus) that recognizes the Neu5Ac alpha2,6 Gal beta1,4 GlcNAc/Glc trisaccharide epitope with high affinity. The lectin does not interact with alpha2,3-linked Neu5Ac or Neu5Ac alpha2,6 GalNAc groups as occur in ovine submaxillary mucin. An unusual lectin with two distinctly different carbohydrate-binding sites is present in tubers of Xanthosoma sagittifolium (L). One species of sites recognizes clusters of oligomannosyl residues. The other type of binding site best accommodates a nonsialylated, triantennary oligosaccharide having LacNAc or Lacto-N-biose (Gal beta1,3GlcNAc) groups at its three nonreducing termini. The banana lectin has also been studied. It recognizes both alpha and beta1,3-linked glucosyl oligosaccharides, generates a precipitin curve with the branched trisaccharide Man alpha1,6[Man alpha1,3]Man, and binds to beta-glucans containing beta1,6-glucosyl end groups.

Relative abundance of oligosaccharides in Candida species as determined by fluorophore-assisted carbohydrate electrophoresis.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual oligomannosyl residues in Candida mannoprotein, the major antigenic determinant located on the outer surface of the yeast cell wall. The single terminal aldehydes of oligomannosyl residues released by hydrolysis were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligomannoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligomannoside moieties in heterogeneous samples. FACE analysis revealed the major oligomannosides released by acid hydrolysis and beta-elimination of Fehling-precipitated mannan from Candida albicans, which were the same as those previously reported in studies based on mass and nuclear magnetic spectroscopic analysis. FACE was also amenable to the analysis of samples obtained by direct hydrolysis of whole yeast cells. Whole-cell acid hydrolysis and whole-cell beta-elimination of two isolates each of C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. rugosa, C. stellatoidea, and C. tropicalis resulted in oligomannoside gel banding patterns that were species and strain specific for the 16 isolates surveyed. Whereas some bands were specific for an individual isolate or species, other bands were shared by two or three species in various groupings. Differences in the mannoprotein composition of C. albicans A9 and four spontaneous cell surface mutants were also detected. Mannan "fingerprints," or banding pattern profiles, derived from the electrophoretic mobilities of individual bands relative to the migration of acid-hydrolyzed dextran (relative migration index) yielded profiles characteristic of individual isolates not revealed by standard assimilation and biochemical profiles. FACE represents an accessible, sensitive, and quantitative analytical tool enabling the characterization of yeast mannan complexity.

Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

The insect stage of the protozoan parasite Leishmania mexicana secretes a phosphomonoesterase in the form of a filamentous complex. The polypeptide subunits of this polymer are modified by phosphoglycans and/or oligomannosyl residues linked to phosphoserine. Based on peptide sequence data of a predominant 100 kDa protein of the filamentous complex, two tandemly arranged, single copy genes, lmsap1 and lmsap2, were cloned and sequenced. lmsap1 predicts a protein with features characteristic of acid phosphatases and a remarkable serine- and threonine-rich region of 32 amino acids close to the C-terminus. In the otherwise identical lmsap2 product, this region is extended to 383 amino acids and is composed of short Ser/Thr-rich repeats. Deletion analysis demonstrates that lmsap1 encodes the major 100 kDa protein of the complex while a minor 200 kDa component is derived from the lmsap2 gene. Null mutants of either gene retain the ability to secrete acid phosphatase filaments, while a deletion of both genes results in Leishmania defective in enzyme formation. The Ser/Thr-rich domains are the targets for phosphoglycan modifications as shown by the expression of secreted fusion proteins composed of these C-terminal regions and the N-terminal domain of a lysosomal acid phosphatase.

Expression of α-1,3 linkage-containing oligomannosyl residues in a cell-wall mannan of Candida tropicalis grown in yeast extract-Sabouraud liquid medium under acidic conditions

We investigated the cell-wall mannan obtained from Candida tropicalis IFO 1647 strain cells grown in yeast extract-Sabouraud medium at pH 3.0 by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The results indicate that the phosphate group and the side chains containing a β-1,2-linked mannopyranose unit decreased compared to those of mannan from cells grown under conventional conditions (pH 5.9) with concomitant expression of α-1,3 linkage-containing oligomannosyl side chains. The results of acetolysis of these mannans indicated that the presence of α-1,3-linked mannopyranose unit existed in side chains corresponding to pentaose and hexaose, Man pα1-3Man pα1-2Man pα1-2Man pα1-2Man, and Man pα1-2Man pα1-3Man pα1-2Man pα1-2Man pα1-2Man, in the mannan from cells grown at pH 3.0.

Structural identification of an epitope of antigenic factor 5 in mannans of Candida albicans NIH B-792 (serotype B) and J-1012 (serotype A) as beta-1,2-linked oligomannosyl residues.

In previous articles, we reported the presence of phosphate-bound beta-1,2-linked oligomannosyl residues in the mannans of strains of Candida albicans serotypes A and B and Candida stellatoidea. To identify the antigenic factor corresponding to this type of oligomannosyl residue, a relationship between chemical structure and antigenic specificity in the mannans of C. albicans NIH B-792 (serotype B, B-strain) and C. albicans J-1012 (serotype A, J-strain) was investigated by using a combination of two-dimensional 1H nuclear magnetic resonance spectroscopy of H-1, H-2, and H-5 regions in the mannans and an enzyme-linked immunosorbent assay that employed concanavalin A-coated microtiter plates. It was shown in the present 1H nuclear magnetic resonance study that an examination of chemical shifts not only in the H-1 region but also in the H-5 region was useful for the quantitative determination of the phosphate-bound beta-1,2-linked oligomannosyl residues. In the enzyme-linked immunosorbent assay using concanavalin A-coated plates, it was revealed that, of factor sera 1, 4, and 5, only factor serum 5 showed a reactivity proportional to the densities of the beta-1,2-linked oligomannosyl residues of the mannan subfractions of different phosphate contents that had been prepared from the bulk B-strain mannan by DEAE-Sephadex chromatography. The above results indicate that the phosphate-bound beta-1,2-linked oligomannosyl residues, Manp beta 1----(2Manp beta 1----)n2Man (n = 0-5), correspond to antigenic factor 5.

Evidence for oligomannosyl residues containing both beta-1,2 and alpha-1,2 linkages as a serotype A-specific epitope(s) in mannans of Candida albicans.

In order to identify the branches containing both beta-1,2 and alpha-1,2 linkages as the serotype A-specific epitope(s) in the mannans of Candida albicans, serotype A strains with oligosaccharides constituting the beta-1,2 linkage, the alpha-1,2 linkage, and both the beta-1,2 and the alpha-1,2 linkages were prepared from the mannans of C. albicans serotype A strains (NIH A-207 and J-1012) and tested for their inhibitory effects in the precipitin and slide agglutination assays. The results indicated that two oligosaccharides containing both beta-1,2 and alpha-1,2 linkages, Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Manp alpha 1-2Man and Manp beta 1-2Manp beta 1-2Manp alpha 1-2Manp alpha 1-2Manp alpha 1-2Man, served as epitopes participating in the serotype A specificity of C. albicans strains.

Rapid determination of immobilized ConA lectin activity.

ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-dimethacrylate and commercially available high-pressure liquid chromatography (HPLC) sorbents Separon HEMA 1000 EL, Separon HEMA 1000 E, and Separon HEMA 1000 EH (Tessek, Prague, CSFR Denmark). Specific, sensitive, and rapid method for determination of immobilized ConA lectin activity was developed. beta-Galactosidase from Aspergilus oryzae oligomannosyl residues was used as specific affinant. After separation of bound and unbound beta-galactosidase, enzyme activity was measured in supernatant and thus immobilized ConA lectin activity was calculated easily. The use of the method for evaluating the properties of immobilized ConA, efficiency of immobilization, specific activity, and thermostability is shown. The method developed could be generalized by using artificially glycosylated enzyme for any lectin.

Structural study of cell wall phosphomannan of Candida albicans NIH B-792 (serotype B) strain, with special reference to 1H and 13C NMR analyses of acid-labile oligomannosyl residues

Chemical structures of manno-oligosaccharides, from biose to heptaose, released from the phosphomannan of Candida albicans NIH B-792 strain (serotype B) by mild acid hydrolysis were investigated. The results of 1H NMR, 13C NMR, and fast atom bombardment mass spectrometry analyses confirmed that these manno-oli gosaccharides belong to a homologous β-1,2-linked series. Although chemical shifts of 1H NMR patterns of these oligosaccharides were considerably too complicated to be assigned, their 13C NMR patterns were sufficiently simple to be interpreted, exhibiting a regular increase of downfield shift of ppm values of the C-1 atom from each mannopyranose residue in proportion to their molecular weights. In order to determine the whole chemical structure of the parent phosphomannan, the acid-stable domain was subjected to acetolysis and then enzymolysis with the Arthrobacter GJM-1 α-mannosidase and the resultant manno-oligosaccharides were investigated for their chemical structures by 1H NMR spectroscopy. The results of a precipitin-inhibition test using the β-1,2-linked manno-oligosaccharides, from biose to hexaose, in comparison with the corresponding isomers containing α-1,2 linkage with small amounts of α-1,3 linkage, indicated that the haptens possessing the former linkage exhibited much higher inhibitory effects than the corresponding isomers containing the latter linkages did. Based on the present findings, a chemical structure of the phosphomannan of this C. albicans strain was proposed.

Ultracytochemical characterization of anionic sites in the wall of brain capillaries.

The chemical nature of anionic sites located on both fronts of the endothelial cells (ECs) and in the basement membrane (BM) of mouse brain capillaries was studied using tissue sections embedded in Lowicryl K4M and cationic colloidal gold. Before labelling with cationic probe, the sections were digested with the following enzymes: trypsin, papain, pronase E, proteinase K, collagenase, chondroitinase ABC, hyaluronidase, heparinase, heparitinase, neuraminidase and endoglycosidase H. The results indicate that the negatively charged surface layer on the luminal front differs in chemical nature from that on the abluminal front of the EC. Anionic sites located on the luminal surface of the plasmalemma of the ECs are mainly contributed by sialic acid residues of acidic glycoproteins. On the contrary, the anionic domains on the abluminal front of the EC represent mixed proteoglycan and acid glycopeptides containing hydrophobic amino acids, sialic acid residues, and are rich in heparan sulphate-bearing glycosaminoglycans. The anionic sites of the BM are contributed in a substantial degree by chondroitin and heparan sulphate-rich glycosaminoglycans. The effect of endoglycosidase H suggests that glycopeptides containing oligomannosyl residues linked to N-acetylglucosamine contribute in small degree in maintenance of the negative charge in the BM, but not on the surfaces of the EC. These results show that brain endothelium bears surface anionic domains differing chemically from those described for some fenestrated and continuous endothelia. The distribution of anionic sites indicates that the discrimination against various negatively charged molecules takes place on both fronts of the ECs as well as in the BM of brain micro-blood vessels. The exact role of these domains in the function of the blood-brain barrier remains to be established.

Preparation of monoclonal antibodies reactive with beta-1,2-linked oligomannosyl residues in the phosphomannan-protein complex of Candida albicans NIH B-792 strain.

Hybridomas obtained by fusing the spleen cells of BALB/c female mice hyperimmunized with heat-killed yeast-form cells of Candida albicans NIH B-792 strain and a mouse myeloma cell line, P3X63Ag8.653, produced antibodies to beta-1,2-linked oligomannosyl residues in the phosphomannan-protein complex of the parent cells. Most of these monoclonal antibodies were IgM, but about 10% of the hybridomas produced IgG1 immunoglobulins. Ascites fluid from BALB/c mice inoculated with an IgG1-producing hybridoma showed different precipitability with the phosphomannan-protein complexes of three representative C. albicans strains, with NIH B-792 (serotype B) greater than NIH A-207 (serotype A) greater than J-1012 (serotype A, formerly serotype C). In contrast, a rabbit polyclonal antiserum to C. albicans NIH B-792 cells was unable to distinguish these same complexes. This ascites fluid agglutinated the heat-killed cells of three Candida strains, but not those of three others or of Torulopsis glabrata IFO 0622. The other ascites fluids, containing antibodies of the IgM class, agglutinated cells from three C. albicans strains and also C. tropicalis IFO 0587 cells.

Teratocarcinoma stem cells have a cell surface carbohydrate-binding component implicated in cell-cell adhesion

Teratocarcinoma stem cells maintained in the undifferentiated state express a carbohydrate-binding component that recognizes oligomannosyl residues. This cell surface molecule is detected by a rosette assay in which the stem cells form rosettes with glutaraldehyde-fixed trypsinized rabbit erythrocytes. Addition of simple sugars to the assay mixture has little effect, but rosette formation is inhibited by a series of mannose-rich glycoproteins (yeast invertase, yeast mannans and horseradish peroxidase). Periodate oxidation eliminates the inhibitory activity of invertase whereas pronase digestion has little effect, indicating that carbohydrate moieties are essential for inhibition. Invertase and its glycopeptide derivatives also inhibit the reaggregation of dispersed stem cells and promote the dissociation of preformed aggregates. These results suggest that intercellular adhesion of teratocarcinoma stem cells may be the consequence of the interaction of a lectin-like component detected in the rosette assay with a complementary oligosaccharide receptor on adjacent cells.

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

Particulate preparations from developing cotyledons of Pisum sativum L. cv. Burpeeana catalyze glycosyl transfer from UDP-[(14)C]N-acetylglucosamine and GDP-[(14)C]mannose. Radioactivity is transferred to lipid components soluble in chloroform-methanol (2:1) and chloroform-methanol-water (1:1:0.3) and into a water-insoluble and lipid-free residue.The chloroform-methanol-soluble component formed from GDP-[(14)C]mannose appears to be a mannosyl lipid, whereas the chloroform-methanol-water-soluble fraction is probably a mixed oligosaccharide-lipid containing N-acetylglucosamine and mannose residues. The chloroform-methanol-soluble component formed from UDP-[(14)C]N-acetylglucosamine appears to be N,N'-diacetylchitibiosyl lipid, which may be incorporated with mannose to form the chloroform-methanol-water-soluble mixed oligosaccharide lipid.The oligosaccharide lipid appears to function as a precursor for the transfer of the oligosaccharide to the peptide moiety in the formation of the glycoproteins. The bulk of the radioactivity, arising from UDP-[(14)C]N-acetylglucosamine, incorporated into the insoluble residue, is associated with glycoprotein. In contrast only a small percentage of radioactivity in the insoluble residue, arising from GDP-[(14)C]mannose incorporation, appears to be associated with glycoprotein. The majority of the radioactivity found in the residue fraction labeled from GDP-[(14)C]mannose appears to be associated with oligomannosyl residues.