Peanut (Arachis hypogaea) lectin recognizes alpha-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galbeta1-->3GalNAc-), but not its sialylated version. However, an additional specificity towards Galbeta1-->4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galbeta1-->4GlcNAc (LacNAc) was ineffective while terminal alpha-linked galactose (TAG) as well as exposed T antigen (Galbeta1-->3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-alpha-galactoside antibody.     

Enzymatic production of N-acetyl- -glucosamine from chitin. Degradation study of N-acetylchitooligosaccharide and the effect of mixing of crude enzymes

N-Acetyl- -glucosamine (GlcNAc) was produced from chitin by use of crude enzyme preparations. The efficient production of GlcNAc by cellulases derived from Trichoderma viride (T) and Acremonium cellulolyticus (A) was observed by HPLC analysis compared to lipase, hemicellulase, and pectinase. β-Chitin showed higher degradability than α-chitin when using cellulase T. The optimum pH of cellulase T was 4.0 on the hydrolysis of β-chitin. The yield of GlcNAc was enhanced by mixing of cellulase T and A.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea

Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the synthesis of an α-(1→4)-linked glucan polymer from sucrose instead of an expensive activated sugar, such as ADP- or UDP-glucose. Transglycosylation reactions mediated by the ASases of Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were applied to the synthesis of salicin glycosides with sucrose serving as the glucopyranosyl donor and salicin as the acceptor molecule. Two salicin glycoside transfer products were detected by TLC and HPLC analyses. The synthesis of salicin glycosides was very efficient with NPAS with a yield of over 90%. In contrast, DGAS specifically synthesized only one salicin transglycosylation product. The transglycosylation products were identified as α-d-glucopyranosyl-(1→4)-salicin (glucosyl salicin) and α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-salicin (maltosyl salicin) by NMR analysis. The ratio between donor and acceptor had a significant effect on the type of product that resulted from the transglycosylation reaction. With more acceptors present in the reaction, more glucosyl salicin and less maltosyl salicin were synthesized.     

Efficient enzymatic synthesis of the sialyl-Lewis tetrasaccharide: A ligand for selectin-type adhesion molecules

Sialyl-Lewis^x (NeuAcα2→3Galβ1→4[Fucαl→3]GlcNAc] has been identified as a ligand for E-selectin, P-selectin and recently also for L-selectin. We have synthesized the sialyl-Lewis^x tetrasaccharide by total enzymatic synthesis from N-acetyllactosamine using a placental α2→3-sialyltransferase specific for type-2 chain acceptors, followed by a cloned human α1→3-fucosyltransferase (FucTV, the ‘plasma-type’ enzyme). This procedure resulted in the tetrasaccharide in a 61% overall yield.     

Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: Differential expression during the miracidium-to-sporocyst transformation

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail–schistosome interactions. Interestingly, Fucα2Fucα3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.     

Enzymatic syntheses of T antigen-containing glycolipid mimicry using the transglycosylation activity of endo-α-N-acetylgalactosaminidase

Thomsen–Friedenreich antigen (T antigen) disaccharide, β- -galactose-(1→3)-α-N-acetyl- -galactosamine (β- -Gal-(1→3)-α- -GalNAc), containing glycolipid mimicry was synthesized using the transglycosylation activity of endo-α-N-acetylgalactosaminidase from Bacillus sp. This enzyme could transfer the disaccharide from a p-nitrophenyl substrate to water-soluble 1-alkanols and other alcohols at a transfer ratio of 70% or more. Although the transfer ratios were lower for water-insoluble than water-soluble alcohols, they were shown to increase by adding sodium cholate to the reaction mixtures. The enzyme also transferred the disaccharide directly from asialofetuin to 1-alkanols. The anomeric bond between the disaccharide and 1-alkanols of the transglycosylation product is in the α configuration as determined by sequential digestion of jack bean β-galactosidase and Acremonium α-N-acetylgalactosaminidase. Since the transglycosylation product, β- -Gal-(1→3)-α- -GalNAc-(1→O)-hexyl, efficiently inhibits the binding of anti-T antigen monoclonal antibody to asialofetuin, it has potential as an agent for blocking T antigen-mediated cancer metastasis.     

Structural characterization of an active polysaccharide from Phellinus ribis

A water-soluble polysaccharide named as PRP was isolated from the fruiting bodies of Phellinus ribis by hot water extraction, DEAE-cellulose and Superdex 30 column chromatography. Its structural characteristics were investigated by FT-IR, NMR spectroscopy, GLC-MS, methylation analysis, periodate oxidation and Smith degradation. Based on the data obtained, PRP was found to be a β-d-glucan containing a (1 → 4), (1 → 6)-linked backbone, with a single β-d-glucose at the C-3 position of (1 → 6)-linked glucosyl residue every eight residues, along the main chain. The glucan has a weight-average molecular weight of about 8.59 kDa by HPGPC determination using dextran samples as the standards. Preliminary activity tests in vitro revealed that PRP could stimulate the proliferation of spleen lymphocyte.     

Morphological and structural characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its anti-exercise fatigue activity

The crude polysaccharide was obtained from Gynostemma pentaphyllum Makino by water extraction followed by ethanol precipitation. The polysaccharide was successively purified by chromatography on DEAE-52 and SephadexG-150 column, and three polysaccharide fractions were obtained and termed GPP1-a, GPP2-b, and GPP3-a, respectively. The administration with GPP1-a markedly prolonged exhaustive exercise time of the mice. Structural features of GPP1-a were investigated by a combination of instrumental and chemical analyses, including atomic force microscope (AFM), scanning electron microscope (SEM), partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, gas chromatography–mass spectrometry (GC–MS) analysis and NMR spectroscopy. The results indicate that GPP1-a has a backbone of (1 → 4)-linked α-d-Glucose residues, which occasionally branches at O-6. The branches are mainly composed of (1 → 6)-linked α-d-Glucose, (1 → 3)-linked β-d-Galactose and (1 → 6)-linked α-d-Galactose residues, and terminated with β-d-Galactose residues and β-l-Arabinose residues.     

High-Performance Capillary Electrophoretic Characterization of Different Types of Oligo- and Polysialic Acid Chains

We have carried out comparative structural analysis of novel oligo- and polysialic acid chains from diverse sources. Controlled acid hydrolysates of (a) colominic acid, α2→8-linked homopolymer ofN-acetylneuraminic acid (Neu5Ac), (b) α2→8-linked oligo/polyNeu5Gc chains present in rainbow trout egg polysialoglycoprotein, and (c) α2→8-linked oligomers of deaminoneuraminic acid (KDN) residues of KDN-rich glycoprotein derived from rainbow trout vitelline envelope were analyzed by high-performance capillary electrophoresis (HPCE). The results showed that three different types of α2→8-linked oligosialic acids having same degree of polymerization can be separated by HPCE. A partial hydrolysate of colominic acid with mild acid was shown by CE to form intramolecular esters during the controlled hydrolysis and the subsequent workup procedure. In contrast, lactonization of (→5-O_glycolyl-Neu5Gcα2→)n, α2→5-O_glycolyl-linked homopolymer ofN-glycolylneuraminic acid (Neu5Gc) present in the egg jelly coat of sea urchin, did not take place as readily as in (→8Neu5Acα2→)n.     

Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man_13–14GlcNAc₂, and in Kloch1 mnn1 mutant, contained oligosaccharide Man_9–11GlcNAc₂, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man_>30GlcNAc₂. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.     

Xanthone O-glycosides from Polygala tenuifolia

Four xanthone O-glycosides, polygalaxanthones IV–VII were isolated from the roots of Polygala tenuifolia Willd., together with eight known compounds. The structures of the four xanthone O-glycosides were established as 6-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1-hydroxy-3,7-dimethoxyxanthone (polygalaxanthone IV), 6-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1,3-dihydroxy-7-methoxyxanthone (polygalaxanthone V), 6-O-(β- -glucopyranosyl)-1,2,3,7-tetramethoxyxanthone (polygalaxanthone VI), and 3-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1,6-dihydroxy-2,7-dimethoxyxanthone (polygalaxanthone VII), respectively, on the basis of analysis of spectroscopic evidence.     

Enzymatic synthesis of N-acetylglucosaminobioses by reverse hydrolysis: characterisation and application of the library of fungal β-N-acetylhexosaminidases

The regioselectivity of 20 extracellular β-N-acetylhexosaminidases of fungal origin was screened in the reverse hydrolysis with 2-acetamido-2-deoxy- -glucopyranose. Most of the enzymes used yielded 2-acetamido-2-deoxy-β- -glucopyranosyl-(1→4)-2-acetamido-2-deoxy- -glucopyranose (3) and 2-acetamido-2-deoxy-β- -glucopyranosyl-(1→6)-2-acetamido-2-deoxy- -glucopyranose (4). So far unknown product of enzymatic condensation, 2-acetamido-2-deoxy-β- -glucopyranosyl-(1→3)-2-acetamido-2-deoxy- -glucopyranose (2) was synthesised using the β-N-acetylhexosaminidases from Penicillium funiculosum CCF 1994, P. funiculosum CCF 2325 and Aspergillus tamarii CCF 1665. Addition of salts ((NH₄)₂SO₄ or MgSO₄ (0.1–1.0 M)) to the reaction increased the yields and also enhanced the β-N-acetylhexosaminidase regioselectivity.     

An Enzyme-Linked Lectin Assay for α1,3-Galactosyltransferase

UDP-Gal:Galβ1-4GlcNAc α1,3-galactosyltransferase (α3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galα1-3Galβ1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of α3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galβ1-4GlcNAc (acceptor) are incubated with α3GalT in the presence of “cold” UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of α3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of α3GalT, no enzymatic conversion of Le^x into GalαLe^x was observed using this assay. Human αGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.     

Saponins from Albizzia lucida

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-α- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[α- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

A spirostane hexaglycoside from Agave cantala fruits

A new steroidal glycoside, agaveside D, isolated from the fruits of Agave cantata was characterized as 3β-{α- -rhamnopyranosyl-(1→2), β- -glycopyranosyl-(1→3)-β- -glucopyranosyl[β- -xylopyransoyl-(1→4)-α- -rhamnopyranosyl-(1→2)]-β- -glucopyranosyl}-25R-5α- spirostane on the basis of chemical degradation and spectrometry.     

Glycoprofile of the different cell types present in the mucosa of the horse guttural pouches

Histochemical characterization of the equine guttural pouches was performed using lectins combined with sialidase digestion and deglycosylation pre-treatments.The goblet cells contained O- and N-linked oligosaccharides with α-Fuc, GlcNAc moieties whereas β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues belonged only to O-linked glycoproteins. The acinar and ductal cells expressed α-Man/α-Glc in N-linked oligosaccharides, GlcNAc in both O- and N-glycoproteins and β-GalNAc, β-Gal-(1–3)-GalNAc, β-Gal-(1–4)-GlcNAc and α-Gal residues included in O-linked glycoproteins. The Golgi area of the epithelial lining expressed α-Fuc in O-linked glycoproteins, internal GlcNAc in N-linked glycoproteins and large amounts of sialic acid residues linked to subterminal β-GalNAc, Galβ1,4GlcNAc and Galβ1,3GalNAc. High amounts of sulpho-carbohydrates and of sialic acids (α2,3–6), linked to-α/β-Gal and sialic acids (α2–6) linked to β-GalNAc, were also demonstrated.Such diversity of the mucin saccharide residues may be implicated in the binding of macromolecules such as those of bacterial or viral etiology, thus playing a role in the organism's host-defense mechanism in the guttural pouches.     

Water-soluble polysaccharide from Bupleurum chinense DC: Isolation, structural features and antioxidant activity

The water-soluble polysaccharide (BCPS-1) was isolated from Bupleurum chinense DC. BCPS-1 (M_w = 29 kDa) was composed of Ara; Gal; Glc with a molar ratio of 2.1:2.5:1. According to FT-IR, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC-MS analysis, the results indicate BCPS-1 had a backbone of (1→5)-linked Ara, (1→4)-linked Gal and (1→3)-linked Gal residues with occasionally branches at O-6. The branches were composed of (1→4)-linked Glc, and terminated with Gal residues. The in vitro antioxidant activity evaluated by DPPH radical scavenging method showed that BCPS-1 had a significant antioxidant effect in a concentration-dependent manner.     

Glycan-binding profile of a D-galactose binding lectin purified from the annelid, Perinereis nuntia ver. vallata

A lectin recognizing D-galactose was purified from the pacific annelid Perinereis nuntia ver. vallata (Polychaeta) by affinity chromatography. Hemagglutinating activity, with a very low titer suggesting the presence of lectin appeared in the supernatant from the homogenization of body with Tris-buffered saline. However, dialyzed supernatant from the precipitate homogenized by galactose in the buffer revealed strong hemagglutinating activity against human erythrocytes. The crude supernatant was applied onto lactosyl–agarose column, and only the supernatant eluted from precipitate with galactose was obtained a galactose-binding lectin with 32 kDa polypeptide was obtained from the supernatant of the precipitate, extracted in presence of galactose. It suggests that the lectin tightly binds with glycoconjugate as endogenous ligand(s) in the tissue. Hemagglutinating activity against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-galactose, N-acetyl-D-galactosamine, lactose, melibiose, and asialofetuin. Glycan-binding profile of the lectin analyzed by frontal affinity chromatography shows that the lectin recognizes branched complex type N-linked oligosaccharides and both type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) lactosamine. The surface plasmon resonance study of the lectin against asialofetuin showed the k_ass and k_diss values are 5.14 × 10⁴ M^− 1 s^− 1 and 2.9 × 10⁻³ s^− 1, respectively. The partial primary structure of the lectin reveals 182 amino acids with novel sequence.