Nonreducing Terminal Residues Research Articles

Thermodynamic binding studies of cell surface carbohydrate epitopes to galectins-1, -3, and -7: Evidence for differential binding specificities

Binding of a series of sialylated and non-sialylated cell surface carbohydrates to bovine heart galectin-1, recombinant murine galectin-3, and recombinant human galectin-7 was investigated by isothermal titration microcalori metry (ITC) and hemagglutination inhibition measurements. Galectin-7 shows nearly equal affinities for lactose and Galbeta(1–4)GlcNAc (LacNAc-II). Galectin-7, however, displays six- and 11-fold weaker affinity for LacNAc-II compared with galectins-1 and -3, respectively. The affinity of galectin-7 for LacNAc-II containing oligosaccharides is also weaker than the other two galectins. ITC measurements show that all three galectins bind to di- and trimeric oligomers of LacNAc-II, which are epitopes found in poly-N-acetyllactosamine chains of glycoprotein receptors, with affinity constants similar to that of LacNAc-II. The binding valencies of the di- and trimeric LacNAc-II oligomers were observed to be one from ITC measurements, indicating formation of 1:1 complexes with all three galectins. Thus, galectins-1, -3, and -7 all possess binding sites that primarily accommodate one LacNAc-II moiety per monomer of protein. Sialylated oligosaccharides show different specificities for the three galectins. While 2,3-sialyl LacNAc-II binds to all three galectins, 2,6-sialyl LacNAc-II fails to bind to any of the galectins; 2,6-sialylated diLacNAc binds well to galectin-3 and galectin-7, but only weakly to galectin-1. Similar results are obtained with 2,6-sialyl lacto-N-neo-tetraose, which has a reducing end lactose moiety. Thus, unlike galectin-1, which predominantly recognizes non-reducing terminal LacNAc-II residues in oligosaccharides, galectins-3 and -7 recognize both non-reducing terminal LacNAc-II residues as well as internal LacNAc-II and lactose residues in sialylated and non-sialylated oligosaccharides.Key words: isothermal titration microcalorimetry, galectins, binding specificities, lectins, carbohydrates.

Mice Deficient in Nervous System-specific Carbohydrate Epitope HNK-1 Exhibit Impaired Synaptic Plasticity and Spatial Learning

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms.

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.

Human N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase cDNA is related to human B cell recombination activating gene-associated gene.

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO(4))) in chondroitin sulfate and dermatan sulfate. We have previously purified the enzyme to apparent homogeneity from the squid cartilage. We report here cloning and characterization of human GalNAc4S-6ST. The strategy for identification of human GalNAc4S-6ST consisted of: 1) determination of the amino acid sequences of peptides derived from the purified squid GalNAc4S-6ST, 2) amplification of squid DNA by polymerase chain reaction, and 3) homology search using the amino acid sequence deduced from the squid DNA. The human GalNAc4S-6ST cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 561 amino acid residues. The recombinant protein expressed from the human GalNAc4S-6ST cDNA transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the nonreducing terminal and internal GalNAc(4SO(4)) residues contained in chondroitin sulfate A and dermatan sulfate. When a trisaccharide and a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine residues were used as acceptors, only nonreducing terminal GalNAc(4SO(4)) residues were sulfated. The nucleotide sequence of the human GalNAc4S-6ST cDNA was nearly identical to the sequence of human B cell recombination activating gene-associated gene.

Rhizobial NodL O -Acetyl Transferase and NodS N -Methyl Transferase Functionally Interfere in Production of Modified Nod Factors

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between nodL and nodS, we have cloned the nodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.

Essential role of the nonreducing terminal alpha-mannosyl residues of the N-linked carbohydrate chain of bovine zona pellucida glycoproteins in sperm-egg binding.

It has been proposed that mammalian sperm bind species-specifically to carbohydrate chains of zona pellucida glycoproteins at fertilization. Although the sperm ligand carbohydrate chains have been characterized in mice and pigs, the existence of the ligands of other mammals remains unclear. In order to explore the bovine sperm ligand, two in vitro competition assay methods were applied. As a result, a high-mannose-type carbohydrate chain, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is the major neutral chain in bovine egg zona glycoproteins, was shown to possess bovine sperm ligand activity. When nonreducing terminal alpha-mannosyl residues were eliminated from the zona glycoproteins by alpha-mannosidase digestion, the ligand activity was reduced, indicating that the alpha-mannosyl residues play an essential role in bovine sperm-egg binding. The number of sperm binding to eggs was reduced to about one-half after fertilization. The ligand-active high-mannose-type chain may be buried after fertilization, since its amount remains unchanged. Pretreatment of bovine sperm with the sperm ligand-carbohydrate chain significantly inhibited penetration of the sperm into oocyte and the male pronucleus formation. Thus, a correlation between the sperm ligand activity and in vitro fertilization rate was observed.

Specificity of Microbial α-Amylase and Amyloglucosidase Hydrolysis of Methyl Amyloses

Methyl amyloses prepared under various conditions were exhaustively digested by means of α-amylase (B. licheniformis) and amyloglucosidase (A. niger). The amount of glucose that was released by the action of the enzymes was determined. Degradation products were further investigated as their O-methyl-O-deuteromethyl derivatives by mass spectrometry. The substituent distribution in the reduced and O-methylated-O-ethylated oligosaccharides was determined, differentiating between non-reducing terminal glucosyl residues, 1→4-linked inner glucosyl units and reducing glucose end groups. The portion of glucose that could be liberated by the enzymes decreased with increasing degree of substitution (DS) but at the same DS it was considerably higher for heterogeneously prepared amylose ethers than for those prepared under homogeneous conditions. Mass spectrometry (fast atom bombardment, FAB- and matrix-assisted laser desorption ionisation, MALDI-MS) gave evidence of different oligomer patterns and average values of degree of substituent/degree of polymerisation (DS/DP) in dependence on the methylation conditions applied. More detailed analysis of the O-methylated positions of the oligosaccharides showed that the reducing glucose end group was usually unsubstituted, while the non-reducing glucosyl residues could be 2-, 6- or even 2,6-di-O-substituted. In contrast, all 3-O-methyl groups were located in the inner 1,4-linked glucosyl units, indicating inhibition of both enzymes. The results of this selective partial degradation are compared with those obtained after chemical random degradation and mass spectrometry.

The occurrence of internal (1→5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1→4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1→4)-linked galactose residues bearing an arabino pyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1→4)-linked galactose residues interspersed with an internal (1→5)-linked arabino furanose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.

Molecular Cloning and Characterization of GalNAc 4-Sulfotransferase Expressed in Human Pituitary Gland

We have previously cloned chondroitin-4-sulfotransferase (C4ST) cDNA from mouse brain. In this paper, we report cloning and characterization of GalNAc 4-sulfotransferase (GalNAc4ST), which transfers sulfate to position 4 of the nonreducing terminal GalNAc residue. The obtained cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 424 amino acid residues. Identity of the amino acid sequence between GalNAc4ST and human C4ST was 30%. When the cDNA was transfected in COS-7 cells, sulfotransferase activity toward carbonic anhydrase VI was overexpressed but no sulfotransferase activity toward chondroitin or desulfated dermatan sulfate was increased over the control. Sulfation of carbonic anhydrase VI by the recombinant GalNAc4ST occurred at position 4 of the GalNAc residue of N-linked oligosaccharides. The recombinant GalNAc4ST transferred sulfate to position 4 of GalNAc residue of p-nitrophenyl GalNAc, indicating that this sulfotransferase transfers sulfate to position 4 at the nonreducing terminal GalNAc residue. Dot blot analysis showed that the message of GalNAc4ST was expressed strongly in the human pituitary, suggesting that the cloned GalNAc4ST may be involved in the synthesis of the nonreducing terminal GalNAc 4-sulfate residues found in the N-linked oligosaccharides of pituitary glycoprotein hormones.

Purification and characterization of α-galactosidase from a thermophilic fungus Thermomyces lanuginosus

An extracellular α-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl α- d-galactosides but efficiently hydrolyzed α-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl α-galactopyranoside and galactosyl 1mannotriose, followed by 1H NMR spectroscopy, pointed out the α-anomer of d-galactose was the primary product of hydrolysis from which the β-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl α-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.

Purification and Characterization ofN-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase from the Squid Cartilage

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate in chondroitin sulfate and dermatan sulfate, was purified 19,600-fold to apparent homogeneity from the squid cartilage. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a broad protein band with a molecular mass of 63 kDa. The protein band coeluted with GalNAc4S-6ST activity from Toyopearl HW-55 around the position of 66 kDa, indicating that the active form of GalNAc4S-6ST may be a monomer. The purified enzyme transferred sulfate from PAPS to chondroitin sulfate A, chondroitin sulfate C, and dermatan sulfate. The transfer of sulfate to chondroitin sulfate A and dermatan sulfate occurred mainly at position 6 of the internal N-acetylgalactosamine 4-sulfate residues. Chondroitin sulfate E, keratan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin were not efficient acceptors of the sulfotransferase. When a trisaccharide or a pentasaccharide having sulfate groups at position 4 of N-acetylgalactosamine was used as acceptor, efficient sulfation of position 6 at the nonreducing terminal N-acetylgalactosamine 4-sulfate residue was observed.

Cell wall and membrane-associated exo-beta-D-glucanases from developing maize seedlings.

A beta-D-glucan exohydrolase was purified from the cell walls of developing maize (Zea mays L.) shoots. The cell wall enzyme preferentially hydrolyzes the non-reducing terminal glucosyl residue from (1-->3)-beta-D-glucans, but also hydrolyzes (1-->2)-, (1-->6)-, and (1-->4)-beta-D-glucosyl units in decreasing order of activity. Polyclonal antisera raised against the purified exo-beta-D-glucanase (ExGase) were used to select partial-length cDNA clones, and the complete sequence of 622 amino acid residues was deduced from the nucleotide sequences of the cDNA and a full-length genomic clone. Northern gel-blot analysis revealed what appeared to be a single transcript, but three distinct polypeptides were detected in immunogel-blot analyses of the ExGases extracted from growing coleoptiles. Two polypeptides appear in the cell wall, where one polypeptide is constitutive, and the second appears at the time of the maximum rate of elongation and reaches peak activity after elongation has ceased. The appearance of the second polypeptide coincides with the disappearance of the mixed-linkage (1-->3), (1-->4)-beta-D-glucan, whose accumulation is associated with cell elongation in grasses. The third polypeptide of the ExGase is an extrinsic protein associated with the exterior surface of the plasma membrane. Although the activity of the membrane-associated ExGase is highest against (1-->3)-beta-D-glucans, the activity against (1-->4)-beta-D-glucan linkages is severely attenuated and, therefore, the enzyme is unlikely to be involved with turnover of the (1-->3), (1-->4)-beta-D-glucan. We propose three potential functions for this novel ExGase at the membrane-wall interface.

Postsource decay fragmentation of N-linked carbohydrates from ovalbumin and related glycoproteins

N-linked glycans were released from chicken ovalbumin by hydrazinolysis and examined by matrix-assisted laser desorption/ionization mass spectrometry. Postsource decay analysis showed that most fragment ions arose as the result of internal glycosidic cleavages involving loss of nonreducing terminal residues from ions that had lost one or both GlcNAc residues from the chitobiose core [GlcNAcβ(1 → 4)GlcNAc]. Cross-ring fragments were abundant from the reducing-terminal GlcNAc but other cross-ring fragments were weak. The ion found to be most useful for determining the composition of the antennae attached to the 3- or 6-linked core mannose residues was an internal cleavage ion formed by loss of both the chitobiose core and the antenna linked to the 3-position of the core branching mannose. This ion was observed to lose water in the absence of a “bisecting” GlcNAc residue (β1 → 4 linked to the core mannose) and to lose a GlcNAc molecule (221 mass units) when a bisecting GlcNAc residue was present.

Nod Factor Requirements for Efficient Stem and Root Nodulation of the Tropical Legume Sesbania rostrata

Azorhizobium caulinodans ORS571 synthesizes mainly pentameric Nod factors with a household fatty acid, an N-methyl, and a 6-O-carbamoyl group at the nonreducing-terminal residue and with a d-arabinosyl, an l-fucosyl group, or both at the reducing-terminal residue. Nodulation on Sesbania rostrata was carried out with a set of bacterial mutants that produce well characterized Nod factor populations. Purified Nod factors were tested for their capacity to induce root hair formation and for their stability in an in vitro degradation assay with extracts of uninfected adventitious rootlets. The glycosylations increased synergistically the nodulation efficiency and the capacity to induce root hairs, and they protected the Nod factor against degradation. The d-arabinosyl group was more important than the l-fucosyl group for nodulation efficiency. Replacement of the 6-O-l-fucosyl group by a 6-O-sulfate ester did not affect Nod factor stability, but reduced nodulation efficiency, indicating that the l-fucosyl group may play a role in recognition. The 6-O-carbamoyl group contributes to nodulation efficiency, biological activity, and protection, but could be replaced by a 6-O-acetyl group for root nodulation. The results demonstrate that none of the studied substitutions is strictly required for triggering normal nodule formation. However, the nodulation efficiency was greatly determined by the synergistic presence of substitutions. Within the range tested, fluctuations of Nod factor amounts had little impact on the symbiotic phenotype.

Inverting character of α-glucuronidase A from Aspergillus tubingensis

α-Glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4- O-methyl- D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4- O-methyl- D-glucuronosyl-α-1,2- D-xylopyranosyl-β-1,4-xylopyranosyl-β-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D 2O reaction mixture by 1H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its β-anomer from which the α-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial α-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).

Differentiation of O-Acetyl and O-Carbamoyl esters of N-Acetyl-Glucosamine by decomposition of their oxonium ions. application to the structure of the nonreducing terminal residue of Nod factors

Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-α-D-glucosamine were synthesized. Oxonium ions (MH − CH 3OH) + were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B 1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B 1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.

Sialic acid: new potential marker of alcohol abuse.

A number of laboratory markers are suggested for the detection and monitoring of alcohol abuse. However, there is still a need to find better indicators of alcohol abuse. Sialic acid (SA) is the name for a series of acyl-derivatives of neuraminic acids that occur as nonreducing terminal residues of glycoproteins or glycolipids in biological fluids and cell membranes. In this study, we investigated the diagnostic value of SA as a marker of alcohol abuse. Sera from social drinkers (n = 38) and alcoholics (n = 77) were analyzed for sialic acid by a colorimetric assay and for carbohydrate-deficient transferrin (CDT) by a radioimmunoassay method. Mean corpuscular volume (MCV), gamma-glutamyltransferase (GGT), aspartate aminotransferase (ASAT), and alanine aminotransferase (ALAT) were determined by using routine methods. The sialic acid levels of both female and male subjects were significantly (p < 0.001) increased among alcoholic subjects when compared with social drinkers. SA levels were decreased after 3 weeks of treatment. The sensitivity and specificity for SA, respectively, were 57.7 and 95.5 for women and 47.8 and 81.3 for men. The respective values for CDT were 57.7 and 95.5 for women and 78.3 and 100.0 for men; for GGT, 60.0 and 95.5 for women and 60.9 and 87.5 for men; for MCV, 52.4 and 95.5 for women and 47.8 and 100.0 for men; for ASAT, 53.8 and 95.5 for women and 43.5 and 100.0 for men; and for ALAT, 38.5 and 90.9 for women and 39.1 and 87.5 for men. Among women, SA and GGT, and among men CDT, showed the largest area under receiver operation curve. This study indicated that sialic acid levels were elevated by high alcohol consumption and reduced during abstinence, especially among women. Thus, sialic acid seems to be an interesting marker that needs further evaluation as a diagnostic tool for alcohol abuse.

Structural characterisation of lipo-chitin oligosaccharides isolated from Bradyrhizobium aspalati, microsymbionts of commercially important South African legumes

The shoots of the South African legume Aspalathus linearis spp. linearis ( A. linearis) are used in the manufacture of an increasingly popular beverage that has acclaimed beneficial effects on health; this important export product is known as Rooibos (or Redbush) tea. Three strains of Bradyrhizobium aspalati, which are the nitrogen-fixing symbionts of Aspalathus carnosa, A. hispida and A. linearis, were tested for the production of lipo-chitin oligosaccharide signal molecules using thin-layer chromatographic analysis after induction with different inducers, including Rooibos tea extract, and radioactive labelling. Large-scale separation, using high-performance liquid chromatography, of lipo-chitin oligosaccharides from B. aspalati isolated from A. carnosa was performed for structural characterisation using fast-atom bombardment mass spectrometry and chemical modifications followed by gas chromatography–mass spectrometric analysis. The strain was shown to secrete a family of unusual lipo-chitin oligosaccharides that are highly substituted on the nonreducing-terminal residue but unsubstituted on the reducing-terminal residue. They have a backbone of three to five β-(1→4)-linked N-acetyl- d-glucosamine residues substituted on the nonreducing terminus with a C16:0, C16:1, C18:0, C18:1, C19:1cy, or C20:1 fatty acyl chain, and are both N-methylated and 4,6-dicarbamoylated.

Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid

A lectin from the fruiting body of Psathyrella velutina has been used as a specific probe for non-reducing terminal N-acetylglucosamine residues. We reveal in this report that P. velutina lectin recognizes a non-reducing terminal N-acetylneuraminic acid residue in glycoproteins and oligosaccharides. Binding of biotinyl P. velutina lectin to N-acetylneuraminic acid residues was prevented by desialylation of glycoconjugates and was distinguished from the binding to N-acetylglucosamine. Sialooligosaccharides were retarded or bound and eluted with N-acetylglucosamine on a P. velutina lectin column, being differentiated from each other and also from the oligosaccharides with non-reducing terminal N-acetylglucosamine which bound more strongly to the column.