Beta Linkage Research Articles

Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides.

A minor component of the major urinary protein complex of the house mouse was chromatographically isolated and ascertained to be a previously suspected glycoprotein. Using highly sensitive mass-spectrometric techniques for sequencing and linkage analysis, the N-linked oligosaccharides of this glycoprotein were characterized. They were determined to be of the complex type with a wide heterogeneity. The heterogeneity was due to both the degree of sialylation and the presence of galactose residues in either beta(1-3) or beta(1-4) linkages. The biantennary structures were the most pronounced glycans, while tri- and tetraantennary entities were minor.

NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans.

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, Ser(GlcpAbeta(1-->3) Galpbeta(1-->3)Galpbeta(1-->4)Xylpbeta)-Gly-Ser-Gly-Se r (GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)Xylp beta)-Gly (1), and the tetrasaccharide dipeptide, Ser(GlcpAbeta(1-->3)Galpbeta(1-->3)Galpbeta(1-->4)X ylpbeta)-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt4 C 1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The straight phi torsion angles of the glycosidic C1'-O1 bonds showed a strict preference for the -sc range whereas the psi torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for beta(1-->3) linkage, +ac for beta(1-->4) linkage). The predominant conformation about the glycopeptide bond is straight phi = -sc and psi = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products.

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.

Molecular dynamics simulation of glycoprotein-glycans of immunoglobulin G and Immunoglobulin M

Glycoprotein-glycans have recently been implicated to play a variety of functional roles. The same glycan chain have been found complexed with proteins of diverse functions. In this article two such glycan chains found attached to Fc regions of immunoglobulin G and immunoglobulin M have been studied. An extensive simulated annealing procedure have been adopted to arrive at a low-energy minimum of the two oligosaccharides. Molecular dynamics simulations have been performed to study the flexibility of the glycosidic linkages. It was found that both glycan chains can undergo conformational transitions and adopt folded and extended conformations. The two beta (1-2) linkages of complex-type glycan had been found to prefer different conformational regime and the terminal fucose linked to the GlcNAc residue drastically modifies the GlcNAc beta (1-4) GlcNAc linkage conformation. In the high-mannose type glycan chain alpha (1-3) linkages can induce flexibility in addition to the alpha (1-6) linkages. The results have been compared with recent experimental nmr and fluorescence energy transfer data.

A functional analysis of the Streptococcus pneumoniae genes involved in the synthesis of type 1 and type 3 capsular polysaccharides.

Type 3 pneumococci produce a capsule composed of cellobiuronic acid units connected in a beta (1-->3) linkage. Cellobiuronic acid is a disaccharide consisting of D-glucuronic acid (GlcA) beta (1-->4) linked to D-glucose (Glc). The genes implicated in the biosynthesis of the type 3 capsule have been cloned, expressed, and biochemically characterized. The three type 3-specific genes--designated as cap3ABC--are transcribed together. However, the two complete open reading frames located upstream of cap3A are not transcribed and, consequently, are not required for capsule formation. The promoter of the cap3 operon was localized by primer extension analysis. The products of cap3A, cap3B, and cap3C were biochemically characterized as a UDP-Glc dehydrogenase, the type 3 polysaccharide synthase, and a Glc-1-P uridyltransferase, respectively. The Cap3B synthase was expressed in Escherichia coli, and pneumococcal type 3 polysaccharide was synthesized in this heterologous system. When a recombinant plasmid (pLSE3B) containing cap3B was introduced by transformation into encapsulated pneumococci of types 1, 2, 5, or 8, the lincomycin-resistant transformants displayed a binary type of capsule, this is, they showed a type 3 capsule in addition to that of the recipient type. Unencapsulated (S2) laboratory strains of S. pneumoniae also synthesized a type 3 capsule when transformed with pLSE3B. On the other hand, we have cloned and sequenced seven type 1-specific genes (designated as cap1A-G), and their functions have been preliminarily assigned based on sequence similarities.

Transplanting two unique beta-glucanase catalytic activities into one multienzyme, which forms glucose.

Endo cellulases of plant pathogenic erwinias degrade cellulose as well as the cellulosic domains of barley (1-3,1-4)-beta-glucan. Depolymerization of the latter substrate is mainly caused by (1-3,1-4)-beta-glucanases, which hydrolyze (1-4)-beta glycosidic linkages adjacent to (1-3)-beta linkages. To construct an enzyme for efficient degradation of barley (1-3,1-4)-beta-glucan, the sequence encoding the catalytic domain and interdomain linker of the cellulase from Erwinia carotovora subspecies atroseptica was fused to that for the heat stable Bacillus hybrid, H(A12-M) delta Y13 (1-3,1-4)-beta glucanase. The chimeric enzyme secreted from Escherichia coli cells did not remain covalently assembled as judged by SDS-PAGE. However, the glycosylated and intact enzyme (denoted CELGLU) is secreted from the yeast Pichia pastoris. CELGLU exhibits both cellulase and (1-3,1-4)-beta-glucanase catalytic activities, and was accordingly classified a true multienzyme. HPLC and NMR analyses revealed that among the products from CELGLU, di- and trimeric oligosaccharides were identical to those produced by the parental cellulase. Tetrameric oligosaccharides, derived from the (1-3,1-4)-beta-glucanase activity of CELGLU, were further degraded by the cellulase moiety to yield glucose and trimers. Compared with the parental enzymes, CELGLU exhibits substantially higher Vmax for degradation of both soluble cellulose and barley (1-3,1-4)-beta-glucan. These findings point to construction of multienzymes as an effective approach for engineering enzymes with novel characteristics.

Preparation of antisera to recombinant, soluble N-acetylglucosaminyltransferase V and its visualization in situ.

N-Acetylglucosaminyltransferase V (GlcNAc-T V) is a glycosyltransferase which transfers N-acetylglucosamine in beta(1,6) linkage to the alpha(1,6)-linked mannose residue of Asn-linked oligosaccharides. This enzyme is characterized by several unusual properties: GlcNAc-T V is the largest lumenal Golgi glycosyltransferase described thus far, and its multiple mRNA transcripts range from 4.5 to about 9.5 kb; GlcNAc-T V mRNA and activity are regulated by the src tyrosine kinase signalling pathway; in brain tissue, large levels of GlcNAc-T V mRNA are present, but only relatively low levels of catalytic activity can be detected; a lectin-resistant cell line, Lec4A, expresses active GlcNAc-T V which is mislocalized intracellularly. In addition, the cell surface oligosaccharide products of this enzyme have been hypothesized to regulate intercellular adhesion. In order to devise specific inhibitors of this enzyme it is necessary to understand its physical structure and how structural changes can influence its activity and localization. We have expressed milligram amounts of a soluble form of recombinant rat GlcNAc-T V, purified it from CHO cell-conditioned media, and used it to prepare specific antisera. This antisera binds selectively to GlcNAc-T V and has been used to visualize B-16 mouse melanoma cell GlcNAc-T V on immunoblots after SDS-PAGE. When the antisera was used in immunofluorescence microscopy experiments on permeabilized B-16 and baby hamster kidney cells, intense, specific staining was observed in intracellular structures which appear to correspond to the Golgi apparatus.

Genomic organization of human complement protein C8 alpha and further examination of its linkage to C8 beta.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8 alpha, C8 beta, C8 gamma) encoded by separate genes. C8 alpha and C8 beta are linked on chromosome 1p32, whereas C8 gamma is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 alpha gene. The gene contains at least 11 exons spanning approximately 70 kb of DNA. When compared to C6, C8 beta and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 alpha and C8 beta linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5 kb) and in a 5' alpha-beta 3' orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.

Architecture of the Yeast Cell Wall: THE LINKAGE BETWEEN CHITIN AND β(1→3)-GLUCAN

To isolate the putative linkage region between chitin and beta(1-->3)-glucan, Saccharomyces cerevisiae cell walls were digested with beta(1-->3)-endoglucanase and the reducing ends of the enzyme-resistant glucose chain stubs were labeled by reduction with borotritide. The radioactive material was further digested with exochitinase to remove the bulk of the chitin, and the liberated oligosaccharides were fractionated on a sizing column. A single peak (compound I) was found to consist of N-acetylglucosamine, glucose, and glucitol residues in the ratio 1:2:1. By digestion with beta-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing terminus, linked to laminaritriitol by a beta(1-->4) bond. Five additional oligosaccharides were recovered, two being analogs of compound I, with 1 or 3 glucose units, respectively; the remaining three were shown to be the reduced analogs of laminaribiose, laminaritriose, and laminaritetraose. The presence of N-acetylglucosamine-containing oligosaccharides arises from the activity of chitinase in cleaving 2 sugar units sequentially in those chains containing an odd number of N-acetylglucosamine residues; correspondingly, oligosaccharides containing only glucose and sorbitol derived from even-numbered chitin chains, a result implying that chitinase can hydrolyze the linkage between N-acetylglucosamine and glucose. It is concluded that the terminal reducing residue of a chitin chain is attached to the nonreducing end of a beta(1-->3)-glucan chain by a beta(1-->4) linkage. Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chitin synthetase 3. The timing and possible mechanism of formation of the chitin-glucan linkage is discussed.

Purification and characterization of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-GlcNAc transferase from mouse kidney.

The Gsl-5 gene mapped on mouse chromosome 19 controls the expression of IV6 beta Gal beta 1-4(Fuc alpha 1-3)GlcNAc, IV3 beta Gal-Gb4Cer in mouse kidney through regulation of the activity of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-Glc-NAc transferase (GNT), which transfers GlcNAc to the C-6 position of GalNAc of IV3 beta Gal-Gb4Cer (GL-X). Here we report that GNT has been purified to apparent homogeneity from mouse kidney by means of preparation of a microsomal fraction, solubilization with Triton X-100, and sequential column chromatographies on CM-Sepharose, UDP-hexanolamine-Sepharose, and Gg4Ose-Aminocellulo-fine. GNT purified 11,000-fold from the microsomal fraction exhibited a specific activity of 26.15 mumol/min/mg protein and an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. The Km values for UDP-GlcNAc and GL-X were 0.36 and 0.11 mM, respectively. Among glycolipids tested as substrates, GL-X was the best and Gg4Cer the next best, but Lc3Cer, Gb4Cer, and GM1 did not act as a substrate. GNT was also able to transfer GlcNAc to Gal beta 1-3GalNAc beta 1-benzyl and Gal beta 1-3GalNAc alpha 1-p-nitrophenyl at C-6 of GalNAc through beta linkage but not to GlcNAc beta-3GalNAc alpha 1-p-nitrophenyl. The purified GNT was digested with lysyl endopeptidase, and four peptides generated were sequenced. The sequence of four peptides spanning 35 amino acid residues in total exhibited 80% homology with that of the reported human core 2 GlcNAc transferase.

The solution conformation of hyaluronan: a combined NMR and molecular dynamics study.

Hyaluronan (HA) is a negatively charged glycosaminoglycan that exhibits a wide variety of biological effects mediated by binding to cell-surface and extracellular matrix proteins (hyaladherins). Short HA oligosaccharides have been shown to retain the specific interactions and biological effects of high molecular weight HA. Although it has a simple disaccharide repeating unit, the aqueous solution conformation of HA has been very difficult to determine because of strong coupling and overlapping resonances. In this study, we propose aqueous solution conformations for an octasaccharide of HA, derived from proton-proton NOE data and restrained molecular dynamics. To overcome spectral overlap and strong coupling, alternate methods for extracting distance restraints were employed. Restrained molecular dynamics calculations yielded one set of interglycosidic angle values for the beta (1,3) linkage (phi 13 = 46 degrees, psi 13 = 24 degrees). In contrast, two sets of values for the beta (1,4) linkage were consistent with the NOE restraints (phi 14 = 24 degrees, psi 14 = -53 degrees or phi 14 = 48 degrees, psi 14 = 8 degrees). The potential difference in flexibility for the two linkages is consistent with unrestrained as well as the restrained molecular dynamics trajectories described here. The conformational parameters obtained from restrained molecular dynamics are used to predict helical parameters of high molecular weight HA and will provide a basis for studies of HA binding to proteins.

Gene XV of Bacteriophage PRD1 Encodes a Lytic Enzyme with Muramidase Activity

Bacteriophage PRD1 is a lipid-containing virus that infects a variety of Gram-negative bacteria, including Escherichia coli. The phage lyses its host by virtue of a virally-encoded lytic enzyme, the synthesis of which has been assigned to gene XV on the basis of complementation analysis and experiments with mutant phages. We report here the cloning of gene XV into an expression plasmid and the purification of its product, protein P15, to near homogeneity. The purified protein P15, identified by N-terminal sequence analysis, showed a strong lytic activity against chloroform-treated Gram-negative cells. No activity against Gram-positive bacterial species could be detected. The pH optimum of the enzyme was between 7.0-8.0. Protein P15 was readily inactivated at temperatures above 4 degrees C, as well as by increasing the ionic strength of the buffers. The analysis of cell wall digests indicated that P15 is a glycosidase that cleaves the beta (1-4) linkage between N-acetylmuramic acid and N-acetylglucosamine, thus displaying muramidase activity.

Enzymatic synthesis of alkyl β‐D‐xylosides by transxylosylation and reverse hydrolysis

The Trichoderma reesei beta-xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl beta-D-xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl beta-D-xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium-controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium-controlled synthesis. The specificity of the beta linkage is confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. (c) 1994 John Wiley & Sons, Inc.

Molecular dynamics simulation of oligosaccharides containing N‐acetyl neuraminic acid

alpha D-N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an alpha(2-->3) or alpha(2-->6) linkage. In the cell surface receptor, sialyl-LewisX, a terminal alpha(2-->3) linkage is present. Previous studies from our laboratory have shown that in solution LewisX adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac alpha(2-->3) Gal beta-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10-50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac alpha(2-->3) Gal beta linkage.

Purification and characterization of UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta-1,3-N-acetylglucosaminyltransferase (poly-N-acetyllactosamine extension enzyme) from calf serum.

UDP-GlcNAc:Gal beta 1-4Glc(NAc) beta-1,3-N-acetylglucosaminyltransferase is involved in the initiation and the extension of poly-N-acetyllactosamine biosynthesis. This enzyme has been purified to about 125,000-fold with a 0.2% yield from calf serum. The purification was achieved by ammonium sulfate precipitation and chromatography on concanavalin A-Sepharose, DEAE-Toyopearl, SP-Toyopearl, Sephacryl S-200, AF-Blue-Toyopearl, and Mono Q columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band corresponding to an apparent M(r) of 70,000. This component was specifically photoaffinity-labeled with 4-thiouridine diphosphate. Exoglycosidase digestion and methylation analysis of the reaction products demonstrated that the enzyme catalyzed the transfer of one N-acetylglucosamine to position C-3 of the terminal galactosyl residue of lactose or N-acetyllactosamine in the beta linkage. The enzyme required Mn2+ ions for its activity and showed a broad pH optimum around 7.0. Apparent Km values for lactose, N-acetyllactosamine, and UDP-GlcNAc were 18.2, 19.6, and 0.129 mM, respectively. Acceptor specificity was tested using several oligosaccharides. The results indicated that terminal Gal beta 1-4Glc(NAc) sequences (type II chains) were preferred substrates for the enzyme. Terminal Gal beta 1-3GlcNAc sequences (type I chains), Lewis X trisaccharides (Gal beta 1-4(Fuc alpha 1-3)GlcNAc), and monosaccharides (galactose) did not serve as substrates.

Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes.

A major problem with pig-to-human-tissue xenograft studies is that humans have natural antibodies to pig cells; these antibodies would cause hyperacute rejection if pig tissues were xenografted to humans. Here we show that most of human IgM antibodies present in the serum of healthy donors and reactive with pig cells react with galactose in an (alpha 1-3) linkage with galactose--i.e., Gal(alpha 1-3)Gal. Absorption studies demonstrated that the antibodies detected the same or similar epitopes on the surface of pig erythrocytes, blood and splenic lymphocytes, and aortic endothelial cells (EC). The antibodies were sensitive to 2-mercaptoethanol (2ME) treatment, did not bind to protein A or G, and were present in the high molecular weight fraction of serum; they are clearly IgM antibodies. Further, the antibodies did not react with human ABO blood group substances and are not related to human blood group A or B, which carry a terminal galactose. The reaction of human serum with pig erythrocytes was specifically inhibited by mono- and disaccharides: D-galactose, melibiose, stachyose, methyl-alpha-D-galactopyranoside, and D-galactosamine but not by D-glucose or methyl-beta-D-galactopyranoside; demonstrating that the reaction is with galactose in an alpha and not a beta linkage. A cDNA clone encoding the murine alpha-1,3-galactosyltransferase (which transfers a terminal galactose residue with an (alpha 1-3) linkage to a subterminal galactose) was isolated by polymerase chain reaction (PCR), cloned, and transfected into COS cells, which are of Old World monkey origin and, like humans, do not express Gal(alpha 1-3)Gal. After transfection, COS cells became strongly reactive with human serum and with IB4 lectin [which reacts only with Gal(alpha 1-3)Gal]; this reactivity could be removed by absorption with pig erythrocytes. As most of the antibody reacting with pig cells can be removed by absorption with either melibiose or Gal(alpha 1-3)Gal+ COS cells, most of these react with Gal(alpha 1-3)Gal. These findings provide the basis for genetic manipulation of the pig alpha-1,3-galactosyltransferase for future transplantation studies.

Preferred conformations and dynamics of five core structures of mucin typeO-glycans determined by NMR spectroscopy and force field calculations

Glycosyltransferases acting on O-glycans have been shown to exhibit distinct specificity for the carbohydrate and the peptide moiety of their substrates. As an approach to study the 3-dimensional interactions between enzymes and O-glycan substrates, we determined the preferred conformations of five oligosaccharide-core structures of mucin type glycoproteins by NMR spectroscopy and by static and dynamic force field calculations. Seven oligosaccharides, representing five basic core structures, were investigated: Gal beta (1-3)GalNAc alpha Bzl (1, core 1), GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha Bzl (2, core 2), GlcNAc beta (1-3)GalNAc alpha Bzl (3, core 3), GlcNAc beta (1-6)[GlcNAc beta (1-3)]GalNAc alpha Bzl (4, core 4), GlcNAc beta (1-6)GalNAc alpha Bzl (5, core 6), the elongated core 2, Gal beta (1-4)GlcNAc beta (1-6)[Gal beta (1-3)]GalNAc alpha pNp (6) and GalNAc alpha-Bzl (7). The dynamic behaviour of the molecules was studied by Metropolis Monte Carlo (MMC) simulations. Experimental coupling constants, chemical shift changes, and NOEs were compared with results from static energy minimizations and dynamic MMC simulations and show a good agreement. MMC simulations show that the (1-6) linkage is much more flexible than the (1-3) or the (1-4) linkages. The preferred conformations of the disaccharides (1) and (3) show only slight differences due to the additional N-acetyl group in (3). The conformational equilibrium of beta (1-3) glycosidic bonds of 1 and 3 was not affected by attaching a beta (1-6) linked GlcNAc unit to the GalNAc residue in 2 and 4. However, experimental and theoretical data show that the beta (1-6) linkages of the trisaccharides 2 and 4, which carry an additional beta (1-3) linked glycosyl residue, change their preferred conformations when compared with (5). The 6-branch also shows significant interactions with the benzyl aglycon altering the preferred conformation of the hydroxymethyl group of the GalNAc to a higher proportion of the gt conformer. The (1-6) linkage of 2, 4, and 6 can have two different families of conformations of which the lower energy state is populated only to about 20% of the time whereas the other state with a relative enthalpy of approximately 4 kcal mol-1 is populated to 80%. This fact demonstrates that the two conformational states have different entropy contents. Entropy is implicitly included in MMC simulations but cannot be derived from energy minimizations.

Partial purification and characterization of mold antigens commonly found in foods

Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.

Heteronuclear NMR studies of 13C-labeled yeast cell wall beta-glucan oligosaccharides.

The structures of uniformly 13C-labeled beta-glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear beta(1-->3) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a beta(1-->6) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain beta(1-->6) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain beta(1-->6) linkages. Since isolated fungal membranes only synthesize linear beta(1-->3) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.

Higher-affinity oligosaccharide ligands for E-selectin.

A series of synthetic oligosaccharides based on sialyl Lewis x (sLex; Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) and sialyl Lewis a (sLea; Neu5Ac alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc) was used to study the binding interactions of selectins. E-selectin-immunoglobulin fusion protein (E-selectin-Ig) bound to immobilized bovine serum albumin (BSA)-neoglycoproteins containing sLex or sLea in a Ca(2+)-dependent manner. Solution-phase sLex tetrasaccharide blocked this interaction by 50% at a concentration of 750 +/- 20 microM (IC50). sLea was more effective (IC50 = 220 +/- 20 microM), while nonsialylated, nonfucosylated derivatives showed little or no activity at concentrations up to 1 mM. Attachment of an 8-methoxycarbonyloctyl aglycone in a beta linkage to the anomeric carbon of the GlcNAc of sLex or sLea increased their blocking activity nearly twofold. Finally, replacement of the 2-N-acetyl substituent of the GlcNAc by an azido or amino group resulted in substantial increases in activity, with the most potent inhibitor being amino substituted sLea, which was 36-fold more active (IC50 = 21 +/- 3 microM) than the reducing tetrasaccharide sLex. In contrast to results obtained with E-selectin-Ig, P-selectin-Ig binding to immobilized BSA-sLea was blocked modestly by most oligosaccharides at 1 mM, with no substantial differences among them. IC50 values of soluble oligosaccharides determined in competitive binding studies accurately predicted blocking of leukocyte adhesion to recombinant E-selectin-Ig and to cytokine-activated endothelium.