Mild tagging procedures for the structural analysis of glycans.

The reductive oxyamination of model glycan structures has been investigated as a mild, alternative tagging procedure to reductive amination using O-(4-nitrobenzyl)-hydroxylamine. Oxime formation was quantitative, but the reduction step did not always go to completion. Novel O- and N-substituted 7-hydroxycoumaryl- and 3-methoxybenzylhydroxylamines were synthesized and shown to couple quantitatively with model saccharides by oxime formation and reductive hydroxyamination, respectively, under very mild, aqueous conditions. The fluorescent derivatives produced show good chromatographic and mass spectrometric properties. Both procedures are suitable for the labeling of carbohydrates and oligosaccharide fragments from glycosaminoglycan structures, such as heparin and heparan sulfate.

Heterocyclic derivatives of sugars: the formation of 1-glycosyl-3-methylpyrazol-5-ones from hydrazones.

Conditions to effect the conversion of monosaccharide and disaccharide hydrazones to 1-glycosyl-3-methylpyrazol-5-ones were examined. The sugar pyrazolone derivatives were sensitive to oxidation, but high yields were achieved with 2,2,2-trifluoroethyl acetoacetate in mildly acidic solution. Azo coupling of the pyrazolones produced highly coloured azopyrazolone derivatives that prevented further degradation, and these may prove useful labels for chromatographic analysis of carbohydrates.

Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production.

Three non-allelic radial swelling mutants (rsw1, rsw2 and rsw3) of Arabidopsis thaliana L. Heynh. were shown to be specifically impaired in cellulose production. Fractionation methods that identify, characterise and quantify some of the major cell wall polysaccharides in small quantities of seedlings demonstrated that changes in the production of cellulose are much more pronounced than changes in the production of non-cellulosic polysaccharides. A crude cell wall pellet was sequentially extracted with chloroform methanol (to recover lipids), dimethyl sulphoxide (starch), ammonium oxalate (pectins) and alkali (hemicelluloses). Crystalline cellulose remained insoluble through subsequent treatments with an acetic/nitric acid mixture and with trifluoroacetic acid. Cetyltrimethylammonium bromide precipitation resolved neutral and acidic polymers in the fractions, and precipitation behaviour, monosaccharide composition and glycosidic linkage patterns identified the major polysaccharides. The deduced composition of the walls of wild-type seedlings and the structure and solubility properties of the major polymers were broadly typical of other dicots. The three temperature-sensitive, radial swelling mutants produced less cellulose in their roots than the wild type when grown at their restrictive temperature (31 degrees C). There were no significant differences at 21 degrees C where no radial swelling occurs. The limited changes seen in the monosaccharide compositions, glycosidic linkage patterns and quantities of non-cellulosic polysaccharides support the view that the RSW1, RSW2 and RSW3 genes are specifically involved in cellulose synthesis. Reduced deposition of cellulose was accompanied by increased accumulation of starch.

A general approach to desalting oligosaccharides released from glycoproteins.

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and beta-eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.

Binding of carbohydrates to solid supports Part 3: Reaction of sugar hydrazones with polystyrene substituted with aromatic aldehydes.

The hydrazones of glucose and N-acetylglucosamine, as models for the residues at the reducing termini of glycans, were covalently and reversibly bound in good yield to hydroxybenzaldehydo ligands attached to a polymer support. The binding, by a sugar azine linkage, occurred within two hours at room temperature at neutral pH, and efficient recoveries of sugars from the beads were achieved by displacement with aqueous hydrazine hydrate, ethanolic benzaldehyde, or aqueous acetone. Enzyme modification of glycans was demonstrated by separation of the products of hydrolysis of lactose hydrazone with beta-galactosidase, using hydroxybenzaldehyde-derivatized polystyrene beads. Addition of a spacer arm to aminopolystyrene beads, for binding of reducing sugars as Amadori compounds to the aromatic amine function, was also investigated.

The relationship between the structures of the O polysaccharides from Escherichia coli O17 and O16.

The chemical structure of the O16 antigen from the lipopolysaccharide of Escherichia coli strain P4 has been determined. Comparison with the structures of other O16 antigens and that of the O17 antigen explains the previously reported cross-reaction of O antigen from the O16 strain K-12 with anti-O17 antibody [D. Liu and P.R. Reeves, Microbiology, 140 (1994) 49-57].

New Rhizobium leguminosarum flavonoid-induced proteins revealed by proteome analysis of differentially displayed proteins.

Proteome analysis was used to establish the first two-dimensional protein map of Rhizobium. R. leguminosarum bv. trifolii strain ANU843 was grown in defined medium in the presence and absence of the flavonoid 7,4'-dihydroxyflavone. Over 1,700 constitutive proteins were resolved, representing about 30% of the estimated genomic output. Proteome analysis of flavonoid-treated cells was done to reveal differentially displayed proteins. The results showed that while the global expression pattern of proteins was largely unaltered by the treatment, four inducible proteins were observed. The four inducible proteins and 20 constitutively expressed proteins were subjected to sequence analysis to provide internal standards for the construction of a two-dimensional Rhizobium protein data base. The identity of 12 proteins, including NodE and NodB, was established. NodE was present throughout the growth of the cells but was diminished in amount in stationary phase cells whereas NodB was not detected in the later stages of growth. Two of the induced proteins sequenced did not match any known nodulation gene product, with one of these being present in mid-late log and stationary phase cells and possessing four consecutive His residues at the N-terminal sequencing was successful with 100 to 200 fmol of protein. Proteome analysis provides a sensitive new tool to examine plant-microbe interactions.

Proteome analysis of glycoforms: a review of strategies for the microcharacterisation of glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis.

Preparative two-dimensional polyacrylamide electrophoresis (2-D PAGE) is a method of separation which for the first time allows protein isoforms to be readily purified for subsequent analysis. The profile of the 2-D separation of the protein complement (proteome) of eukaryotic cells and tissues typically contains obvious 'trains' of spots which differ in pI and/or apparent molecular mass. These are usually isoforms of the same protein and result from post-translational modifications. There is growing evidence that alterations to the glycosylation and/or phosphorylation of a protein can be correlated with developmental and pathological changes; these changes can be visualised on the 2-D separation. It is not clear, however, how these modifications alter the structural properties of the protein and affect their migration in this mode of separation. Strategies need to be developed to obtain a more detailed understanding of the reason for the appearance of isoforms as discrete spots on 2-D PAGE. Standard proteins, fetuin and ovalbumin, were used to monitor the effect of the removal of glycans and phosphates on the migration of the glycoproteins in the 2-D system. The isoforms were not simply explained by the presence or absence of a single modification. To further investigate the reasons for the different migration of the isoforms it is necessary to characterise the modifications in more detail. Unlike protein analysis, until recently the available methodology for the analysis of the glycans attached to proteins has not been sensitive enough to allow analysis of single spots in gels or blots resulting from 2-D electrophoresis. In this paper we review current and future strategies for characterisation of protein modifications using single spots from 2-D gels.

Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis.

Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding. We report a means of determining monosaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel. The sensitivity of the method depends on the degree of glycosylation of the protein. We show that bovine fetuin can be analyzed and identified at the level of 100 pmol. 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences. Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized pH gradient. Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis. The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.

Binding of carbohydrates to solid supports, Part 2: Reaction of sugar hydrazones with isothiocyanate-substituted polystyrene.

The binding of sugars to a polymer support as thiosemicarbazones has been investigated as a means of immobilizing glycans. Hydrazones of glucose and N-acetylglucosamine were prepared by reaction with hydrazine hydrate, and successfully reacted with isothiocyanate-substituted polystyrene by incubation at room temperature and neutral pH. The binding was efficient and stable in aqueous buffers over a range of pH conditions. The bound sugars were recovered in moderate yield by treatment of the beads with hydrazine hydrate, benzaldehyde or acetone. Direct binding of reducing sugars to thiosemicarbazide-substituted polystyrene was not successful because of the unfavourable thermodynamics.

Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine kappa-casein.

kappa-Casein is the major glycoprotein in bovine milk. It has a proteinase-sensitive (chymosin) site which cleaves the glycoprotein into two segments: N-terminal para-kappa-casein domain and the C-terminal kappa-casein macroglycopeptide domain which is highly heterogeneous in oligosaccharide content. We have identified six sites of O-glycosylation on the macroglycopeptide by solid-phase Edman degradation: Thr121, Thr131, Thr133, Thr136 (A variant only), Thr142 and Thr165. No Ser residues are glycosylated. The glycosylation status of 15 of 17 potential O-glycosylation sites in the B variant was accurately predicted using the four peptide motifis previously proposed for the glycosylation of human glycophorin A (Pisano, A., Redmond, J.W., Williams, K.L. and Gooley, A.A., Glycobiology, 3, 429-435, 1993), provided one additional assumption is made concerning an inhibitory role for a nearby Ile.

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster.

Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation.

The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as the O-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.

The elimination of O-linked glycans from glycoproteins under non-reducing conditions.

A simple procedure is described for the elimination of O-linked glycans from bovine submaxillary mucin under non-reducing conditions, using triethylamine in aqueous hydrazine. The glycans were isolated as the hydrazones, which were converted to the reducing glycans by exchange with acetone in neutral aqueous solution. The glycan alditols obtained after reduction corresponded to those obtained by the reductive beta-elimination of O-glycans.

Binding of carbohydrates to solid supports: evaluation of a prototype system.

Mono- and disaccharides were covalently and irreversibly bound to aminopolystyrene beads in good yield by heating in dilute aqueous solution. The degree and stability of sugar binding were determined by chemical and radiochemical methods and the accessibility of the bound sugars was demonstrated by exoglycosidase hydrolysis and by an enzyme-linked lectin-binding assay using Concanavalin A.

Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A.

The human red blood cell sialoglycoprotein, glycophorin A (GpA), contains a 'mucin-like' extensively O-glycosylated extracellular domain which carries the MN blood group antigens. We have revised the sites of O-glycosylation in the extracellular domain of GpA by automated solid-phase Edman degradation, which allowed positive identification and quantitation of O-glycosylated Ser and Thr residues, as well as the single N-glycosylation site. One N-linked and 16 O-linked sites were identified. Carbohydrate was absent on Ser1, Ser14, Ser15, Ser23, Thr28 and Thr58 in GpA. We propose that the glycosyltransferases present in erythrocytes recognize specific flanking sequences around potential O-glycosylation sites. All 16 O-glycosylation sites are explained on the basis of four motifs. Three motifs are associated with Thr-glycosylation: Xaa-Pro-Xaa-Xaa where at least one Xaa = Thr; Thr-Xaa-Xaa-Xaa where at least one Xaa = Thr; Xaa-Xaa-Thr-Xaa where at least one X = Arg or Lys. The fourth motif is associated with Ser-glycosylation: Ser-Xaa-Xaa-Xaa where at least one Xaa = Ser. These simple rules explain the glycosylation (or lack of it) on 21 of 22 Ser/Thr in the extracellular domain of GpA.

Post-translational modifications of the Dictyostelium discoideum glycoprotein PsA. Glycosylphosphatidylinositol membrane anchor and composition of O-linked oligosaccharides.

Prespore-specific antigen (PsA) is a cell-surface glycoprotein isolated from Dictyostelium discoideum, which is post-translationally modified by addition of carbohydrate to threonine residues of the carboxy-terminal peptide domain, and a glycosylphosphatidylinositol (GPI) anchor which attaches the glycoprotein to the cell membrane. The GPI anchor was isolated by proteolytic cleavage of the protein, and the structure of the lipid and glycan portions of the anchor were determined. The lipid moiety of the anchor is an inositolphosphoceramide which contains C18:0 phytosphingosine as a long chain base, and a mixture of fatty acids with a C18:1 mono-unsaturated fatty acid as the major component. The purified GPI anchor was susceptible to digestion by a bacterial phosphatidylinositol-specific phospholipase-C enzyme. The glycan of the GPI anchor consisted of two molecular species present in the ratio 55:45, the structures of which were determined by exoglycosidase sequencing and found to be Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2 and Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine in both structures is glycosidically linked to the inositol ring of the inositolphosphoceramide. The GPI glycan structures are consistent with the conserved core structure of all characterised GPI anchors, and the structure of the D. discoideum GPI moiety has features in common with structures from yeast, protozoa and higher eukaryotes. Compositional analysis of the carbohydrate attached to threonine residues in the carboxy-terminal peptide domain is also presented. The oligosaccharides bind to wheat germ agglutinin, and contain glucosamine and fucose as the major constituents.

Characterisation of oligosaccharides from a glycoprotein variant of human serum albumin (albumin Casebrook) using high-performance anion-exchange chromatography and nuclear magnetic resonance spectroscopy.

The characterisation of oligosaccharides present on albumin Casebrook, a glycoprotein variant of human serum albumin, which contains an N-linked oligosaccharide at an attachment site formed by a point mutation of 494 Asp-->Asn, is described. The monosaccharide compositional analysis of purified glycopeptides suggested the presence of complex biantennary carbohydrate structures. The oligosaccharides which were released by N-glycosidase-F appeared to be a single molecular species according to their retention on high-performance anion-exchange chromatography. The structure of the oligosaccharide was suggested by sequential exoglycosidase digestions and confirmed by proton nuclear magnetic resonance spectroscopy. It was concluded that the oligosaccharides were essentially homogeneous and consisted of an alpha(2-6)-desialylated complex biantennary glycan.

Synthesis of a PAF immunogen and production of PAF-specific antibodies.

Platelet activating factor (PAF), a naturally occurring phospholipid with many potent physiological and pharmacological activities, is implicated as a mediator of many diseases. An immunoassay for PAF would greatly improve quantitation, and hence PAF-specific antibodies were required. Chemically-reactive analogs of PAF, containing an aldehyde group at the end of the 1-O-alkyl chain (hexyl or dodecyl), were synthesized from readily available materials. During the multi-step synthetic procedure, the aldehyde group was protected as an acetal, which was converted by mild acidic hydrolysis to the aldehyde immediately prior to protein coupling. These analogs were coupled to methylated bovine serum albumin and the resultant conjugates were injected into rabbits. Antibodies to PAF were detected using a solid phase radioimmunoassay based on Protein A-Sepharose. The dodecyl PAF conjugate proved to be the more immunogenic conjugate with more than half of the rabbits producing significant levels of antibodies (at least a 10-fold increase in radioactive uptake over pre-immune levels). Results from solid phase immunoassays employing nitrocellulose discs impregnated with PAF, lysoPAF, lecithin, lysolecithin and 2-O-methyl-lysoPAF indicated that the antibodies recognized only PAF. PAF-specific antibodies were isolated by affinity chromatography using a column of PAF-poly(lysine) conjugated to carboxy-activated polyacrylamide. The antibodies may be employed in a sensitive and specific immunoassay for PAF and for many other studies involving PAF.