Synthesis of the disaccharide 6-O-beta-D-galactopyranosyl-2-acetamido-2-deoxy-D-galactose using immobilized beta-galactosidase

The disaccharide 6-O-beta-D-galactopyranosyl-2-acetamido-2-deoxy-D-galactose has been synthesized by transfer of the beta-D-galactopyranosyl residue from lactose to 2-acetamido-2-deoxy-D-galactose utilizing the transferase activity of beta-galactosidase from E. coli. To make the enzyme reusable, it was applied in an immobilized form covalently bound to Sepharose CL-4B. The yield of the disaccharide was about 20%, calculated on the amount of acetamido-deoxy-D-galactose added. The disaccharide could also be obtained by reversal of the hydrolytic activity of the enzyme, using D-galactose and 2-acetamido-2-deoxy-D-galactose as substrate. The yield in this reaction, however, was only 2-3% under the conditions applied.     

Structural studies on the specific type VII pneumococcal polysaccharide

The specific type VII pneumococcal polysaccharide was isolated from the crude capsular material by precipitative and chromatographic methods. It contained D-galactose, D-glucose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose in the molar ratio of 3.5:2.3:3.0:2.1:1.0. Some of its structural features were revealed by methylation studies, time-lapse hydrolysis, periodate oxidation, and enzymic hydrolysis. The polysaccharide is branched at residues of D-galactose and 2-acetamido-2-deoxy-D-galactose. Non-reducing end groups consisted of D-galactopyranose and 2-acetamido-2-deoxy-D-glucopyranose residues, with the former predominating. Major components of the linear chains were (1→3)-linked L-rhamnose and (1→4)-linked D-glucose; the minor ones were (1→2)-linked L-rhamnose, (1→6)-linked D-galactose, and (1→6)-linked 2-acetamido-2-deoxy-D-glucopyranose. The (1→4)-linked D-glucose components may be present as cellobiose residues. The results are in accord with structural features deduced from the serological cross-reactivity of this polysaccharide.     

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

The 13C-N.M.R. spectra of disaccharides of D-glucose, D-galactose, and L-rhamnose as models for immunological polysaccharides.

Complete assignments of the 13C-n.m.r. spectra of disaccharides having beta-glycosidic linkages are presented and discussed. The disaccharides of D-glucose, D-galactose, L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose are model compounds for 13C-n.m.r. studies of immunological polysaccharides. Changing the nature of the reducing glucopyranose rings (D-glucose to L-rhamnose) has no important influence on the chemical shifts of the carbons of the non-reducing glucopyranose ring (D-glucose). The converse is also true: the chemical shifts of the carbons of the reducing glucopyranose ring (L-rhamnose) are not noticeably affected by a change of the non-reducing unit (D-glucose to D-galactose or 2-acetamido-2-deoxy-D-glucose).     

Structural studies on O-specific polysaccharides of lipopolysaccharides from Yersinia enterocolitica serovars O:5 and O:5,27

Lipopolysaccharides of Yersinia enterocolitica serovars O:5 and O:5,27 were shown to have a similar sugar composition, consisting of L-rhamnose, D-glucose, D-galactose, D- and L-glycero-D-manno-heptose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 3-deoxy-D-manno-octulosonate and D-threo-pent-2-ulose (D-xylulose). Partial hydrolysis of lipopolysaccharides with acetic acid produced rhamnans with the following repeating unit: ----3)-L-Rha rho(alpha 1----3)-L-Rha rho(alpha 1----3)-L-Rha rho(beta 1----. 13C-NMR and methylation studies of the lipopolysaccharides gave the following structure for the repeating unit of the two O-specific polysaccharides: ----3)-L-Rha rho(alpha 1----3)-L-Rha rho(alpha 1----3)-L-Rha rho(beta 1----. (formula; see text)     

Determination of the degree of polymerisation of xylans

Aspartoylglycosylamines and their analogs having structures similar to 2-acetamido-1-N-(L-aspart-4-oyl)-2-deoxy-β-D-glucopyranosylamine, in which aspartic acid is linked to glycosylamines from 2-acetamido-2-deoxy-D-galactose, D-galactose, and D-mannose, and to cyclohexylamine and aniline, respectively, were synthesized. These syntheses were accomplished by condensing O-acetylglycosylamines, aniline, and cyclohexylamine with an aspartic acid derivative having blocked 1-carboxyl and 2-amino groups, followed by removal of the protecting groups. The stability of the synthetic compounds in acidic and alkaline media was investigated.     

Structural studies on O-specific polysaccharides of lipopolysaccharides from Yersinia enterocolitica serovars O:1,2a,3, O:2a,2b,3 and O:3

Lipopolysaccharides from Yersinia enterocolitica serovars O:1,2a,3, O:2a,2b,3 and O:3 have been isolated and characterized. 6-Deoxy-L-altrose residues were shown to be the main constituents of lipopolysaccharides isolated in addition to residues of L-rhamnose, D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid being minor components of sugar chains. Mild hydrolysis of lipopolysaccharides with acetic acid furnished O-specific polysaccharides, which are composed of 6-deoxy-L-altrose. Using 13C-NMR spectroscopy and methylation data, the structural features of backbones have been elucidated as follows: ----2)-6d-L-Altp(beta 1----2)-6d-L-Altp(beta 1----3)-6d-L-Altp)(beta 1----for serovars O:1,2a,3 and O:2a,2b,3;----2)-6d-L-Altp(beta 1----for serovar O:3. In addition, O-polysaccharide of serovar O:2a,2b,3 was found to contain an O-acetyl group at the C-3 position of some 1,2-linked sugar residues.     

The central domain of bovine submaxillary mucin consists of over 50 tandem repeats of 329 amino acids. Chromosomal localization of the BSM1 gene and relations to ovine and porcine counterparts.

We previously elucidated five distinct protein domains (I-V) for bovine submaxillary mucin, which is encoded by two genes, BSM1 and BSM2. Using Southern blot analysis, genomic cloning and sequencing of the BSM1 gene, we now show that the central domain (V) consists of approximately 55 tandem repeats of 329 amino acids and that domains III-V are encoded by a 58.4-kb exon, the largest exon known for all genes to date. The BSM1 gene was mapped by fluorescence in situ hybridization to the proximal half of chromosome 5 at bands q2. 2-q2.3. The amino-acid sequence of six tandem repeats (two full and four partial) were found to have only 92-94% identities. We propose that the variability in the amino-acid sequences of the mucin tandem repeat is important for generating the combinatorial library of saccharides that are necessary for the protective function of mucins. The deduced peptide sequences of the central domain match those determined from the purified bovine submaxillary mucin and also show 68-94% identity to published peptide sequences of ovine submaxillary mucin. This indicates that the core protein of ovine submaxillary mucin is closely related to that of bovine submaxillary mucin and contains similar tandem repeats in the central domain. In contrast, the central domain of porcine submaxillary mucin is reported to consist of 81-amino-acid tandem repeats. However, both bovine submaxillary mucin and porcine submaxillary mucin contain similar N-terminal and C-terminal domains and the corresponding genes are in the conserved linkage regions of the respective genomes.     

Application of high-resolution n.m.r. spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F

The specific capsular polysaccharide of Streptococcus pneumoniae type 7F (American type 51) is a high-molecular-weight neutral polymer composed of 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, L-rhamnose, and 2-O-acetyl-L-rhamnose residues. N.m.r. spectroscopy (1H and 13C), in conjunction with composition and methylation analyses, and periodate oxidation data, showed the polysaccharide to be a branched polymer with a repeating heptasaccharide unit having the following structure. (formula; see text)     

Quantitative precipitin studies on the specificity of an extract from tridacna maxima (röding)

Haemolymph from the clam Tridacna maxima precipitated with purified H-blood-group substances, Helix pomatia galactogen, and pneumococcus type XIV polysaccharide. Although gel diffusion, gel electrophoresis, and inhibition experiments indicated that only a single precipitating lectin was present in the haemolymph, quantitative precipitin and haemagglutination results suggested that a second agglutinin with anti-H-like specificity was also present. Evidence obtained from hapten inhibition experiments indicated that the precipitin that reacts with pneumococcus type XIV polysaccharide can be inhibited by a number of simple sugars. Of the compounds tested, 2-acetamido-2-deoxy-D-galactose was the best inhibitor of precipitation with pneumococcus type XIV polysaccharide and of haemagglutination with human erythrocytes, but the inhibition experiments showed that the extract was also markedly inhibited by D-galactosamine hydrochloride, D-galactose, lactose, and p-nitrophenyl beta-D-galactopyranoside. The latter compound was more active than its parent sugar, which was in turn a more potent inhibitor than p-nitrophenyl alpha-D-galactopyranoside. Melibiose, raffinose, and stachyose, compounds which each contain terminal alpha-linked D-galactopyranosyl residues, were relatively weak inhibitors. The combining sites of the lectin that reacts with pneumococcus type XIV polysaccharide appear, therefore, to be most complementary to 2-acetamido-2-deoxy-D-galactopyranosyl residues, probably in beta linkage.     

Structural analysis of the specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72)

The specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72) was found to be a high molecular weight polymer composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-fucose, L-rhamnose, glycerol, and phosphate (2:1:1:1:1:1:1). Partial hydrolysis, dephosphorylation, methylation analysis, periodate oxidation studies, and one- and two-dimensional 1H and 13C high-field nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a 1-phosphoglycerol-substituted hexasaccharide repeating unit having the structure: (formula; see text).     

The structure of the antigenic O-polysaccharide of the lipopolysaccharide of Edwardsiella ictaluri strain MT104

The structural characterization of the antigenic O-polysaccharide component of the lipopolysaccharide produced by the fish pathogenic bacterium Edwardsiella ictaluri MT104 was undertaken by the application of NMR spectroscopy and chemical analysis. The O-chain was found to be a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in a 1:2:1 ratio having the structure: [carbohydrate structure]; see text.     

Mucin-specific bark lectin from elderberry Sambucus sieboldiana and its applications to the affinity chromatography of mucin

Three bark lectins were isolated from elderberry Sambucus sieboldiana using fetuin-Sepharose 4B and mucin-Sepharose 4B, and were studied comparatively for their binding to glycoprotein and to clarify various physicochemical features. For each, a unique pattern on isoelectric focusing was noted and their affinity toward various glycoproteins differed, indicating the structures of their carbohydrate binding sites possibly differ. One bark lectin showed specific binding toward porcine mucin. The purity of mucin from a crude porcine stomach mucin or an extract of porcine submaxillary glands could be improved by affinity chromatography on immobilized lectin having binding specificity toward mucin.     

Subunit structure of porcine submaxillary mucin

The structure of a high molecular weight fraction of porcine submaxillary mucin was studied by using degradative techniques. Reduction of disulfide linkages released mucin subunits together with an associated protein(s) of approximately 140 kDa. The molecular weights of the subunits ranged from approximately 0.5 x 10(6) to 2.5 x 10(6). Trypsinization of subunits generated glycosylated domains and small, poorly glycosylated or nonglycosylated tryptic peptides. The glycosylated domains, which have an average molecular weight of approximately 270K, possess an unusual amino acid composition containing only nine different amino acids. The minor amino acids which are absent from the glycosylated domains but which are consistently present in both the mucin and the mucin subunits were recovered in the tryptic peptides. Pronase digestion of the glycosylated domains generated smaller fragments of approximately 17 kDa. Comparing these results to the partial cDNA sequence for porcine submaxillary mucin reported by Timpte et al. [(1988) J. Biol. Chem. 263, 1081-1088] suggests that the glycosylated domains consist of variable numbers of the 81 amino acid tandem repeat observed in the cDNA sequence. Further, the fact that porcine submaxillary mucin contains subunits, link proteins, and glycosylated domains suggests that its structure is similar to that described for cervical and intestinal mucins. Intact mucin, mucin "subunits", and the glycosylated domains are all polydisperse with respect to molecular weight, indicating that mucin polydispersity is due to variability in the number of units linked together as well as to variability in the size of the units.     

Structural characterization of the O-polysaccharide antigen of Edwardsiella tarda MT 108

Edwardsiella tarda, a Gram-negative bacterium, is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. The lipopolysaccharide produced by the fish pathogenic strain E. tarda MT 108 was isolated and the structure of its antigenic O-polysaccharide component determined by the application of chemical analyses, high-resolution 1D and 2D nuclear magnetic resonance spectroscopy, and mass spectrometry. The polysaccharide was found to be a polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), D-galactose (D-Gal), L-rhamnose (L-Rha), D-galacturonic acid (D-GalA) and (2S,3R)-threonine (1:1:1:1:1:1) having the structure: [structure: see text].     

Transport and phosphorylation of 2-deoxy-D-galactase in renal cortical cells.

The transport and phosphorylation of 2-deoxy-D-[3H]galactose in rabbit renal cortical cells was studied. 1. The uptake of 2-deoxy-galactose by cortical slices is associated with an appearance of both free and phosphorylated sugar in the cells. At 1 mM external sugar the cells establish a steady-state gradient of free 2-deoxy-galactose of 3.97 +/- 0.15 (23 animals). 2. The acid-labile sugar phosphate accumulated in the tissue has been identified by a combination of paper and radio-chromatography, as well as on the basis of some of its chemical properties, as 2-deoxy-D-galactose 1-phosphate. Ice-cold trichloroacetic acid produces a decomposition of this compound. 3. Increasing external pH (6-8) brings about a decrease in the steady-state levels of both free and phosphorylated sugar in slices. On the other hand, increasing pH activates the phosphorylation of 2-deoxy-D-galactose by a crude kinase in a tissue extract. 4. Sugar phosphate accumulated in the cells is dephosphorylated by the action of a Zn2+ -activated phosphatase. 5. The efflux of 2-deoxy-D-galactose from the cells is rather slow compared with that found for D-galactose. The efflux is associated with some dephosphorylation of cellular sugar phosphate, and some loss of 2-deoxy-galactose phosphate into the wash-out medium takes place. 6. An inhibition analysis of the uptake of 2-deoxy-D-galactose by the slices indicates that the transport site is shared by D-galactose. The following points of interaction between the sugar molecule and the carrier are identified: C1-OH, C3-OH and C4-OH (both axial) and C6-OH. A (pyranose) ring structure is also essential. A close packing between the substrate and the carrier in the vicinity of C2 is indicated. 7. The data suggest that the above transport system is localized predominantly at the antiluminal (basolateral) face of the renal tubular cells. While the detailed mechanism of the actual transport step (i.e. active transport of the free sugar, or by the action of a phosphotransferase) is still unclear, the data present evidence that both galactokinase and a Zn2+ -activated phosphatase participate in the maintenance of an intracellular steady state of the transported sugar.     

Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced by Actinobacillus pleuropneumoniae serotype 15.

The specific capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 15 was determined to be a high-molecular-mass polymer having [alpha]D + 69 degrees (water) and composed of a linear backbone of phosphate diester linked disaccharide units of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc) and 2-acetamido-2-deoxy-D-galactose (D-GalNAc) residues (1:1). Thirty percent of the D-GalNAc residues were substituted at O-4 by beta-D-galactopyranose (beta-D-Galp) residues. Through the application of chemical and NMR methods, the capsule, which defines the serotype specificity of the bacterium, was found to have the structure [structure: see text]. The O-polysaccharide (O-PS) component of the A. pleuro pneumoniae serotype 15 lipopolysaccharide (LPS) was characterized as a linear unbranched polymer of repeating pentasaccharide units composed of D-glucose (2 parts) and D-galactose (3 parts), shown to have the structure [structure: see text]. The O-PS was chemically identical with the O-antigen previously identified in the LPSs produced by A. pleuro pneumoniae serotypes 3 and 8.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

Structure of an acidic O-specific polysaccharide of the marine bacterium <Emphasis Type="Italic">Pseudoalteromonas agarivorans</Emphasis> KMM 232 (R-form)

An acidic O-specific polysaccharide containing L-rhamnose, 2-acetamido-2-deoxy-D-galactose, 2,6-dideoxy-2-(N-acetyl-L-threonine)amino-D-galactose, and 2-acetamido-2-deoxy-D-mannuronic acid was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Pseudoalteromonas agarivorans KMM 232 (R-form) followed by gel-permeation chromatography. The polysaccharide was subjected to Smith degradation to give a modified polysaccharide with trisaccharide repeating unit containing L-threonine. The initial and modified polysaccharides were studied by sugar analysis and ¹H- and ¹³C-NMR spectroscopy, including COSY, TOCSY, ROESY, and HSQC experiments, and the structure of the branched tetrasaccharide repeating unit of the polysaccharide was established.     

Characterization of the lipopolysaccharide O-antigen of Cronobacter turicensis HPB3287 as a polysaccharide containing a 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid) residue

Cronobacter turicensis, previously known as Enterobacter sakazakii, is a Gram-negative opportunistic food-borne pathogen that has been reported as a cause of life-threatening neonatal infections. From chemical and physical analyses involving composition analysis, methylation, two-dimensional high-resolution nuclear magnetic resonance, and mass spectrometry methods, the antigenic O-polysaccharide in the smooth-type lipopolysaccharide of C. turicensis (strain HPB 3287) was determined to be a high molecular mass polymer of a repeating pentasaccharide unit composed of D-galactose, D-glucose, 2-acetamido-2-deoxy-D-galactose, and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (legionaminic acid), in a molar ratio 2:1:1:1, and having the structure: [see formula in text].