Facile synthesis of arabinomannose penta- and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, Rhodococcus equi

Pentasaccharide repeating unit 20 of the lipoarabinomannan from the equine pathogen, Rhodococcus equi, and its dimer 31, were synthesized. The pentasaccharide was obtained by assembling a benzoylated 2,6-branched mannosyl trisaccharide acceptor 13 with a free hydroxyl group at C-2' of the mannose residue attached to the core mannose residue by (1 --> 6)-linkage, followed by coupling with 2,3,5-tri-O-benzoyl-alpha-D-arabinofuranosyl-(1 --> 2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (18), and by deacylation. Meanwhile, the decamer 31 was obtained by firstly preparing a benzoylated mannose (1 --> 6)-linked tetrasaccharide backbone 26 with 2-, 2"-O-ClAc, and 2'-, 2'-O-Ac groups, respectively, then by dechloroacetylation and subsequent condensation with perbenzoylated trichloroacetimidate, and then by deacetylation and subsequent coupling with 18, and finally, by deacylation.     

Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N-acetylgalactosaminidase from Streptomyces sp.

Gal beta-(1-->3)-GalNAc-linked hexapeptide was synthesized by a transglycosylation using Gal beta-(1-->3)-GalNAc beta-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Gal beta-(1-->3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.     

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-Xyl,a part of the common linkage region of a glycosaminoglycan

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->OMe) was achieved by coupling of methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate with a trisaccharide acceptor. The trisaccharide acceptor was obtained by condensation of 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with methyl 2,3-di-O-benzoyl-beta-D-xylopyranoside, followed by deallylation. The beta-(1-->3)-linked disaccharide was prepared readily with p-methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside as the key synthon. The alpha-(1-->3)-linkage was formed in considerable amount with galactose mono- and disaccharide trichloroacetimidate donors with C-2 neighboring group participation.     

An enzymatically produced novel cyclomaltopentaose cyclized from amylose by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}

A bacterial strain AM7, isolated from soil and identified as Bacillus circulans, produced two kinds of novel cyclic oligosaccharides. The cyclic oligosaccharides were produced from amylose using a culture supernatant of the strain as the enzyme preparation. The major product was a cyclomaltopentaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. The other minor product was cyclomaltohexaose cyclized by an alpha-(1-->6)-linkage, cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}. We propose the names isocyclomaltopentaose (ICG5) and isocyclomaltohexaose (ICG6) for these novel cyclic maltooligosaccharides having one alpha-(1-->6)-linkage. ICG5 was digested by alpha-amylase derived from Aspergillus oryzae, cyclomaltodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus, and maltogenic alpha-amylase. On the other hand, ICG6 was digested by CGTase from B. stearothermophilus and B. circulans, and maltogenic alpha-amylase. This is the first report of enzymatically produced cyclomaltopentaose and cyclomaltohexaose, which have an alpha-(1-->6)-linkage in their molecules.     

Determination of rate constants for β-linkage isomerization of three specific aspartyl residues in recombinant human αA-crystallin protein by reversed-phase HPLC

The major soluble eye lens protein, αA-crystallin, has a very long half-life. Thus, many post-translational modifications, including stereoinversion, have been found in its constituent amino acids. We determine the rates of β-linkage isomerization, which is the main reaction through the formation of a succinimide intermediate, of specific Asp residues of recombinant human αA-crystallin protein by simple RP-HPLC method. Kinetic analyses of the β-linkage isomerization were performed on the three Asp residues of αA-crystallin, (58)Asp, (84)Asp, and (151)Asp, because the d/l ratios of both the (58)Asp and (151)Asp residues were higher than 1.0 in the αA-crystallin isolated from aged human eye lens. The β-linkage isomerizations of both the (58)Asp and (84)Asp residues were suppressed in the recombinant protein by approximately 0.4-0.5 times compared to those in the synthetic peptide below 50 °C, whereas the isomerization of the (151)Asp residue occurred at the same rate for the whole protein and synthetic fragmentary peptide. The suppression of (58)Asp isomerization in the recombinant protein relaxed to some extent when the αA-crystallin protein was incubated at a high temperature. The far-UV CD spectra showed that the secondary structure of the protein was partially disordered at temperatures greater than 60 °C in the recombinant αA-crystallin protein. These results suggest that the (58)Asp residue was restrained from forming the succinimide intermediate by the higher order structure of the αA-crystallin protein, and that the structural environment around the (151)Asp residue of the αA-crystallin was similar to that of the synthetic fragmentary peptide with respect to succinimide formation. The difference in the influence of the secondary structure of the αA-crystallin protein inverts the order of the succinimide formations of the (58)Asp and (151)Asp residues in the recombinant protein as compared with the order in the synthetic fragmentary peptides.     

Type I arabinogalactan contains beta-D-Galp-(1-->3)-beta-D-Galp structural elements

Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a beta-D-Galp-(1-->4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all (1)H and (13)C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a beta-(1-->3)-linkage instead of the expected beta-(1-->4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->4)-alpha/beta-Galp-OH and beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->3)-alpha/beta-Galp-OH. The fact that the deviating beta-(1-->3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The beta-(1-->3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion).     

High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.

We have expressed the Neisseria meningitidis lgtA gene at a high level in Escherichia coli. The encoded beta-N-acetylglucosaminyltransferase, referred to as LgtA, which in the bacterium is involved in the synthesis of the lacto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransferase appeared to be unusual in that it displays a broad acceptor specificity toward both alpha- and beta-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Product analysis by one- and two-dimensional 400 MHz 1H- and 13C-NMR spectroscopy reveals that LgtA catalyzes the introduction of GlcNAc from UDP-GlcNAc in a beta 1-->3-linkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha/beta-R beta 3-N-acetylglucosaminyltransferase. Although lactose is a highly preferred acceptor substrate the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures in enzyme assisted procedures. Furthermore, LgtA shows a high donor promiscuity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze the introduction of GalNAc in beta 1-->3-linkage to alpha- or beta-Gal in the acceptor structures at moderate rates. LgtA therefore shows promise to be a useful catalyst in the preparative synthesis of both GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.     

Application of NMR,molecular simulation,and hydrodynamics to conformational analysis of trisaccharides

The preferred conformations and conformational flexibilities of the trisaccharides alpha-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe (I) and alpha-D-Glcp-(1-->3)[beta-D-Glcp-(1-->4)]-alpha-D-Glcp-OMe (II) in aqueous solution were determined using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) and Langevin dynamics (LD) simulations, and hydrodynamics calculations. Both trisaccharides have a vicinal substitution pattern in which long range (nonsequential) interactions may play an important role. LD simulation at 600 K indicated that the all-syn conformation predominated, though other conformations were apparent. NOE data and MD and LD simulations at 298 K all indicated that trisaccharide I is a single all-syn conformer in solution. Given that previous studies showed evidence of anti-conformers in beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe, this result provides an example of how changing the anomeric configuration of one residue from beta to alpha can make an oligosaccharide more rigid. Discrepancies in inter-ring distances obtained by experiment and by simulation of the all-syn conformer suggest the presence of an anti-psi conformation at the beta-(1-->4)-linkage for II. A combined analysis of measured and calculated translational diffusion constants and (13)C T(1) relaxation times yield order parameters of 0.9 for each trisaccharide. This implies that any interconversion among conformations is significantly slower than tumbling. Anisotropies of approximately 1.6 and 1.3 calculated for I and II, respectively, are consistent with the observed relatively flat T(1) profiles because the tumbling is not in the motional narrowing regime.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I

Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.     

A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis

Alignment of 15 vertebrate alpha1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis alpha1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha1, 4-linkage. The single amino acid substitution Trp111 --> Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional mutation of Asp112 --> Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast, substitution of Arg115 in bovine futb-encoded alpha1, 3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 acceptors. However, the double mutation Arg115 --> Trp/Glu116 --> Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.     

Cloning, sequencing, and expression of the genes encoding an isocyclomaltooligosaccharide glucanotransferase and an alpha-amylase from a Bacillus circulans strain

The gene for a novel glucanotransferase, isocyclomaltooligosaccharide glucanotransferase (IgtY), involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}] from starch, was cloned from the genome of B. circulans AM7. The IgtY gene, designated igtY, consisted of 2,985 bp encoding a signal peptide of 35 amino acids and a mature protein of 960 amino acids with a calculated molecular mass of 102,071 Da. The deduced amino-acid sequence showed similarities to 6-alpha-maltosyltransferase, alpha-amylase, and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in this enzyme, indicating that this enzyme should be assigned to this family. The DNA sequence of 8,325-bp analyzed in this study contained two open reading frames (ORFs) downstream of igtY. The first ORF, designated igtZ, formed a gene cluster, igtYZ. The amino-acid sequence deduced from igtZ exhibited no similarity to any proteins with known or unknown functions. IgtZ was expressed in Escherichia coli, and the enzyme was purified. The enzyme acted on maltooligosaccharides that have a degree of polymerization (DP) of 4 or more, amylose, and soluble starch to produce glucose and maltooligosaccharides up to DP5 by a hydrolysis reaction. The enzyme (IgtZ), which has a novel amino-acid sequence, should be assigned to alpha-amylase. It is notable that both IgtY and IgtZ have a tandem sequence similar to a carbohydrate-binding module belonging to a family 25. These two enzymes jointly acted on raw starch, and efficiently generated ICG5.     

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

Synthesis and photolytic cleavage of bovine insulin B22-30 on a nitrobenzoylglycyl-poly (ethylene glycol) support

The synthesis of the C-terminal nonapeptide of bovine insulin B-chain is described. 4-(Bromomethyl)-3-nitrobenzoylglycyl-poly(ethylene glycol) Mr = 15,000) was used as soluble support. The C-terminal alanine was first converted to Boc-Ala-O-(2-nitro-4-carboxy) benzyl ester which was then coupled to Gly-PEG via DCC activation. The synthesis was performed using the in situ symmetrical anhydride coupling method. Cleavage of the protected peptide from the polymeric support was achieved by photolysis. The product was then chromatographed on a column of Sephadex LH-20. All the protecting groups of a sample were removed with liquid HF and the unprotected crude peptide was purified by ion-exchange chromatography on CM-Sephadex to obtain an electrophoretically and chromatographically pure peptide. The identity of this peptide was confirmed by field desorption mass spectrometry and amino acid analysis. Circular dichroism measurement suggests that the free nonapeptide possesses a disordered conformation. The nonapeptide was tested for the racemization of the individual amino acids by gas chromatography and the results showed that no residue was significantly racemized.     

A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195

Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein. The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end. CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-(1-->4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan. Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch.     

PRESENCE OF 2′,5′-LINKAGES IN A HOMOPYRIMIDINE DNA OLIGONUCLEOTIDE PROMOTES STABLE TRIPLEX FORMATION UNDER PHYSIOLOGICAL CONDITIONS

We prepared 15-mer homopyrimidine oligonucleotides containing three or four 2′,5′-linked DNA units, and their ability as a triplex-forming oligonucleotide (TFO) was analyzed in detail. UV melting experiments showed that replacement of a 3′,5′-linkage by a 2′,5′-linkage at every third or fourth residue in TFO significantly promoted stable triplex formation under physiological conditions.     

Isolation and structural characterization of low-molecular-mass monosialyl oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis

The carbohydrate chains of the respiratory-mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive straight-phase high-performance liquid chromatography (HPLC) on a silica-based alkylamine column, and reverse-phase HPLC on a silica-based octadecyl column, respectively. The structures of the oligosaccharidealditols were determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are: (formula; see text) 23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631-642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643-654]. The sialic acid residue was found to occur either in alpha (2----3)- or alpha (2----6)-linkage to a galactosyl residue or in alpha (2----6)-linkage to GalNAc-ol.     

Molecular characterization of a novel beta1,3-galactosyltransferase for capsular polysaccharide synthesis by Streptococcus agalactiae type Ib

A group B streptococcus, Streptococcus agalactiae type Ib, produces a high-molecular-weight polysaccharide consisting of the following pentasaccharide repeating unit: -->4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. The type-specific capsular polysaccharide (CP) synthesis (cps) genes of this strain were cloned and analyzed. A cloned 10-kb DNA fragment contained cpsIbE to L and neu (neuraminic acid synthesis gene) B. Comparison of the gene products with those of S. agalactiae type Ia, which has a similar but distinct CP, showed that the translation products of cpsIa and cpsIb genes exhibited very high homology except for those of cpsJ and K. In the type Ia strain, cpsIaJ encodes beta1,4-galactosyltransferase, which catalyzes the transfer of galactose as the fourth monosaccharide of the sugar repeating unit. In the type Ib CP, this galactose forms a beta1,3-linkage to GlcNAc. The low homology between the type Ia and Ib CpsJs seems to reflect this difference. By enzymatic activity measurement, the cpsIbJ product was found to display beta1,3-galactosyltransferase activity. Furthermore, hydrophobic cluster analysis clarified the similarities and differences of the structures in N-terminal regions, including the DXD motif, between the galactosyltransferases.