Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

One-pot enzymatic synthesis of the Galα1→3Galβ1→4GlcNAc sequence within situ UDP-Gal regeneration

The trisaccharide Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4-epimerase, UDP-Gal:GlcNAc 4-galactosyltransferase and UDP-Gal:Gal14GlcNAc 3-galactosyltransferase, Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was formed in a 2.2 µmol ml^–1 yield starting from the acceptor GlcNAc1O-(CH₂)₈COOCH₃. This is an efficient and convenient method for the synthesis of the Gal13Gal14GlcNAc epitope which plays an important role in various biological and immunological processes.     

Comparative structural analysis of snail galactans by a radioimmunoassay to elucidate species-specific determinants

Summary Galactans, the storage polysaccharides in the perivitelline fluid of many snails showed a high degree of species-specificity as revealed by quantitative precipitin formations with lectins, polyclonal antisera, myeloma proteins as well as by the reactivity with the enzyme galactose oxidase. However, their chemical compositions were remarkably similar since thed-Gal residues were all linked 13 and 16 glycosidically.The specificity seemed to be related to the different degrees of branching in the various galactans but could also be due to some other minor constituents in some galactans such asl-galactose or phosphate.In this study a Radioimmunoassay was developed using the galactan of the snailLymnaea stagnalis to elucidate those differences which were only related to a unique distribution of the 13 and 16 linkages, since this galactan was composed exclusively ofd-galactose residues. The galactan was labeled by sequential oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Inhibition of the binding of the labeled galactan to insolubilized antibodies was investigated by galactans of different species, their chemically modified products, andd-galactose-composed oligosaccharides of unambiguously identified structures.Inhibition byLymnaea stagnalis galactan was about 45 000 times that ofHelix pomatia galactan. The most complementary oligosaccharide found was -d-Gal13[-d-Gal16]-d-Gal11l-Gro, being about 200 times more effective thand-Gal. However, a fraction with molecular weights between 700 and 1000 isolated from the partially hydrolized galactan was still seven times more effective. From the reactivity of the antiserum with the different oligosaccharides tested the following structure was inferred which most likely represented the complete determinant recognized by the antiserum: -d-Gal13[-d-Gal16]-d-Gal16[-d-Gal13]-d-Gal1. This determinant seemed to be most common inLymnaea stagnalis galactan and its frequency of occurrence appears to correspond to the inhibitory potency of other snail galactans.     

Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones

Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human 1 and constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by¹H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (1 6)-flucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc12, Gal1 4GlcNAc1 2 or Gal1 3G11 4GlcNAc1 2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (1 2)-linked GlcNAc residues. Galactosylation of the GlcNAc1 2Man1 6 branch occurs four times more frequently than that of the GlcNAc1 2Man1 3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carryingN-acetylneuraminic acid (Neu5Ac) orN-glycolylneuraminic acid (Neu5Gc), exclusively (2 6)-linked to Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two chimeric antibodies, but that the relative amounts of the major oligosaccharide components, the degree of sialylation and the molar ratio of Neu5Ac to Neu5Gc varies with the SP2/0 cell subclone, and only slightly with cell age.     

A clinicopathological evaluation of anti-fucosylated antigens antibody (YB-2) in colorectal carcinoma

A newly generated monoclonal antibody, YB-2, reacts simultaneously with Y (Fuc12Gal14[Fuc13]GlcNAc), Le^b (fuc12Gal13[Fuc14]GlcNAc) and H type 2 (Fuc12Gal14GlcNAc) antigens (Jpn J Cancer Res 1993: 84; 641-8). Since these antigens have been reported to be expressed strongly in malignant colorectal tissues, we investigated the usefulness of this antibody as an immunochemical tool for diagnosis of colorectal cancer. The rate of positive staining with YB-2 antibody in colorectal carcinoma (n=101), adenoma (n=26) and normal tissues (n=25) was 95.0, 50.0 and 12.0%, respectively. The specimens with negative staining were restricted in Dukes' A patients but 75% of Dukes' C patients were strongly positive. The intensity of positive staining with YB-2 antibody was also significantly related to the clinico-pathological features such as the depth of invasion, metastasis, histological types and tumor location. Moreover, the 5-year survival in patients whose tumors were positive with YB-2 antibody was found to be significantly low. Therefore, YB-2 antibody could be useful for immunodiagnosis and, possibly, immunotherapy of colorectal carcinoma.     

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride β-glucosidase

A new -glucosidase was partially purified from Trichoderma viride cellulase. This -glucosidase catalyzed a transglycosylation reaction of cellobiose to give -D-Glc-(16)--D-Glc-(14)-D-Glc (1, yield: 18.8%) and -D-Glc-(16)--D-Glc-(16)--D-Glc-(14)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into -D-Glc-(16)--D-Glc-(13)-D-Glc (3, 15.3%) and -D-Glc-(16)--D-Glc-(16)-D-Glc (4, 20.2%), respectively. The structures (1–4) of the products were determined by ¹H and ¹³C NMR spectroscopies. This high regio- and stereoselectively of the -glucosidase could be applied for oligosaccharide synthesis.     

Possible complex translocation t(9; 14; 13)(q12;pl?;q31) in mother of a child with 9p-trisomy syndrome

Summary A mentally retarded boy with trisomy 9p is described. This trisomy arose through aberrant segregation of translocation chromosome during meiosis in his mother, who has a complex translocation involving chromosomes 9, 13, and 14. Based on both G-, Q-banding, and DNA replication patterns, the patient's karyotype was identified as 47,XY,-13, +(9;13) (9pter9q12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter). We suppose his mother's karyotype to be 46,XX,-9,-13,-14,+t(9;13) (9pterq12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter), +t(9;14) (9qter9q12::14pl?14qter). His phenotypically normal brother and sister are also carriers, having the same translocation chromosome as their mother. Clinical findings of the patient included peculiar face with hypertelorism, prominent nasal bridge and deformed helix, marked delay of osseous development, hypoplastic phalangia in fingers and toes, dysplastic nails and absence of digital triradii.     

Determination in situ of neutral and acidic fucose-containing oligosaccharides in the bovine submandibular gland

Lectins conjugated to horseradish peroxidase in combination with selected exoglycosidase digestion procedures were used to localize fucoglycoconjugates in the bovine submandibular gland. In particular, sequential treatments were employed to determine the distribution of neutral and acidic fucose-containing oligosaccharides that were previously shown to be present by biochemical techniques. Information was obtained on the distribution of the acidic oligosaccharide A-1a, -Fuc(12)--Gal-(14)--GlcNAc-(13)-[-NeuAc-(26)]-GalNAc-ol, which was sequenced in situ and localized in acinar cells.     

A Comparison of the Lipopolysaccharides and O-Specific Polysaccharides of Azospirillum brasilense Sp245 and Its Omegon-Km Mutants KM018 and KM252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas–liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units 2)--D-Rhap-(1 3)--D-Rhap-(1 3)--D-Rhap-(1 2)--D-Rhap-(1 2)--D-Rhap-(1 as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical composition.     

Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase

Summary The chemical structure of oligosaccharides produced by Zymomonas mobilis levansucrase (EC 2.4.1.10) was determined using enzymatic hydrolysis, mass spectroscopy, and ¹³C-NMR spectroscopy. The major oligosaccharide (98% of total oligomers) produced from transfructosylation reactions with -sucrose was identified as 1-kestose (O--D-glucopyranosyl-(12)-O--D-fructofuranosyl-(12)--D-fructofuranoside).     

Cyclic nucleotide phosphodiesterase and 5′-nucleotidase: A coupled system

Evidence is presented that multiple forms of cyclic nucleotide phophodiesterase (PDE) activity chromatographically separated from the soluble fraction of bovine hypothalamus are co-eluted with multiple forms of 5-nucleotidase (5N) activity. The enzymes could not be resolved from each other by anion-exchange chromatography on DEAE-TSK; by affinity chromatography on phenyl-, blue-, concanavalin A-, 5 AMP-sepharose, cAMP-silica gel; or by gel filtration on sephacryl S-200. The catalytic activities were found to be associated with the tetrameric, dimeric, and monomeric forms of the enzymes. The molecular weights determined by gel filtration or by SDS-gel electrophoresis were 220, 114, and 57 kDa, respectively. Kinetic analysis revealed that the first-order rate constant for 5 AMP hydrolysis measured in the reactions: cAMP5AMPadenosine was 100 times higher than that in the reaction: 5AMPadenosine. Thus, functional interrelation between PDE and 5N was expressed in drastic acceleration of the consecutive reactions: cAMP 5AMPadenosine. The results confirm the conclusion about the existence of a multienzyme system involving PDE and 5N or of a single bifunctional enzyme in brain tissue.This revised version was published online in June 2005 with corrections to the author name Gurvits.     

Total synthesis of a cholinergic neuron-specific ganglioside GT1aα: A high affinity ligand for myelin-associated glycoprotein (MAG)

An efficient total synthesis of a cholinergic neuron-specific ganglioside GT1a (IV³NeuAcIII⁶NeuAcII³NeuAc-GgOse4Cer) is described. The suitably protected sialyl-(26)-gangliotriose (III⁶NeuAc-GgOse3) derivative was glycosylated with the phenyl 2-thioglycoside of sialic acid in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) in acetonitrile medium, giving the disialogangliotriose (III⁶NeuAcII³NeuAc-GgOse3) derivative which contains both sialyl-(26)-GalNAc and sialyl-(23)-Gal structures (Route I). This pentasaccharide was efficiently synthesized also by the coupling of (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(26)-2-deoxy-3,4--isopropylidene-2-phthalimido-D-galactopyranosyl trichloroacetimidate with 2-(trimethylsilyl)ethyl (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(23)-(2,6-di--benzyl--D-galactopyranosyl)-(14)-2,3,6-tri--benzyl--D-glucopyranoside, followed by conversion of the phthalimido group to the acetamido group (Route II). -Deisopropylidenation and further glycosylation with methyl (methyl 5-acetamido-4,7,8,9-tetra--acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonate)-(23)-2,4,6-tri--benzoyl-1-thio--D-galactopyranoside, promoted by dimethyl(methylthio)sulfonium triflate (DMTST), gave the desired trisialogangliotetraose (IV³NeuAcIII⁶NeuAcII³NeuAc-GgOse4) derivative, which was converted stepwise into the title ganglioside GT1a by the introduction of the ceramide part and then complete deprotection. The ganglioside obtained was shown to be identical with the native GT1a on TLC-immunostaining.     

On the action of hydroxylamine,hydrazine and their derivatives on the water-oxidizing complex

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S₀ S₁, S₁ S₂, S₂ S₃, S₃ (S₄) S₀. State S₁ is most stable in the dark. Oxygen is released during S₃ (S₄) S₀. Hydroxylamine and hydrazine interact with S₁. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S₁ S₂ : S₂ S₃ : S₃ (S₄) S₀ : S₀ S₁ :... in the absence of the agents, to S₁ ^* S₀ : S₀ S₁ : S₁ S₂ : S₂ S₃ :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H(S₃ S₀) : 1H⁺(S₀ S₁) :... to 2H⁺(S₁ ^* S₀) : 1H⁺(S₀ S₁) : OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H⁺(S₃ S₀) :... The 2H⁺ were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53–57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n3 molecules of hydroxylamine and of n2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl^- or OH^-, which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S₁.Abbreviations DNP-INT dinitrophenylether of iodonitrothymol - FWHM full width at half maximum - NR neutral red (3-amino-7-dimethylamino-2-methylphenazine-HCI) - PS II photosystem II - WOC or (in formulas:) W water-oxidizing complex Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Linkage of sugar chains to a fragment peptide of FGF-5S by a chemoenzymatic strategy and changes in the rate of proteolytic hydrolysis

Various O-linked and N-linked sugar chains were linked enzymatically to a fragment peptide (Leu-Ser-Gln(or Asn)-Val-His-Arg) of FGF-5S. First, galactose was linked with -(13)-linkage to GalNAc-linked peptide by a transglycosylation using -galactosidase from Bacillus circulans (recombinant). Then sialic acid was linked with the aid of sialyltransferase from rat liver (recombinant) to give NeuAc-(23)-Gal-(13)-GalNAc-linked hexapeptide. Further, a sialylated 2-chain biantennary sugar chain was linked by a transglycosylation using endo N-acetyl--D-glucosaminidase from Mucor hiemalis (endo M, recombinant). The activity of DNA synthesis in a fibroblast cell line was increased by this glycosylation. The resistance of the obtained glycopeptides towards proteolytic hydrolysis by rat serum and by five proteases was compared with that of original peptide. The resistance was remarkably enhanced by the glycosylation.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Synthesis of Aminoethyl Glycosides of the Carbohydrate Chains of Glycolipids Gb3, Gb4, and Gb5

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with ethyl 2,3,4,6-tetra-O-benzyl- and ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio--D-galactopyranoside in the presence of methyl trifluoromethanesulfonate led to trisaccharide 2-azidoethyl (2,3,4,6-tetra-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside and its 3"-O-acetylated analogue, 2-azidoethyl (3-O-acetyl-2,4,6-tri-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside in yields of 85 and 83%, respectively. Deacetylation of the latter compound and subsequent glycosylation with 4-trichloroacetamidophenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-trichloroacetamido--D-galactopyranoside and 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in the corresponding selectively protected derivatives of the tetrasaccharide GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ and the pentasaccharide Gal(13)GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ in 88 and 73% yields, respectively. Removal of O-protecting groups, substitution of acetyl group for the N-trichloroacetyl group, and reduction of the aglycone azide group resulted in the target 2-aminoethyl globo-tri-, -tetra-, and -pentasaccharide, respectively.