Efficient Synthesis of O-Linked Glycopeptide by a Transglycosylation Using Endo α-N-Acetylgalactosaminidase from Streptomyces sp.

Galβ-(1→3)-GalNAc-linked hexapeptide was synthe-sized by a transglycosylation using Galβ-(1→3)-Gal-NAcβ-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Galβ-(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.     

Synthesis of 3-O-sulfoglucuronyl lacto-N-neotetraose 2-aminoethyl glycoside and biotinylated neoglycoconjugates thereof

The 2-aminoethyl glycoside of pentasaccharide 3-O-sulfo-GlcA(beta-1-->3)Gal(beta-1-->4)GlcNAc(beta-1-->3)Gal(beta-1--> 4)Glc(beta (1) and its conjugates with biotin and biotinylated polyacrylic acid were synthesized as molecular probes to investigate the recognition of the HNK-1 epitope containing carbohydrates by proteins. Key steps in the first of two investigated schemes for the preparation of the target compound 1 were (a) assembling of the pentasaccharide backbone (compound 10) by glycosylation of selectively substituted allyl glycoside of the trisaccharide GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta with glucuronyl-galactose glycosyl donor, (b) transformation of the allyl aglycon in 10 into 2-azidoethyl one (to give 11), (c) selective deprotection of the OH group at C-3 of the GlcA residue in 11 via saponification, intramolecular formation of 6,3-lacton (13) and its methanolysis, and (d) subsequent O-sulfation. The alternative scheme with the use of 2-azido-ethyl glycoside of the trisaccharide GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta instead of the allyl glycoside 6 was less effective due to smaller yield at the step of pentasaccharide synthesis. Additionally to 1 the 2-aminoethyl glycosides of the oligosaccharides GlcA(beta-1-->3)Gal(beta-1-->4)GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta, 3-O-sulfo-GlcA(beta-1-->3)Gal(beta, and GlcA(beta-1-->3)Gal(beta were also synthesized.     

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: a novel N-acetylgalactosaminyltransferase in human blood plasma.

Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.     

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) seedlings

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.     

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.     

Enzymatic synthesis of sulfated disaccharides using beta-D-galactosidase-catalyzed transglycosylation.

We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans beta-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo beta-D-galactopyranoside (S6Gal beta-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6'-sulfo N-acetyllactosamine (S6Gal beta1-4GlcNAc) and its positional isomer 6'-sulfo N-acetylisolactosamine (S6Gal beta1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Gal beta1-2Glc was mainly produced along with beta-(1-1)alpha, beta-(1-3), beta-(1-6) isomers in 74% total yield. When methyl alpha-D-glucopyranoside (Glc alpha-OMe) was an acceptor, the enzyme also formed mainly S6Gal beta1-2Glc alpha-OMe with its beta-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of beta-(1-2)-linked disaccharides. In contrast, with the corresponding methyl beta-D-glucopyranoside (Glc beta-OMe) acceptor, S6Gal beta1-3Glc beta-OMe and S6Gal beta1-6Glc beta-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the beta-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.     

Mucin biosynthesis: characterization of rabbit small intestinal UDP-N-acetylglucosamine:galactose beta-3-N-acetylgalactosaminide (N-acetylglucosamine----N-acetylgalactosamine) beta-6-N-acetylglucosaminyltransferase

We have characterized a UDP-GlcNAc:Gal beta-3-GalNAc (GlcNAc----GalNAc) beta-6-N-acetylglucosaminyltransferase from rabbit small intestinal epithelium by using freezing point depression glycoprotein as the acceptor. Optimal enzyme activity was obtained at pH 7.0-7.5, at 3 mM MnCl2, and at 0.08% Triton X-100. Ca2+, Mg2+, and Ba2+ also enhanced enzyme activity. The apparent Michaelis constant was 4.80 mM for freezing point depression glycoprotein, 0.59 mM for periodate-treated porcine submaxillary mucin, 0.49 mM for Gal beta 1----3 GalNAc alpha Ph, and 1.03 mM for UDP-GlcNAc. No enzyme activity was observed when asialo ovine submaxillary mucin was used as the acceptor. The 14C-labeled oligosaccharide obtained by alkaline borohydride treatment of the product was shown to be a homogeneous trisaccharide by compositional analysis, Bio-Gel P-4 gel filtration, and high-performance liquid chromatography. The structure of the trisaccharide was identified as Gal beta 1----3-(GlcNAc beta 1----6)GalNAc-H2 by (a) identification of 2,3,4,6-tetramethyl-1,5-diacetylgalactitol and 1,4,5-trimethyl-3,6-diacetyl-2-N-methylacetamidogalactitol by gas-liquid chromatography-mass spectrometry and (b) the complete cleavage of the newly formed glycosidic bond by jack bean beta-hexosaminidase. The structure of the trisaccharide was confirmed by 1H nuclear magnetic resonance (270 MHz) and also by periodate oxidation of the trisaccharide followed by NaBH4 reduction, 4 N HCl hydrolysis, a second NaBH4 reduction, and the identification of threosaminitol on an amino acid analyzer. By acceptor competition studies, the enzyme activity was shown to be a much N-acetylglucosaminyltransferase. We postulate that this glycosyltransferase may play a key role in the regulation of mucin oligosaccharide synthesis.     

Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma

ASGP-1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O-linked oligosaccharide chains. We have used this system to investigate the O-glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S-1 and S-2) were isolated as their oligosaccharitols by alkaline borohydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide -O4S-GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and S-2 its sialylated derivative -O4S-GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP-1 intermediate in size between immature and mature sialomucin. Pulse-chase analyses showed that the intermediate could be chased into mature ASGP-1. The concomitant conversion of S-1 into S-2 had a half-time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and sialylated derivative GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNAc. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with beta-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as beta-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient synthesis of a sialyl T-antigen-linked glycopeptide by the chemoenzymatic method

A sialyl T-antigen-linked tetrapeptide was prepared by the combined method of chemical synthesis and enzymatic synthesis. The GalNAc-linked peptide was first obtained by using a commercial peptide synthesizer, and then a galactose residue was attached with beta-(1-->3)-linkage by transglycosylating with a recombinant beta-galactosidase from Bacillus circulans. The sialic acid residue was then combined by alpha-(2-->3)-linkage with sialytransferase from rat liver.     

Polysaccharides from Gracilaria corticata: sulfation, chemical characterization and anti-HSV activities

In this study, we have analyzed water-extracted polysaccharides of Gracilaria corticata. The water extract (WE), a galactan-containing sub-fraction (F3) and their hyper sulfated derivatives (WES1, WES2, F3S1 and F3S2) had anti-HSV activity with inhibitory concentration 50% (IC50) from 1.1 to 27.4 microg/ml. Sub-fraction F3, which has a molecular mass of 30 kDa, consists of a backbone of beta-(1-->3) and alpha-(1-->4)-linked-galactopyranosyl residues. This linear galactan contained Gal2Xyl1, Gal2AnGal2, Gal4 and Me-Gal3AnGal2 as oligomeric building subunits. Sulfate group was located at C-4 of (1-->3)-linked galactopyranosyl residues of the native galactan, and appeared to be very important for the anti-herpetic activity.     

Structural characterization and in vitro antitumor activity of a novel polysaccharide isolated from the fruiting bodies of Pleurotus ostreatus

A novel water-soluble polysaccharide (POPS-1) was obtained from the fruiting bodies of Pleurotus ostreatus by hot water extraction, ethanol precipitation, and fractionated by DEAE-cellulose ion exchange chromatography and sepharose CL-6B gel filtration chromatography using an ATKA explore 100 purifier. The structure characterization and antitumor activity of the POPS-1 were evaluated in this paper. According to GC analysis, HPGPC, FT-IR, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC-MS analysis, the results indicate POPS-1 (M(w)=31 kDa) was composed of Man; Gal; Glc with a molar ratio of 1:2.1:7.9, it had a backbone of beta-(1-->3)-linked glucose residues, which occasionally branches at O-6. The branches were composed of (1-->3)-linked Glc, (1-->4)-linked Gal, (1-->4)-linked Man, and terminated with Glc and Gal residues. Cytotoxicity assay showed POPS-1 presented significantly higher antitumor activity against Hela tumor cell in vitro, in a dose-dependent manner, and exhibited significantly lower cytotoxicity to human embryo kidney 293T cells than Hela tumor cells compared with 5-Fu. The results suggest POPS-1 may be considered as a potential candidate for developing a novel low toxicity antitumor agent.     

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.     

Enzymatic transfer of a preassembled trisaccharide antigen to cell surfaces using a fucosyltransferase.

The Lewis alpha (1-->3/4)-fucosyltransferase (Le-FucT) is known to fucosylate both Type I (beta Gal(1-->3) beta GlcNAc) and Type II (beta Gal(1-->4) beta GlcNAc) sequences even when these are sialylated at OH-3 or fucosylated at OH-2 of the terminal Gal residues. These acceptor sequences are ubiquitous on mammalian cell-surface glycoproteins and glycolipids. The Le-FucT enzyme is therefore a potential candidate as a universal reagent for the modification of cell surfaces. We have found that a readily accessible, partially purified Le-FucT from human milk, which normally uses GDP-fucose (a 6-deoxy sugar) as the donor for the transfer of a single fucose residue, will also transfer a fucose residue substituted on C-6 by a very large sterically demanding structure, in this instance, a synthetic blood group antigen. As a demonstration of the ability of the Le-FucT to modify glycoconjugates in a mild and specific manner, we chemically synthesized the complex sugar-nucleotide alpha Gal(1-->3) [alpha Fuc(1-->2)]-beta Gal-O-(CH2)8COHN(6)-beta-L-fucose-GDP (13) which is a GDP-fucose analog where the human blood group B trisaccharide antigen is covalently linked to C-6 of fucose through an amino group. It is shown that, in enzyme-linked immunosorbent assays, the Le-FucT uses both immobilized beta Gal(1-->3) beta GlcNAc-bovine serum albumin conjugates and fetuin as acceptor substrates and renders them blood group B-active as detected by a monoclonal anti-B blood-grouping antibody. The fucose residue to which the B-trisaccharide is linked therefore becomes covalently attached to the acceptor oligosaccharide chains of those glycoproteins. Incubation of type "O" erythrocytes with the Le-FucT and complex donor 13 results in the covalent transfer of alpha Gal(1-->3) [alpha Fuc(1-->2)] beta Gal-O-(CH2)8COHN(6)-beta-L-Fuc to cell-surface acceptors since the cells become phenotypically "B" and are agglutinated by the same antibody. It is proposed that the Le-FucT represents a powerful new tool with the ability to label animal cell surfaces with preassembled oligosaccharide and possibly also other complex recognition markers.     

Molecular cloning and characterization of a novel human galactose 3-O-sulfotransferase that transfers sulfate to gal beta 1-->3galNAc residue in O-glycans

We have identified a novel galactose 3-O-sulfotransferase, termed Gal3ST-4, by analysis of an expression sequence tag using the amino acid sequence of human cerebroside 3'-sulfotransferase (Gal3ST-1). The isolated cDNA contains a single open reading frame coding for a protein of 486 amino acids with a type II transmembrane topology. The amino acid sequence of Gal3ST-4 revealed 33%, 39%, and 30% identity to human Gal3ST-1, Gal beta 1-->3/4GlcNAc:-->3'-sulfotransferase (Gal3ST-2) and Gal beta 1-->4GlcNAc:-->3'-sulfotransferase (Gal3ST-3), respectively. The Gal3ST-4 gene comprised at least four exons and was located on human chromosome 7q22. Expression of Gal3ST-4 in COS-7 cells produced a sulfotransferase activity that catalyzes the transfer of [(35)S]sulfate to the C-3' position of Gal beta 1-->3GalNAc alpha 1-O-Bn. Gal3ST-4 recognizes Gal beta 1-->3GalNAc and Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc as good substrates, but not Gal beta 1-->3GalNAc(OH) or Gal beta 1-->3/4GlcNAc. Asialofetuin is also a good substrate, and the sulfation was found exclusively in O-linked glycans that consist of the Gal beta 1-->3GalNAc moiety, suggesting that the enzyme is specific for O-linked glycans. Northern blot analysis revealed that 2.5-kilobase mRNA for the enzyme is expressed extensively in various tissues. These results suggest that Gal3ST-4 is the fourth member of a Gal:-->3-sulfotransferase family and that the four members, Gal3ST-1, Gal3ST-2, Gal3ST-3, and Gal3ST-4, are responsible for sulfation of different acceptor substrates.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

The aspartimide problem in Fmoc-based SPPS. Part I.

A variety of Asp beta-carboxy protecting groups, Hmb backbone protection and a range of Fmoc cleavage protocols have been employed in syntheses of the model hexapeptide H-VKDGYI-OH to investigate the aspartimide problem in more detail. The extent of formation of aspartimide and aspartimide-related by-products was determined by RP-HPLC. This study included three new Fmoc-Asp-OH derivatives: the beta-(4-pyridyl-diphenylmethyl) and beta-(9-phenyl-fluoren-9-yl) esters and also the orthoester Fmoc-beta-(4-methyl-2,6,7-trioxabicyclo[2.2.2]-oct-1-yl)-alanine. 3-Methylpent-3-yl protection of the Asp side chain resulted in significant improvements with respect to aspartimide formation. Complete suppression was achieved using the combination OtBu side chain protection and Hmb backbone protection for the preceding Gly residue.     

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.     

The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases

The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.