Structural studies on glycolipids of shellfish. V. Gala-6 series glycosphingolipids of the marine snail, Chlorostoma argyrostoma turbinatum

A series of glycosphingolipids and phosphonoglycosphingolipid containing only galactose as the sugar component were isolated from the marine snail, Chlorostoma argyrostoma turbinatum. The structures of these lipids were studied by methylation analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, and characterized as follows: the glycosphingolipids galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide and galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide, and phosphonoglycosphingolipid N-methylaminoethylphosphonyl galactosyl(1-1)ceramide. The main molecular species of the ceramide moiety were hexadecanoyl-octadecasphingenine and hydroxyhexadecanoyl-octadecasphingadienine in all of these sphingolipids.     

American Leishmania spp. and Trypanosoma cruzi: galactosyl alpha(1-3) galactose epitope localization by colloidal gold immunocytochemistry and lectin cytochemistry.

Patients with Chagas' disease or different clinical forms of leishmaniasis (cutaneous or visceral) have elevated galactosyl alpha (1-3)galactose antibodies. Using colloidal gold immunocytochemistry--monoclonal antibody gal-13 (specific for lipid-linked galactosyl alpha (1-3)galactose residues) and anti-nidogen antibodies and lectin cytochemistry (Bandeiraea simplicifolia IB4), both techniques specific for demonstrating galactosyl alpha (1-3)galactose residues--we have found terminal disaccharide residues on the Trypanosoma cruzi external surface of Vero cell-derived trypomastigotes but not in intact epimastigotes (although disrupted epimastigotes strongly stained), in the lips of the flagellar pocket, and on the parasitic side exactly opposite to the flagellar pocket in amastigote and promastigote forms of American Leishmania. These results resemble those obtained using anti-laminin antibodies in both trypanosomatids. In addition, results obtained with anti-nidogen antibodies seem to recognize in Trypanosoma cruzi and American Leishmania culture forms another different unknown terminal disaccharide. These results confirm the presence of terminal galactosyl alpha (1-3)galactose residues in both trypanosomatids, and that rabbit anti-laminin antibodies are indeed also recognizing galactosyl alpha (1-3)galactose residues as demonstrated for human circulating antibody. The presence of abundant galactosyl alpha (1-3)galactose residues on Trypanosomatid family members suggests a specific unknown role in parasite physiology for this terminal disaccharide.     

Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation.

Vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus, which were produced by cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars, were found to be less stable in human serum than those from alphaGal-negative cells, indicating that galactosyl(alpha1-3)galactosylation sensitizes these viruses as well as mammalian type C oncoviruses (Rother et al., J. Exp. Med. 182:1345-1355, 1995; Takeuchi et al., Nature (London) 379:85-88, 1996) to complement killing via natural anti-alphaGal antibodies. Thus, virus killing mediated by anti-alphaGal antibodies may play a role as a barrier to animal-to-human infection of various enveloped viruses. Virus vectors for human in vivo gene therapy based on the viruses mentioned above should be produced from alphaGal-negative cells.     

Chemical structure of monosialoganglioside GM1b biosynthesized in vitro.

The structure of a neuraminidase-labile monosialoganglioside which is formed in vivo from asialoganglioside (galactosyl (beta, 1 in equilibrium 3) N-acetylgalactosaminyl (beta, 1 in equilibrium 4) galactosyl (beta, 1 in equilibrium 4) glucosyl (1 in equilibrium 1) ceramide) and cytidine-5'-monophospho-N-acetylneuraminic acid in the presence of young rat brain sialytransferase has been established. This monosialoganglioside contains a neuraminidase-labile N-acetylneuraminyl group which is linked at position C-3 of the terminal galactosyl unit. This result was obtained by ultramicro scale permethylation of radioactive neuraminidase-labile monosialoganglioside biosynthesized from asialoganglioside labeled with tritium in the terminal galactose.     

Purification and characterization of stachyose synthase from lentil (Lens culinaris) seeds: galactopinitol and stachyose synthesis.

Stachyose synthase (STS) (EC 2.4.1.67) was purified 313-fold from mature seeds of lentil. The final preparation had a specific activity of 9.09 nkat stachyose formed per milligram of protein. The enzyme was a monomeric protein with a molecular mass of 88.6 kDa (SDS-PAGE) and an isoelectric point of 4.8 (chromatofocusing). Western analysis revealed cross-reactivity of polyclonal antibodies raised against STS from adzuki bean with the lentil enzyme. The purified enzyme catalyzed a range of different galactosyl transfer reactions. In addition to the genuine STS reaction (raffinose + galactinol --> stachyose + myo-inositol), the enzyme catalyzed the reversible galactosyl transfer from galactinol to d-pinitol (1d-3-O-methyl-chiro-inositol), yielding galactopinitol A (O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol) and myo-inositol. Galactopinitol A could be further galactosylated by STS to give ciceritol (O-alpha-d-galactopyranosyl-(1 --> 6)-O-alpha-d-galactopyranosyl-(1 --> 2)-4-O-methyl-d-chiro-inositol). Enzymatic synthesis of galactopinitol A and ciceritol is a new observation. However, STS was not only able to utilize galactopinitol A as galactosyl acceptor, but also as galactosyl donor to form stachyose from raffinose. The role of STS in the metabolism of galactosyl cyclitols and oligosaccharides in plant seeds is discussed.     

猴头菌水溶性多糖的分离纯化与结构表征

从猴头菌子实体中分离得到一种新型的水溶性杂多糖HEPF2,分子量大小为1.66′104Da,该多糖由岩藻糖、半乳糖和葡萄糖以1.00:3.69:5.42比例构成,同时也含有微量的3-O-甲基鼠李糖。进一步利用傅立叶变换红外光谱法、糖组成分析、甲基化分析、部分酸水解法和核磁共振法等方法进行结构鉴定,检测结果表明,该杂多糖中包含1→4、1→6结合的葡萄糖和1→6结合的半乳糖残基,连接于主链的侧链残基,包括岩藻糖残基、少数的端基葡萄糖和半乳糖残基。核磁共振法检测结果还表明,1→4结合葡萄糖为β构型,(1→6)结合半乳糖、(1→2,6)结合半乳糖和端基葡萄糖均为α构型。     

Structure of oligosaccharide side chains of an intestinal immune system modulating arabinogalactan isolated from rhizomes of Atractylodes lancea DC

An intestinal immune system modulating arabino-3,6-galactan (ALR-5IIa-1-1) has been found in rhizomes of Atractylodes lancea DC. [Planta Medica 1998, 64, 714-719; Carbohydr. Polyms. 2001, 46, 147-156], however other arabino-3,6-galactans from Larix and Acacia failed to express the modulating activity. Degradation of the galactosyl side chains in Araf-side chain-trimmed ALR-5IIa-1-1 (AF-ALR-5IIa-1-1) with an endo-beta-D-(1-->6)-galactanase remarkably decreased the activity of AF-ALR-5IIa-1-1. Structural analysis indicated that the major endo-beta-D-(1-->6)-galactanase-digestable side chains in ALR-5IIa-1-1 are composed of beta-D-(1-->6)-galactopyranosyl oligosaccharides having d.p. 1-8. Because degradation of the beta-D-(1-->3)-galactan backbone in AF-ALR-5IIa-1-1 also significantly reduced its activity, some of these galactosyl side chains attached to beta-D-(1-->3)-galactan backbone are suggested to be responsible for expression of the activity of ALR-5IIa-1-1.     

Structural studies on glycolipid of shellfish. III. Novel glycolipids from Turbo cornutus

Five kinds of sphingoglycolipids were isolated from Turbo cornutus. Four of them were a series of novel glycolipids consisting only of galactose. The structures of these glycolipids were studied by methylation analysis, periodate oxidation, enzymatic degradation, and proton magnetic resonance spectroscopy. Three glycolipids were characterized as galactosyl(beta 1 leads to 1)ceramide, galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 1)ceramide, and galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 1)ceramide. Data indicating that the 4th glycolipid might be the tetragalactosyl derivative of this series were obtained. The carbohydrate moiety of the 5th glycolipid, in contrast, was composed of fucose, galactose, glucose and N-acetylglycosamine in a molar ratio of 1 : 2 : 1 : 1.     

PmST2: a novel Pasteurella multocida glycolipid α2-3-sialyltransferase

Pasteurella multocida (Pm) is a multi-species pathogen that causes diseases in animals and humans. Sialyltransferase activity has been detected in multiple Pm strains and sialylation has been shown to be important for the pathogenesis of Pm. Three putative sialyltransferase genes have been identified in Pm genomic strain Pm70. We have reported previously that a Pm0188 gene homolog in Pm strain P-1059 (ATCC 15742) encodes a multifunctional sialyltransferase (PmST1). We demonstrate here that while PmST1 prefers to use oligosaccharides as acceptors, PmST2 encoded by the Pm0508 gene homolog in the same Pm strain is a novel glycolipid α2-3-sialyltransferase that prefers to use lactosyl lipids as acceptor substrates. PmST2 and PmST1 thus complement each other for an efficient synthesis of α2-3-linked sialosides with or without lipid portion. In addition, β1-4-linked galactosyl lipids are better PmST2 substrates than β1-3-linked galactosyl lipids. PmST2 has been used successfully in the preparative scale synthesis of sialyllactosyl sphingosine (lyso-GM3), which is an important glycolipid and an intermediate for synthesizing more complex glycolipids such as gangliosides.     

Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.     

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase,partial purification of the enzyme from maturing seeds,and steady-state kinetic analysis of raffinose synthesis

Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea ( Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol ( O-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.     

Characterization of major glycolipids in bovine erythrocyte membrane

Several neutral glycolipids and gangliosides were isolated from bovine erythrocyte stroma. Their structures were determined by partial acid hydrolysis, methylation analysis, periodate oxidation and CrO3 oxidation. Two major neutral glycolipids were characterized as lactosylceramide and galactosyl(alpha1--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Two major gangliosides were N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide and N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Minor glycolipids were glucosyl- and galactosylceramide, glucosamine-containing tri- and tetraglycosylceramide, glucosamine-containing disialosylhexaglycosylceramide, and gangliosides containing N-acetylneuraminic acid. The ceramide moiety of each glycolipid contained perdominantly d18:1 sphingosine, and normal fatty acids of C16:0, C22:0, C24:0, and C24:1.     

Treatment of rhamnogalacturonan I with lithium in ethylenediamine

Rhamnogalacturonan I is a pectic polysaccharide that is solubilized from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus) by the action of a highly purified endo-1,4-α-polygalacturonanase. Rhamnogalacturonan I has a linear backbone consisting of the diglycosyl repeating unit, →4)-α-d-GalpA-(1→2)-α-l-Rhap-(1→. Approximately half of the α-l-rhamnosyl residues of the backbone are branched at O-4. Selective cleavage at the galactosyluronic acid residues of the backbone by treatment of rhamnogalacturonan I wit lithium in ethylenediamine resulted in the release of the neutral glycosyl-residue sidechains that had been attached to the backbone. Various analytical techniques, including combined liquid chromatography-mass spectrometry, combined gas-liquid chromatography-mass spectrometry, and ¹H-nuclear magnetic resonance spectroscopy, were used to determine the structure of the side chains. The majority of the sidechains were isolated as oligoglycosylalditols, with rhamnitol at the “reducing” end. Terminal 2-, 4-, or 6-linked galactosyl residues were found attached to O-4 of the rhamnitol residues The 2-, 4-, and 6-linked galactosyl residues had terminal or 2-linked arabinosyl, or additional galactosyl, residues attached to them. Based on the results of fast-atom-bombardment mass spectrometry, the side chains were found to range in size from one to fourteen glycosyl residues. The side-chain structures suggest that there are four or more distinct families of side chains attached to the backbone of rhamnogalacturonan I.     

SPHINGOGLYCOLIPIDS IN THE NERVOUS SYSTEM IN FABRY'S DISEASE

Abstract— Two glycolipids, accumulated in the spinal ganglia of a patient with Fabry's disease were identified as: galactosyl (α1 → 4) galactosyl (β1 → 4) glucosyl(1 → 1) ceramide (CTH) and galactosyl (α1 → 4) galactosyl(1 → 1) ceramide (CDG). Only one glycolipid which had the same structure as the CTH in the spinal ganglia accumulated in the sympathetic ganglia of the patient. In the nervous system, CTH contained behenic acid (C_22:0) as the major fatty acid. In the spinal ganglia, CDG also contained behenic acid as the major fatty acid.     

Complete 1H and 13C NMR assignment of mono-sulfated galactosylceramides with four types of ceramides from human kidney

The full assignment of 1H and 13C NMR signals of galactosylceramide 3-sulfate (galactosyl sulfatide) and 1H signals of galactosylceramide 6-sulfate was achieved by using 1H-1H DQF-COSY and 1H-13C heteronuclear COSY. Analyses were performed on a mixture of galactosyl sulfatides with four representative ceramide types consisting of a combination of non-hydroxy or 2-hydroxy fatty acids and sphingenine or 4D-hydroxysphinganine (trihydroxysphinganine) as the long-chain bases. The 1H and 13C NMR parameters of galactosyl sulfatide with 4-hydroxysphinganine as well as 13C signals of complex lipids with 4-hydroxysphinganine were elucidated for the first time. Not only sulfation of the galactosyl residue, but also modification of the aglycon, including hydroxylation of fatty acids and hydration of the double bond in sphingoid bases, altered the chemical shifts substantially. In addition, the unique long-range coupling constants, 4J(H,H) and 5J(H,H), in the galactosyl residue of galactosyl sulfatide could be determined.     

Synthesis of some N-galactosides of 3-aryl-5-benzyl (or substituted benzyl)-1,2,4-triazin-6(1H)-/ones or thiones of expected biological activity

The 1-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-3-aryl-5-benzyl (or substituted benzyl)-1,2,4-triazin-6(1H)-/ones or thiones were prepared via galactosidation of 3-aryl-5-benzyl (or substituted benzyl)-1,2,4-triazin-6(1H)-/ones or thiones with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide. The structure of the new galactosyl derivatives was based on both spectroscopic and chemical evidences.     

Glycoconjugate distribution in gastric fundic mucosa of Umbrina cirrosa L. revealed by lectin histochemistry

The stomach of adult shi drum Umbrina cirrosa was investigated using a battery of nine horseradish peroxidase‐conjugated lectins combined with enzymatic treatment, in order to distinguish glycoconjugate sugar residues. Epithelial cells showed the presence of galactosyl(β1→4)N‐acetylglucosamine, mannose, N‐acetylgalactosamine, N‐acetylglucosamine, fucose and sialic acid‐galactosyl(β1→3)N‐acetylgalactosamine residues. Gastric pits had similar sugar residues with the exception of N‐acetylgalactosamine which was less diffused. Gastric glands were characterized by the presence of glycoconjugates containing galactosyl(β1→3)N‐acetylgalactosamine, N‐acetylglucosamine, galactosyl(β1→4) N‐acetylglucosamine, N‐acetylgalactosamine and a small amount of sialic acid linked to N‐acetylgalactosamine.     

Structural studies on teichoic acids in cell walls of several serotypes of Listeria monocytogenes

Structural studies were carried out on the teichoic acids in cell walls of Listeria monocytogenes serotypes 3a, 4b, 4f, 6, and 7. The structure of the dephosphorylated repeating units, obtained by treatment with 46% hydrogen fluoride or alkaline hydrolysis, was examined by methylation analysis, acetolysis, and 1H-NMR spectroscopy. The results of Smith degradation of the teichoic acids and 13C-NMR spectroscopy led to the following most likely structures of the repeating units of the teichoic acids:----1-[N-acetylglucosaminyl(alpha 1----4)]ribitol-5-phosphate----for serotype 3a,----4-[galactosyl(alpha 1----6)][glucosyl(beta 1----3)]N -acetylglucosaminyl(beta 1----2)ribitol-5-phosphate----for serotype 4b,----4-[galactosyl(alpha 1----6)][N -acetylglucosaminyl(alpha 1----3)]N-acetylglucosaminyl(beta 1----2)ribitol -5-phosphate----for serotype 4f,----4-N-acetylglucosaminyl(beta 1----4)ribitol -5-phosphate----for serotype 6, and----1-ribitol-5-phosphate----for serotype 7. About 40% of the repeating units of the teichoic acid from serotype 4f were not substituted at C-3 of beta-N-acetylglucosaminyl residues.     

Lectin specificity and binding characteristics of human C-reactive protein.

C-reactive protein (CRP) is thought to play an important role in immunomodulation. The exact biologic function of this pentraxin protein is, however, still unclear. Here we report experiments designed to further characterize the binding properties of CRP. Using purified human CRP it could be shown that CRP immobilized onto polystyrene surfaces or onto latex beads binds distinct plasma glycoproteins including IgG, asialofetuin, asialo-beta 2-glycoprotein I and, likewise, synthetic glycoproteins as a lectin, exhibiting binding specificity for terminal galactosyl residues of the glycoprotein glycans. Binding of CRP to IgA, IgM, IgG, asialofetuin, asialo-beta 2-glycoprotein I and to synthetic glycoproteins requires immobilization onto surfaces of both CRP and the ligand. Fibronectin and fibrinogen are bound by surface-immobilized CRP also in soluble phase. Comparing various mono-, di-, and trisaccharides as competitive inhibitors of the lectin binding activity of CRP, only beta-D-Gal-(1-3)-D-GalNAc, beta-D-Gal-(1-4)-D-GalNAc, and beta-D-Gal-(1-4)-beta-D-Gal-(1-4)-D-GlcNAc had significant inhibitory power at a concentration of 8 mmol/liter. Binding activity of CRP was pH-dependent with an optimum at pH 5 to 6 and was reduced by 90% when pH was shifted from 6 to the physiologic pH value of 7.4. CRP exhibited lectin-like properties with binding specificity for galactosyl residues also when bound to K-562 erythroleukemia cells. It is therefore suggested that CRP immobilized onto surfaces exhibits lectin activity toward galactosyl groups preferentially in a mildly acidic environment as present at sites of inflammation.     

Synthesis of methyl α-d-glucopyranosyl-(1–2)-α-d-galactopyranosyl-(1-3)-α-d-glucopyranoside and an acyclic analogue thereof for probing the carbohydrate-binding specificity of bacteriophage ϕX 174

The title trisaccharide was synthesized using methyl 1-thioglycoside building blocks. An acyclic analogue, methyl 3-O-(α-D-glycopyranosyl-oxyethyl)-α-D-glucopyranoside, which has an ethylene bridge in place of the galactosyl residue, was also synthesized.