Type Sugar Chains Research Articles

Therapeutic Application and Structural Features of Adeno-Associated Virus Vector.

Adeno-associated virus (AAV) is characterized by non-pathogenicity, long-term infection, and broad tropism and is actively developed as a vector virus for gene therapy products. AAV is classified into more than 100 serotypes based on differences in the amino acid sequence of the capsid protein. Endocytosis involves the uptake of viral particles by AAV and accessory receptors during AAV infection. After entry into the cell, they are transported to the nucleus through the nuclear pore complex. AAVs mainly use proteoglycans as receptors to enter cells, but the types of sugar chains in proteoglycans that have binding ability are different. Therefore, it is necessary to properly evaluate the primary structure of receptor proteins, such as amino acid sequences and post-translational modifications, including glycosylation, and the higher-order structure of proteins, such as the folding of the entire capsid structure and the three-dimensional (3D) structure of functional domains, to ensure the efficacy and safety of biopharmaceuticals. To further enhance safety, it is necessary to further improve the efficiency of gene transfer into target cells, reduce the amount of vector administered, and prevent infection of non-target cells.

Control of Cell-Substrate Binding Related to Cell Proliferation Cycle Status Using a Cytocompatible Phospholipid Polymer Bearing Phenylboronic Acid Groups.

To provide high-quality cellular raw materials for cell engineering and pharmaceutical engineering, a polymer substrate is prepared for cell separation focusing on the cell proliferation cycle. There are many types of sugar chains on cell membranes, which function as signaling molecules to control interactions with the exterior of the cell; their abundance changes during the cell-proliferation cycle. In this study, a phenylboronic acid group, which has affinity for sugar chains, is introduced into a polymer containing a phosphorylcholine group that does not induce cell activation. On the surface of this polymer, human promyelocytic leukemia cells can adhere. The adhesion rate is increased by pretreating the substrate with an alkaline solution. Moreover, cell adhesion is dependent on the sugar additive in the culture medium. Therefore, cell adhesion is governed by reactions between the sugar chain on the cell membrane and the phenylboronic acid groups on the substrate. It is revealed that the adhesion rate changes depending on the expression level of sugar chains related to the cell-proliferation cycle. Based on this, it may be proposed a cell proliferation cycle-specific separation process using the polymer substrate based on cell adhesion depending on sugar chain density.

Anti-influenza triterpenoid saponins (saikosaponins) from the roots of Bupleurum marginatum var. stenophyllum

This study to investigate antiviral components from the roots of Bupleurum marginatum var. stenophyllum led to the isolation of five novel saikosaponins, namely 6″-O-crotonyl-saikosaponin a (1), tibesaikosaponin I (2), tibesaikosaponin II (3), tibesaikosaponin III (4), tibesaikosaponin IV (5), along with 9 known analogues (6–14). Their structures were established by spectral data analyses (IR, MS, 1D and 2D NMR) and by comparison of spectral data with those of the related known compounds. Antiviral testing of all compounds against influenza A virus A/WSN/33 (H1N1) in 293TGluc cells showed that nepasaikosaponin k (12), saikosaponin n (13) and saikosaponin h (14) behaved more potent inhibitory activity and selectivity than the positive control, Ribavirin. The preliminary structure-activity relationship studies suggest that the 13, 28-epoxy group, the type of sugar chain and the type of olefinic bonds are significant for antiviral activity and selectivity.

Evaluation of novel saponins from Psammosilene tunicoides and their analogs as immunomodulators

Two original oleanane-type triterpenoid saponins, tunicosaponin A (TSA) and tunicosaponin E (TSE) were isolated from the roots of Psammosilene tunicoides. Four semi-synthetic saponin derivatives, TSA1, TSA2, TSA3, and TSA4, were synthesized from TSA; two derivatives TSE1 and TSE2 were prepared from TSE. Through comparing their hemolytic activity and effects on ovalbumin (OVA)-induced IgG response with those of Quil A, TSA2 was selected as a lead candidate to evaluate acute and hepatotoxic toxicities and adjuvant potentials on the cellular and humoral immune responses of ICR mice against OVA. TSA2 had lower hemolytic activity than Quil A and TSA (P<0.001). Furthermore, TSA2 did not cause any mortality and side effects when mice were administered subcutaneously at a dose up to 1.6mg. Moreover, no significant hepatotoxic effect was observed in TSA2 groups in doses ranging from 0.05mg to 0.8mg. The Con A-, LPS-, and OVA-induced splenocyte proliferation, OVA-specific antibody levels (IgG, IgG1, IgG2a and IgG2b) and IFN-γ, TNF-α, IL-2, IL-4 and IL-5 in serum were significantly enhanced by TSA2 (25μg/mouse). The present study of structure–activity relationship indicates that the hydrophobicity of the ester/amide chains bonds to the carboxyl group of the glucuronic acid residue at position C-3 of the triterpene aglycone and the type of sugar chain at C-28 position of saponin could affect the potential of toxicity and adjuvant. Our findings demonstrate that TSA2 possesses higher adjuvant activities with less adverse effects and should be further explored as an immunomodulator for immune responses.

Structural Basis of the Anti-HIV Activity of the Cyanobacterial Oscillatoria Agardhii Agglutinin

The cyanobacterial Oscillatory Agardhii agglutinin (OAA) is a recently discovered HIV-inactivating lectin that interacts with high-mannose sugars. Nuclear magnetic resonance (NMR) binding studies between OAA and α3,α6-mannopentaose (Manα(1-3)[Manα(1-3)[Manα(1-6)]Manα(1-6)]Man), the branched core unit of Man-9, revealed two binding sites at opposite ends of the protein, exhibiting essentially identical affinities. Atomic details of the specific protein-sugar contacts in the recognition loops of OAA were delineated in the high-resolution crystal structures of free and glycan-complexed protein. No major changes in the overall protein structure are induced by carbohydrate binding, with essentially identical apo- and sugar-bound conformations in binding site 1. A single peptide bond flip at W77-G78 is seen in binding site 2. Our combined NMR and crystallographic results provide structural insights into the mechanism by which OAA specifically recognizes the branched Man-9 core, distinctly different from the recognition of the D1 and D3 arms at the nonreducing end of high-mannose carbohydrates by other antiviral lectins.

Regulatory Mechanism of Silkworm Hemocyte Adhesion to Organs

Circulating hemocytes in the body fluid of the silkworm are increased during the larval-larval molting period. We investigated hemocyte adhesion to organs mediating the selectin-selectin ligands during the feeding period and the larval-larval molting period using the lectin staining method, sugar chain digestion test with glycoside hydrolases, and the hemocyte adhesion inhibition test using monosaccharides. The results of these tests suggested that the selectin ligand involved in hemocyte adhesion was the Sialyl Lewis x-type, and the structure was changed from the feeding period to the larval-larval molting period. Beta-galactosidase appears to be an enzyme that eliminates N-acetylgalactosamine and sialylated N-acetylgalactosamine from the terminal of Sialyl Lewis x. Beta-galactosidase activation in skin basement membranes, muscle, fat bodies, midguts, and hemocytes increased markedly during the larval-larval molting period, and at that time, hemocytes were detached from organs. Adding 20-hydroxyecdysone or its analog, tebufenozide to cultured fat bodies increased β-galactosidase activity in these tissues. Therefore, 20-hydroxyecdysone may induce a structural change in Sialyl Lewis x type sugar chains on the cell surface of silkworm's organs by increasing the β-galactosidase activity to detach hemocytes from organs and increase the number of circulating hemocytes during the larval-larval molting period.

Pyridylamination as a means of analyzing complex sugar chains

Herein, I describe pyridylamination for versatile analysis of sugar chains. The reducing ends of the sugar chains are tagged with 2-aminopyridine and the resultant chemically stable fluorescent derivatives are used for structural/functional analysis. Pyridylamination is an effective “operating system” for increasing sensitivity and simplifying the analytical procedures including mass spectrometry and NMR. Excellent separation of isomers is achieved by reversed-phase HPLC. However, separation is further improved by two-dimensional HPLC, which involves a combination of reversed-phase HPLC and size-fractionation HPLC. Moreover, a two-dimensional HPLC map is also useful for structural analysis. I describe a simple procedure for preparing homogeneous pyridylamino sugar chains that is less laborious than existing techniques and can be used for functional analysis (e.g., sugar-protein interaction). This novel approach was applied and some of the results are described: i) a glucosyl-serine type sugar chain found in blood coagulation factors; ii) discovery of endo-β-mannosidase (EC 3.2.1.152) and a new type plant α1,2-l-fucosidase; and iii) novel substrate specificity of a cytosolic α-mannosidase. Moreover, using homogeneous sugar chains of a size similar to in vivo substrates we were able to analyze interactions between sugar chains and proteins such as enzymes and lectins in detail. Interestingly, our studies reveal that some enzymes recognize a wider region of the substrate than anticipated.

The N‐glycan profile of mouse myelin, a specialized central nervous system membrane

Understanding the rich complement of sugar chains found in cellular membranes is impeded by the complexity of cell types and membrane diversity. To overcome this, we have analyzed the N-linked sugar chain composition of the glycoproteins of CNS myelin, an elaboration of the plasma membranes of oligodendrocytes (OLs) that result in a multilamellar wrapping of neuronal axons, facilitating nerve conduction with dramatic savings of space and energy. Due to an usually high lipid to protein ratio, myelin can be separated readily from other heavier membranes on sucrose gradients and further fractionated into subdomains related to myelin structure and function, including compact myelin and myelin-associated axolemmal membrane (Menon et al. 2003). We analyzed these fractions for N-linked sugar chains, using 2D HPLC following hydrazinolysis and pyridylamination. Our results indicate that compared with total brain homogenate, the amount of N-glycans is 1.3-fold higher in the myelin-associated axolemmal membranes, but it is 0.5-fold less in CM. M5 [Manalpha1-3((Manalpha1-3)(Manalpha1-6)Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc] is the most abundant sugar chain in total brain homogenate, compact myelin, and myelin-associated axolemma, constituting approximately 20% of sugar chains. Although the types of sugar chains are similar among the fractions, their expression levels vary significantly. In addition to high mannose type oligosaccharides, the core fucosylated, biantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc) residue, A2G1(3)FB [Galbeta1-4GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc], A2G1(6)FB [GlcNAcbeta1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4 (Fucalpha1-6)GlcNAc] and BA-1 [Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc], and A1(6)G0F [Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6) GlcNAc] are also present in relatively large proportions in compact myelin. We suggest that these differences may be related to myelin-axolemmal function.

Efficient and rapid purification of recombinant human α-galactosidase A by affinity column chromatography

The lysosomal enzyme α-galactosidase A (α-Gal A) metabolizes neutral glycosphingolipids that possess α-galactoside residues at the non-reducing terminus, and inherited defects in the activity of α-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant α-Gal A by sequential Concanavalin A (Con A)–Sepharose and immobilized thio-α-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human α-Gal A. We recommend the use of a mixture of 0.9 M methyl α-mannoside and 0.9 M methyl α-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate–phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute α-Gal A. This procedure is especially useful for the purification of mutant forms of α-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant α-Gal A (M279I) expressed in COS-7 cells within 6 h at 62% overall yield is presented.

Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120

We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen. nov., sp. nov. This paper deals with the mechanism of action of the anti-HIV activity of AH. AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2. AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein. The investigation of the effects of various sugars on AH–gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC 50=3.0 μg/ml). Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain. The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.

Monitoring of the tissue distribution of fibroblast growth factor containing a high mannose-type sugar chain produced in mutant yeast.

Most therapeutic glycoproteins have been produced in mammalian cell lines. However, the mammalian cell culture system has various disadvantages, i.e., a high culture cost, difficulty in performing a large scale-up because of complicated handling requirements, and the risk of contamination by prion or other unknown pathogenic components through cultivation in the presence of bovine serum. There is thus a growing need for other host cells in which the recombinant glycoproteins can be produced. Recently, we successfully developed a mutant yeast strain engineered in a glycosylation system. The sugar chain produced in the mutant yeast is not immunogenic to the human immuno-surveillance system. In the present study, we selected fibroblast growth factor (FGF) as a model glycoprotein and assessed the bioactivity of FGF produced in yeast in terms of its proliferating activity and tissue distribution in mammalian cells and in the whole body. Structural changes in the sugar chains of FGFs derived from mutant yeast, as compared with those from mammalian cells, did not affect the proliferating activity remarkably. However, the tissue distribution in the mouse differed significantly; a high-mannose type sugar chain was the major determinant of the specific distribution of FGF to the kidney. The mechanism of this phenomenon is still unclear, but our observations suggest that recombinant glycoproteins derived from mutant yeasts producing high-mannose type sugar chains would be applicable for tissue-targeting therapy.

Chemo-enzymatic synthesis of the glycosylated α-mating factor of Saccharomyces cerevisiae and analysis of its biological activity

The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae α-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated α-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl α-mating factor (Trp–His–Trp–Leu–Gln(GlcNAc)–Leu–Lys–Pro–Gly–Gln–Pro–Met–Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-β- N-acetylglucosaminidase, we synthesized glycosylated α-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of α-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated α-mating factor was lower than that of native α-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated α-mating factor, its bioactivity was recovered. Glycosylated α-mating factor exhibited higher resistance against proteolysis than native α-mating factor. It was found that the bioactivity of N-acetylglucosaminyl α-mating factor was higher than that of α-mating factor. Circular dichroism studies indicated that a slight change in the structure of α-mating factor may influence its activity.

Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity.

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.

The Trimannosyl Cores of N-Glycans are Important for the Procoagulant Protease-Inhibitory Activity of Urinary Protein C Inhibitor

We investigated the relationship between the procoagulant protease-inhibitory activity and the N-glycan structures in urinary protein C inhibitor (uPCI) by sequential exoglycosidase digestions based on the N-glycan structures elucidated in this report. uPCI was glycosylated on the three potential N-glycosylation sites, asparagines 230, 243 and 319 (N230, N243 and N319) in the molecule and had four biantennary complex type sugar chains. The inhibitory activities of uPCI toward thrombin and plasma kallikrein were little changed by the sequential removal of N-acetylneuraminic acid and galactose residues from the termini and N-acetylglucosamine residues from the branches of the N-glycans. However, the inhibitory activities were markedly decreased by further removing α-mannose residues from the trimannosyl cores of the N-glycans. These results suggest that the trimannosyl cores of N-glycans are important for uPCI to inhibit the procoagulant protease.

Glycan residues of N- and O-linked oligosaccharides in the premeiotic spermatogenetic cells of the urodele amphibianpleurodeles waltl characterized by means of lectin histochemistry

The aim of this work was the characterization of the glycoconjugates of the premeiotic spermatogenetic cells of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. In the cytoplasm of the primordial germ cells, primary and secondary spermatogonia and primary spermatocytes, a granular structure can be observed close to the nucleus. These granules contain four types of sugar chains according to their appearance during the differentiation process: 1. some oligosaccharides that are identified in all the four cell types above mentioned, which include N-linked oligosaccharides with Fuc, Galβ1,4GlcNAc and Neu5Acα2,3Galβ1,4GlcNAc and O-linked oligosaccharides with Galβ1,4GlcNAc and Neu5Acα2,3Galβ1,4GlcNAc; 2. other glycan chains that are not present in the primary spermatocytes (N-linked oligosaccharides with DBA-positive GalNAc, GlcNAc, and a slight amount of Neu5Acα2,6Gal/GalNAc and O-linked oligosaccharides with WGA-positive GlcNAc); 3. the sugar chains that are not in the earliest step of spermatogenesis (formed by both N-linked and O-linked oligosaccharides with Glc); and 4. other that appear at the earliest and latest stages, but not in the intermediate ones, (N-linked oligosaccharides with Man and O-linked oligosaccharides with SBA- and HPA-positive GalNAc and PNA-positive Galβ1,3GalNAc). This structure could be related with the Drosophila spectrosome and fusome, unusual cytoplasmic organelles implicated in cystic germ cell development. Data from the present work, as compared with those from mammals and other vertebrates, suggest that, although no dramatic changes in the glycosilation pattern are observed, some cell glycoconjugates are modified in a predetermined way during the early steps of the spermatogenetic differentiation process.

The N-glycan acceptor specificity of a glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope.

The acceptor specificity of a rat brain glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope on glycoproteins, was investigated using asialoorosomucoid as a model acceptor substrate. Structural analysis of N-linked oligosaccharides, to which glucuronic acid was transferred by GlcAT-P, by means of two-dimensional mapping of pyridylamino-oligosaccharides and MS spectrometry, demonstrated that the enzyme transferred glucuronic acid to bi-, tri-, and tetra-antennary complex type sugar chains, with almost equal efficiency, indicating that the enzyme has no preference as to the number of acceptor sugar branches. Next, we studied the branch specificity of this enzyme by means of the selective branch scission method involving two step exoglycosidase digestion using authentic pyridylamino-oligosaccharides. The GlcAT-P is highly specific for the terminal N-acetyllactosamine structure and no glucuronic acid was incorporated into a Galbeta1-3GlcNAc moiety. The GlcAT-P transferred glucuronic acid to the galactose residues in the N-acetyllactosamine branches of bi-, tri-, and tetra-antennary oligosaccharide chains, with different efficiencies and most preferentially to those in the Galbeta1-4GlcNAcbeta1-4Manalpha1-3 branch.

A journey to the world of glycobiology.

Finding of the deletion phenomenon of certain oligosaccharides in human milk and its correlation to the blood types of the donors opened a way to elucidate the biochemical basis of blood types in man. This success led to the idea of establishing reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins. N-Linked sugar chains were first released quantitatively as oligosaccharides by enzymatic and chemical means, and labelled by reduction with NaB3H4. After fractionation, structures of the radioactive oligosaccharides were determined by a series of methods developed for the studies of milk oligosaccharides. By using such techniques, structural rules hidden in the N-linked sugar chains, and organ- and species-specific N-glycosylation of glycoproteins, which afforded a firm basis to the development of glycobiology, were elucidated. Finding of galactose deficiency in the N-linked sugar chains of serum IgG from patients with rheumatoid arthritis, and malignant alteration of N-glycosylation in various tumors opened a new research world called glycopathology. However, recent studies revealed that several structural exceptions occur in the sugar chains of particular glycoproteins. Finding of the occurrence of the Galbeta1-4Fucalpha1- group linked at the C-6 position of the proximal N-acetylglucosamine residue of the hybrid type sugar chains of octopus rhodopsin is one of such examples. This finding indicated that the fucosyl residue of the fucosylated trimannosyl core should no more be considered as a stop signal as has long been believed. Furthermore, recent studies on dystroglycan revealed that the sugar chains, which do not fall into the current classification of N and O-linked sugar chains, are essential for the expression of the functional role of this glycoprotein. It was found that expression of many glycoproteins is altered by aging. Among the alterations of the glycoprotein patterns found in the brain nervous system, the most prominent evidence was found in P0. This protein is produced in non-glycosylated form in the spinal cord of young mammals. However, it starts to be N-glycosylated in the spinal cord of aged animals. These evidences indicate that various unusual sugar chains occur as minor components in mammals, and play important roles in particular tissues.

Beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

Conversion of Brain-Specific Complex Type Sugar Chains by N-Acetyl- -D-Hexosaminidase B

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.