One-pot enzymatic synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc sequence with in situ UDP-Gal regeneration

The trisaccharide Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was enzymatically synthesized, with in situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4'-epimerase, UDP-Gal:GlcNAc beta 4-galactosyltransferase and UDP-Gal:Gal beta 1-->4GlcNAc alpha 3-galactosyltransferase, Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was formed in a 2.2 mumol ml-1 yield starting from the acceptor GlcNAc beta 1-->O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc epitope which pays an important role in various biological and immunological processes.     

Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 2. Characterization of E-selectin binding fractions, and structural requirements for physiological binding to E-selectin

E-selectin binding gangliosides were isolated from myelogenous leukemia HL60 cells, and the E-selectin binding pattern was compared with that of human neutrophils as described in the preceding paper in this issue. The binding fractions were identified as monosialogangliosides having a series of unbranched polylactosamine cores. Structures of fractions 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were characterized by functional group analysis by application of monoclonal antibodies, 1H-NMR, FAB-MS, and electrospray mass spectrometry with collision-induced dissociation of permethylated fractions. Fractions 12-3, 13-1, and 13-2 were characterized by the presence of a major ganglioside with the following structure: NeuAc alpha 2-->3Gal beta 1-->4 GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer. Fractions 12-3 and 13-2 contained, in addition, small quantities (10-15%) of extended SLex with internally fucosylated structures: NeuAc alpha 2-->3 Gal beta 1-->4-(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3) GlcNAc beta 1-->3 Gal beta 1-->4 (+/- Fuc alpha 1-->3)GlcNA c beta 1-->3 Gal beta beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->Glc Beta Cer. Fraction 13-1, showing stronger E-selectin binding activity than 12-3 and 13-2, contained only a trace quantity (< 1%) of SLex. Fraction 14, which also showed clear binding to E-selectin, was characterized by the presence of the following structures, in addition to two internally monofucosylated structures (XX and XXI, Table 2, text): NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 GlcNAc beta 1-->3 Gal beta 1-->4 Glc beta Cer; andNeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4 (Fuc alpha 1--3)-GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1--4Glc beta Cer. SLex determinant was completely absent. Thus, the E-selectin binding epitope in HL60 cells is carried by unbranched terminally alpha 2-->3 sialylated polylactosamine having at least 10 monosaccharide units (4 N-acetyllactosamine units) with internal multiple fucosylation at GlcNAc. These structures are hereby collectively called "myeloglycan". Monosialogangliosides from normal human neutrophils showed an essentially identical pattern of gangliosides with selectin binding property. Myeloglycan, rather than SLex, provides a major physiological epitope in E-selectin-dependent binding of leukocytes and HL60 cells.     

Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.     

Reduction of the major swine xenoantigen, the alpha-galactosyl epitope by transfection of the alpha2,3-sialyltransferase gene

alpha2,3-Sialyltransferase represents a putative enzyme that reduces the Galalpha1-3Gal beta1-4GlcNAc-R (the alpha-galactosyl epitope) by intracellular competition with alpha1,3-galactosyltransferase for a common acceptor substrate. This study demonstrates that the overexpression of the alpha2,3-sialyltransferase gene suppresses the antigenicity of swine endothelial cells to human natural antibodies by 77% relative to control cells and by 30% relative to cells transfected with alpha1,2-fucosyltransferase, and in addition, it reduces the complement-mediated cell lysis by 75% compared with control cells and by 22% compared with cells transfected with alpha1, 2-fucosyltransferase. The mechanism by which the alpha-galactosyl epitope was reduced was also studied. Suppression of alpha1, 3-galactosyltransferase activity by 30-63% was observed in the transfectants with alpha2,3-sialyltransferase, and mRNA expression of the alpha1,3-galactosyltransferase gene was reduced as well. The data suggest that the alpha2,3-sialyltransferase effectively reduced the alpha-galactosyl epitope as well as or better than the alpha1, 2-fucosyltransferase did and that the reduction of the alpha-galactosyl epitope is due not only to substrate competition but also to an overall reduction of endogenous alpha1, 3-galactosyltransferase enzyme activity.     

Immunochemical characterization of anti-H monoclonal antibodies obtained from a mouse immunized with human saliva

Three IgM class anti-H monoclonal antibodies (1E3, 1E5 and 3H1) were obtained from a BALB/c mouse immunized with human O type saliva. These antibodies were found to agglutinate red cells from O group and A and B subgroups but not from Bombay and para-Bombay individuals whose H antigen was barely detected by anti-H reagents. The agglutination reactions of these antibodies were inhibited by H antigens from human tissues. It was also demonstrated that both 1E3 and 3H1 reacted with H disaccharide (Fuc alpha 1-->2Gal beta), H type 1 (Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta), H type 2 (Fuc alpha 1-->2Gal beta 1-->4GlcNAc beta), H type 3 (Fuc alpha 1-->2Gal beta 1-->3GalNAc alpha) and H type 4 (Fuc alpha 1-->2Gal beta 1-->3GalNAc beta) but not with Lea (Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), Leb (Fuc alpha 1-->2Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), X (Gal beta 1-->4[Fuc alpha-->3]GlcNAc beta) or Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta). On the other hand, 1E5 was found to react with H type 1, H type 2, Leb and Y. Because of the unique reactivities against various fucosyl linkages these monoclonal antibodies could be useful not only as anti-H reagents but also as reagents for the structural analysis of fucosylated glycoconjugates.     

Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.     

Synthesis of oligosaccharides related to HNK-1 antigen. 2. Synthesis of 3'-O-(3-O-sulfo-beta-D-glucuronopyranosyl)-lacto-N-neotetrao se beta-propylglycoside

A derivative of allyl 3"-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-lactoside with a free OH group at C-4GlcNAc was glycosylated with trichloroacetimidate of selectively protected GlcA(beta 1-->3)Gal alpha disaccharide in dichloromethane in the presence of trimethylsilyl triflate resulting in a pentasaccharide product with an 82% yield. This product was converted to monohydroxy derivative with a free OH group at C-3GlcA via the formation and the subsequent opening of the 6,3-lactone ring in the glucuronic acid residue. The 3"'-O-sulfation of the monohydroxy derivative, the removal of the protective groups, and the reduction of the allyl aglycon yielded the pentasaccharide propyl glycoside NaSO3-3GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-Opr comprising the oligosaccharide chain of the SGGL-1 glycolipid, which is recognized by HNK-1 antibodies. NaSO3-3GlcA(beta 1--> 3)Gal beta OAll, GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-OPr and GlcA(beta 1-->3)Gal beta OAll were synthesized in a similar way.     

Automatic and effortful processes in auditory memory reflected by event-related potentials. Age-related findings

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man alpha1 --> 6 (Man alpha1 --> 3) Man beta1 --> 4GlcNAc beta1 --> 4GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal beta1 --> 3Gal beta1 --> 4GlcNAc beta1 --> 2Man alpha1 --> 6 (Gal beta1 --> 3Gal beta1 --> 4GlcNAc beta1 --> 2Man alpha1 --> 3) Man beta1 --> 4GlcNAc beta1 --> 4 (Fuc alpha1 --> 6)GlcNAc-PA; 6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively.     

Enzymatic synthesis of N-linked oligosaccharides terminating in multiple sialyl-Lewis(x) and GalNAc-Lewis(x) determinants: clustered glycosides for studying selectin interactions

Galactosyltransferase, sialyltransferase, and fucosyltransferase were used to create a panel of complex oligosaccharides that possess multiple terminal sialyl-Le(x) (NeuAc alpha 2-3Gal[Fuc alpha 1-3] beta 1-4GlcNAc) and GalNAc-Le(x) (GalNAc[Fuc alpha 1-3]beta 1-4GlcNAc). The enzymatic synthesis of tyrosinamide biantennary, triantennary, and tetraantennary N-linked oligosaccharides bearing multiple terminal sialyl-Le(x) was accomplished on the 0.5 mumol scale and the purified products were characterized by electrospray MS and 1H NMR. Likewise, biantennary and triantennary tyrosinamide oligosaccharides bearing multiple terminal GalNAc-Le(x) determinants were synthesized and similarly characterized. The transfer kinetics of human milk alpha 3/4-fucosyltransferase were compared for biantennary oligosaccharide acceptor substrates possessing Gal beta 1-4GlcNAc, GalNAc beta 1-4GlcNAc, and NeuAc alpha 2-3Gal beta 1-4GlcNAc which established NeuAc alpha 2-3Gal beta 1-4GlcNAc as the most efficient acceptor substrate. The resulting complex oligosaccharides were chemically tethered through the tyrosinamide aglycone to the surface of liposomes containing phosphatidylthioethanol, resulting in the generation of glycoliposomes probe which will be useful to study relationships between binding affinity and the micro- and macro-clustering of selectin ligand.     

In vitro biosynthesis of a decasaccharide prototype of multiply branched polylactosaminoglycan backbones

Multiply branched polylactosaminoglycans are expressed in glycoproteins and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their ability to carry multiple lectin-binding determinants, which makes them prominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (5), where LacNAc is the disaccharide Gal beta1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc (1) with UDP-GlcNAc and alpha midchain beta1,6-GlcNAc transferase activity (GlcNAc to Gal), present in rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched octasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAc beta1-3'LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (2) and LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'LacNAc (3), were formed first at comparable rates. Later, both intermediates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest that, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galactoses. The enzyme-binding epitope of 1 is probably LacNAc beta1-3'LacNAc, because the trisaccharides GlcNAc beta1-3'LacNAc and LacNAc beta1-3Gal as well as the tetrasaccharide GlcNAc beta1-3'LacNAc beta1-3Gal were poor acceptors, while LacNAc beta1-3'LacNAc was a good one. Midchain beta1,6-GlcNAc transferase activities present in serum of several mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. We suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.     

Chemoenzymatic synthesis of a sialylated diantennary N-glycan linked to asparagine

A partial structure of many glycoproteins, a glycosylated asparagine carrying a complex type undecasaccharide N-glycan (Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man alpha 1-3) [Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4) GlcNAc(beta 1-4)GlcNAc-Asn) was obtained by total synthesis. As a starting material served a chemically synthesized diantennary heptasaccharide azide which was deprotected in a three-step sequence in high yield. The reduction of the anomeric azide was accomplished with propanedithiol in methanol-ethyldiisopropylamine. Coupling of the glycosyl amine to an activated aspartic acid gave the benzyl protected asparagine conjugate. After removal of the six benzyl functions the resulting free heptasaccharide asparagine was elongated enzymatically in the oligosaccharide part. The use of beta-1,4-galactosyltransferase and alpha-2,6-sialytransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in a full length N-glycan, a sialyated diantennary undecasaccharide-asparagine of the complex type.     

Reduction of metastatic properties of BL6 melanoma cells expressing terminal fucose(alpha)1-2-galactose after alpha1,2-fucosyltransferase cDNA transfection

We reported previously that transfection of BL6 melanoma cells that do not express the alpha1,3-galactosyltransferase (alpha1,3GT) gene with the alpha1,3GT cDNA resulted in synthesis and expression of alpha-galactosyl epitopes (Gal(alpha)1-3Gal(beta)1-4GlcNAc-R) and an impairment of their metastatic potentials. It was of interest to test whether inhibition of metastatic properties of BL6 melanoma cells is specifically associated with the appearance of the terminal alpha-Gal or whether capping N-acetyllactosamine with another oligosaccharide would also affect the metastatic properties of BL6 melanoma cells. For this purpose, BL6-2 clone isolated from B16BL6 melanoma was transfected with the alpha1,2-fucosyltransferase (alpha1,2FT) cDNA. The alpha1,2FT catalyzes a transglycosylation reaction, resulting in syntheses of the Fuc(alpha)1-2Gal(beta)1-4GlcNAc-R structure, which is known as the H antigen of O blood group in humans and is also synthesized in some cells of mice. Transfection of BL6 melanoma cells with the alpha1,2FT cDNA resulted in the appearance of the terminal Fuc(alpha)1-2Gal(beta)1-4GlcNAc-R epitopes reacting with the Ulex europaeus agglutinin lectin. In parallel, the transfected cells showed a decrease in N-acetyllactosamine sialylation. Decline in sialylation of the transfected cells is likely to be the result of competition between alphal,2FT and alpha2,3- or alpha2,6-sialyltransferases for the common substrate N-acetyllactosamine (Gal(beta)1-4GlcNAc-R) on N-linked carbohydrate chains of glycoproteins and glycolipids. The alpha1,2FT-transfected BL6-2 cells showed an increase in homotypic aggregation. In parallel, metastatic ability of the alpha1,2FT-transfected BL6-2 cells was reduced significantly in the immunocompetent as well as immunosuppressed (X-irradiated) mice. Thus, these data imply that capping N-acetyllactosamine with alphaGal or alphaFuc and the corresponding reduction in sialylation of BL6-2 melanoma cells were associated with reduction of their metastatic potential.     

Enzymatic synthesis of oligosaccharides containing Gal beta-->4Gal disaccharide at the non-reducing end using beta-galactanase from Penicillium citrinum

The transglycosylation reaction was done with a beta-galactanase from Penicillium citrinum. The regioselectivity in the transglycosylation reaction was studied using soy bean arabinogalactan as a donor and mono- or disaccharide derivatives containing beta-galactosyl residue as acceptors. We also synthesized oligosaccharides containing Gal beta 1-->4Gal sequence such as Gal beta 1-->4Gal beta1-->4Glc, Gal beta 1-->4Gal beta 1-->3GlcNac, Gal beta 1-->4Gal beta 1-->4GlcNAc, Gal beta 1-->4Gal beta 1-->6GlcNAc, and Gal beta 1-->4Gal beta 1-->3GalNAc for use in the total synthesis of complex sugar chains.     

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.     

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galbeta1,3GalNAc alpha-O-Al, Galbeta1,4GlcNAcbeta-O-Al, Galbeta1,3GlcNAcbeta-O-Al, and mucin-type Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Galbeta1, 3GalNAc alpha- linkage and the other (Group B) was directed toward the Galbeta1,4GlcNAc branch beta1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galbeta-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward beta1,3 linked Gal, particularly, the T-hapten without beta1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galbeta1,3GalNAc alpha-O-Al and Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-O-sulfotransferase activities by fucose, sulfate, and other substituents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galbeta1,3GalNAc alpha-O-Al to act as an acceptor for Group A. (2) An alpha1,3-fucosyl residue on the beta1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked beta1,3 to GalNAc alpha-. (3) Lewis x and Lewis a terminals did not serve as acceptors for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the beta1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked beta1,3 to GalNAc alpha. (5) Group A activity on Gal linked beta1,3 to GalNAc remained unaffected by 3'-sulfation of the beta1,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Galbeta1,4GlcNAcbeta-O-Al and Galbeta1,3GlcNAcbeta-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5- to 7-fold active, respectively, in their ability to serve as acceptors for Group B.     

Cardiovascular effects of microinjections of opioid agonists into the ''Depressor Region'' of the ventrolateral periaqueductal gray region

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.     

The structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage

Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion-exchange chromatography. Their structures have been determined by high-field 1H-NMR spectroscopy and high-pH anion-exchange chromatography. Both alpha(2-6)- and alpha(2-3)-linked N-acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is alpha(1-3)-linked to N-acetylglucosamine has been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N-linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both alpha(2-6)-linked N-acetylneuraminic acid and alpha(1-3)-linked fucose. It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non-reducing end of the chain. The galactose residue adjacent to the non-reducing cap is sulphated in only 1-3% of chains, compared with a sulphation level of over 40% closer to the reducing end. This highlights the difference between the chain termini and the repeat region in terms of structure and points to the potential for functional importance. The repeat region and capping fragments of the N-linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have the following general structure: NeuAc-(alpha 2-3/6)Gal[6SO3-](beta 1-4)GlcNAc6SO3-(beta 1-3)Gal[6SO3-] (beta 1-4)?[Fuc(alpha 1-3)]0-1GlcNAc6SO3-(beta 1-3)Gal-[6SO3-](beta 1-4)? 6-7GlcNAc6SO3-.     

Expression of alpha-1,3-galactose and other type 2 oligosaccharide structures in a porcine endothelial cell line transfected with human alpha-1,2-fucosyltransferase cDNA

The binding of xenoreactive natural antibodies to the Galalpha1-3Galbeta1-4GlcNAc (alpha-galactose) oligosaccharide epitope on pig cells activates the recipient's complement system in pig to primate xenotransplantation. Expression of human alpha-1, 2-fucosyltransferase in pigs has been proposed as a strategy for reducing the expression level of the alpha-galactose epitope, thereby rendering the pig organs more suitable for transplantation into humans. The aim of this study was to examine how the cell surface expression of alpha-galactose, H, and related fucosylated and sialylated structures on a pig liver endothelial cell line is affected by transfection of human alpha-1,2-fucosyltransferase cDNA. Nontransfected and mock-transfected cells expressed alpha-galactose, alpha-2,3-sialylated, and alpha-2,6-sialylated epitopes strongly, with low level expression of type 2 H and LewisX. By contrast, expression of the H epitope was increased 5-8-fold in transfected cells with a 40% reduction in the expression of alpha-galactose epitope and a 50% decrease in sialylation, as measured by binding of Maackia amurensis and Sambuccus nigra agglutinins. LewisX expression was reduced to background levels, while the LewisY neoepitope was induced in human alpha-1,2-fucosyltransferase-expressing pig cells. The activities of endogenous alpha-1,3-galactosyltransferase, alpha-1,3-fucosyltransferases, and alpha-2,3- and alpha-2, 6-sialyltransferases acting on lactosamine were unaffected. Our results show that a reduction in alpha-galactose epitope expression in porcine endothelial cells transfected with human alpha-1, 2-fucosyltransferase cDNA may be achieved but at the expense of considerable distortion of the overall cell surface glycosylation profile, including the appearance of carbohydrate epitopes that are absent from the parent cells.     

Cloning of a human UDP-galactose:2-acetamido-2-deoxy-D-glucose 3beta-galactosyltransferase catalyzing the formation of type 1 chains

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human beta3-galactosyltransferase distinct from the melanoma enzyme. The new beta3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(beta1-3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.