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.     

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.     

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.     

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 10(9) primary bovine aortic endothelial cells and structurally characterized by immunological and chemical methods. Glucosyl- and lactosylceramide were detected as the main neutral glycosphingolipids (28% and 40% of total orcinol stain, respectively); LcOse3Cer and nLcOse4Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse6Cer occurred only in trace quantities. No neutral glycosphingolipids of the ganglio-series (GgOse3Cer and GgOse4Cer) and the globo-series (GbOse4Cer and the Forssman antigen) have been detected; only traces of GbOse3Cer were identified by TLC immunostaining. Positive CD15 bands obtained by TLC overlay with anti-Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-R antibody indicated the presence of lipid bound Lewisx antigen, whereas the isomeric Lewis(a) structure (Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-R) was not detectable. G(M3) substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole ganglioside fraction. G(M3)-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives. C18-sphingosine was the only long chain base, whereas variation occurred due to C(24:0,24:1) and C16 fatty acids. Terminally alpha2-3 sialylated neolacto-series gangliosides with nLcOse4- and nLcOse6Cer (<5% of total resorcinol stain) were found in almost equal quantities, whereas no alpha2-6 sialylated counterparts were detected. Fucosylated gangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis[x], sialyl Lewisa, and VIM-2 antigen) and sulfoglucuronylneolacto series structures with HNK-1 epitope were not detectable in the acidic glycosphingolipid fraction by TLC immunostaining. Gangliotetraose-type gangliosides G(M1) and G(D1a) (<1 % of total resorcinol stain) as well as traces of G(D1b) and G(T1b) have been distinctly identified by combined choleragenoid-TLC-immunostaining and previous neuraminidase treatment. The expression of dominant glycosphingolipids lactosylceramide and G(M3)(Neu5Gc) was proved by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The results provide the basis for investigation of the role of glycosphingolipids as cell surface antigens of cellular interaction as well as receptors for blood components and macromolecules of the extracellular matrix.     

The lipooligosaccharides of Haemophilus ducreyi are highly sialylated

The major lipooligosaccharides of the sexually transmitted pathogen Haemophilus ducreyi 35000 have been previously found to terminate in N-acetyllactosamine and sialyl-N-acetyllactosamine, Neu5Ac alpha 2-->3Gal beta 1-->4GlcNAc (W. Melaugh, N. J. Phillips, A. A. Campagnari, M. V. Tullius, and B. W. Gibson, Biochemistry 33: 13070-13078, 1994). In this study, mass spectrometry and composition analyses have shown that the lipooligosaccharides from three other H. ducreyi strains also contain N-acetyllactosamine and are highly sialylated (approximately 30 to 50%), although one African strain was found to contain neither of these structural features.     

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.     

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.     

A comparative assessment of TLC overlay technique and microwell adsorption assay in the examination of influenza A and Sendai virus specificities towards oligosaccharides and sialic acid linkages of gangliosides

Influenza A and Sendai viruses bind to neolacto-series gangliosides isolated from human granulocytes. Differences in receptor specificity of influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and parainfluenza Sendai virus (HNF1, Z-strain) were determined by two direct solid phase binding assays: the overlay technique, which combines high-resolution in the separation of gangliosides on thin-layer chromatograms with direct binding; and the microwell adsorption assay as a convenient binding assay which is performed in microtitre wells to estimate the avidity of binding to an isolated ganglioside. Both methods were applied for comparative binding studies. Viruses were found to exhibit specificity for oligosaccharides and sialic acids as well as for chain length of the neutral carbohydrate backbone, whereas differing fatty acids (C24:1 and C16:0) in the ceramide portion had no impact on virus adsorption. Terminal sialyloligosaccharides Neu5Ac alpha 2-3Gal beta 1-4Glc-R of GM3, and Neu5Ac alpha 2-3Gal beta 1-4GlcNAc-R as well as Neu5Ac alpha 2-6Gal beta 1-4GlcNAc-R of neolacto-series gangliosides with nLcOse4Cer and nLcOse6Cer backbone, exhibited significant specific receptor activity towards the different viruses. To compare the data revealed from both test systems, values of virus binding were ascertained by a non-parametric statistical approach based on rank correlation. The rank correlation coefficient rs was calculated according to Spearman from each virus binding towards GM3, IV3Neu5Ac-nLcOse4Cer, IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6SCer. The rank correlation coefficients 0.74, 0.95 and 0.92, which were determined for A/PR/8/34 (H1N1), A/X-31 (H3N2) and Sendai virus (HNF1, Z-strain), respectively, indicated that both assays generate highly correlated experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration of a crystalloid fluid preload does not prevent the decrease in arterial blood pressure after induction of anaesthesia with propofol and fentanyl

Five oligosaccharide alpha1-phosphates and one sulfated glycopeptide have been isolated from the hemofiltrate of one patient with end-stage renal disease. Isolation of these compounds has been achieved using reverse osmosis, ion-exchange and size-exclusion chromatography and high performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H and 31P NMR spectroscopy. The chemical structures were determined to be: 1 NeuAc alpha2-3Gal alpha1-OPO3H2; 2 NeuAc alpha2-6Galbeta1-4GlcNAc alpha1-OPO3H2; 3 NeuAc alpha2-3Galbeta1-3GalNAc alpha1-OPO3H2; 4 NeuAc alpha2-3Galbeta1-3[NeuAc alpha2-6]GalNAc alpha1-OPO3H2 (proposed structure); 5 Fuc alpha1-2Galbeta1-4[Fuc alpha1-3]GlcNAc alpha1-OPO3H2; 6 HOSO3-4Fuc alpha1-6GlcNAcbeta1-NAsn. While 2 and 3 have been previously characterized as compounds of urine and hemofiltrate, the oligosaccharide alpha1-phosphates 1, 4, and 5 could be isolated--to our knowledge--for the first time from biological material. Compound 6 is the first glycopeptide reported to contain a 4-sulfated fucose residue.     

Molecular species analysis of glycosphingolipids from small intestine of Japanese quail, Coturnix coturnix Japonica by HPLC/FAB/MS

Neutral glycosphingolipids were isolated from quail small intestine and their structures were analysed. They contained: Gal beta 1-4GlcCer(LacCer), Gal alpha 1-4GalCer(Ga2Cer), Gal alpha 1-4Gal beta 1-4GlcCer(Gb3Cer), GlcNAc beta 1-3Gal beta 1-4GlcCer(Lc3Cer), GalNAc beta 1-4Gal beta 1-4GlcCer(Gg3Cer), GalNAc beta 1-4[GalNAc beta 1-3] Gal beta 1-4GlcCer(LcGg4Cer), and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer (Forssman glycolipid) as well as glucosylceramide, galactosylceramide (Nishimura K et al. 1984) Biochim Biophys Acta 796:269-76) and the LeX glycolipid, III3 Fuc alpha-nLc4Cer (Nishimura K et al. (1989) J. Biochem (Tokyo) 101:1315-18). The molecular species compositions of these glycosphingolipids were examined using fast atom bombardment-mass spectrometry linked with reversed-phase high-performance liquid chromatography. By such analysis, we could classify the quail glycosphingolipids into at least three classes: glycolipids rich in species having four hydroxyl groups in the ceramides (GalCer, Gg3Cer, LcGg4Cer and LeX), those rich in the ceramides of N-acyl trihydroxysphinganine with normal fatty acids (Lc3Cer), and glycolipids rich in the ceramides of N-acyl sphingenine with normal fatty acids (LacCer, Gb3Cer and Forssman glycolipid). Immunohistochemical observation implies that the differences in the hydrophobic moieties specified the localization of glycosphingolipids in the tissue.     

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.     

Enzymatic synthesis of the alpha 3-galactosyl-Lex tetrasaccharide: a potential ligand for selectin-type adhesion molecules

Using recombinant UDP-Gal:Gal beta 1-->4GlcNAc alpha 1,3-galactosyltransferase and human milk alpha 1,3-fucosyltransferase the disaccharide Gal beta 1-->4GlcNAc has been converted in vitro into a tetrasaccharide product. The product has been characterized by gel filtration chromatography and HPLC and was analyzed using 1H-NMR. Based on NMR spectral data along with the known linkage specificity of the alpha 1,3-galactosyltransferase and the alpha 1,3-fucosyltransferase used, the chromatographic behaviour of the product, and the 1:1 molar ratios of the galactose and fucose residues calculated from incorporated radioactivity, it is concluded that the structure of the tetrasaccharide product is Gal alpha 1-->3Gal beta 1--4[Fuc alpha 1-->3]-GlcNAc. The tetrasaccharide is a non-charged analogue of the sialyl-Lex determinant that potentially may act as a ligand structure in selectin-mediated cell-cell adhesion.     

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.     

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-.     

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.     

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.     

A novel method to co-localize glycosaminoglycan-core oligosaccharide glycosyltransferases in rat liver Golgi. Co-localization of galactosyltransferase I with a sialyltransferase

4-Methylumbelliferyl-beta-xyloside (Xyl beta MU) primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of 2 galactoses and 1 glucuronic acid residue to make the typical core structure, GlcUA beta 1, 3Gal beta 1,3Gal beta 1,4Xyl beta MU. To investigate the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with Xyl beta MU and 1 microM UDP-[3H]Gal and then chased with 5 microM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labeling depends entirely upon transporter-mediated delivery of the labeled sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isolated from the incubation medium and from within the Golgi and their structures analyzed by C18, anion-exchange, and amine adsorption high performance liquid chromatography in combination with glycosidase digestions. Surprisingly, the major products within the Golgi were two sialylated xylosides (Sia alpha 2,3Gal beta 1,4Xyl-beta MU and Sia alpha 2,8Sia alpha 2,3Gal beta 1,4Xyl beta MU) rather than the expected group of partially completed GAG core structures. Less than 10% of the products within the Golgi are the expected core structures containing a second Gal residue or, in addition, GlcUA. The amount of the sialylated products is only partially decreased if the chase is omitted or if the chase is done in the absence of added CMP-Sia, suggesting a pool of previously transported CMP-Sia drives synthesis of the major products. Conversely, when detergent permeabilized vesicles are provided with high concentration of the same sugar nucleotides, the ratio of sialylated products is reduced and replaced by an increase in GAG-like products. These results argue that GAG core-specific Ga1 transferase I and II are not extensively co-localized within the same Golgi compartment. By contrast, glycosaminoglycan core Gal transferase I is substantially co-localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialyltransferase. Incubating intact Golgi vesicles with exogenous diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases of multiple pathways within the Golgi compartments.     

Conformational studies on the selectin and natural killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses (SuLNFPII and SLNFPII) using NMR spectroscopy and molecular dynamics simulations. Comparisons with the nonacidic parent molecule LNFPII

This investigation is focused on the conformational behavior of the blood group Lewisa (Le(a)-active pentasaccharide lacto-N-fucopentaose II (LNFPII) and its sulfated and sialylated analogs, SuLNFPII and SLNFPII. The latter two are more potent oligosaccharide ligands for the animal lectins, E- and L-selectin, and the natural killer cell receptor, NKR-P1, than are the shorter chain analogs based on the trisaccharide Le(a) domain. We report here that the three oligosaccharides based on the fucopentasaccharide have very similar average solution conformations as determined from NMR spectroscopical parameters, in particular 13C chemical shift differences. From restrained simulated annealing and restrained molecular dynamics (MD) simulations performed in order to determine the most probable conformational distributions around the glycosidic linkages we derive models for these oligosaccharides that are in good agreement with experimental parameters, such as rotating-frame Overhauser effects (ROE's) and long-range 1H,13C coupling constants across the glycosidic linkages. In these model structures the Le(a) domain at the non-reducing end of the longer chain oligosaccharides approximates the same rigid structure as in the shorter analogs. The Gal beta 1-4Glc linkage at the reducing end is also rather rigid, showing only little more flexibility than the Le(a) domain. However, the NeuAc alpha 2-3Gal linkage in SLNFPII, and the GlcNAc beta 1-3Gal linkage in all three oligosaccharides are flexible, in each case fluctuating mainly between two minimum energy structures: (phi = -81 degrees, psi = 8 degrees) and (phi = -160 degrees, psi = -20 degrees) for the NeuAc alpha 2-3Gal linkage, as reported previously for the isomeric sequence 3'-sialyl Le(x), and (phi = -25 degrees, psi = -26 degrees) and (phi = 20 degrees, psi = 24 degrees) for the GlcNAc beta 1-3Gal linkage. The flexibility of the latter linkage may allow the lactosyl domain at the reducing end to fit with little strain into extended carbohydrate binding sites on the recognition proteins, and, for the purposes of drug designs, it will be important to establish which conformational distribution is assumed for the GlcNAc beta 1-3Gal linkage in these longer chain oligosaccharides in the bound state.     

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.