3Gal Beta Research Articles

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide.

A locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) has been cloned from gonococcal strain F62. The locus contains five open reading frames. The first and second reading frames are homologous, but not identical, to the fourth and fifth reading frames, respectively. Interposed is an additional reading frame which has distant homology to the Escherichia coli rfaI and rfaI genes, both glucosyl transferases involved in lipopolysaccharide core biosynthesis. The second and fifth reading frames show strong homology to the lex-1 or lic2A gene of Haemophilus influenzae, but do not contain the CAAT repeats found in this gene. Deletions of each of these five genes, of combinations of genes, and of the entire locus were constructed and introduced into parental gonococcal strain F62 by transformation. The LOS phenotypes were then analyzed by SDS-PAGE and reactivity with monoclonal antibodies. Analysis of the gonococcal mutants indicates that four of these genes are the glycosyl transferases that add GalNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3 Gal beta 1--4 to the substrate Glc beta 1-->4Hep--R of the inner core region. The gene with homology to E. coli rfaI/rfaI is involved with the addition of the alpha-linked galactose residue in the biosynthesis of the alternative LOS structure Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->4Hep-->R. Since these genes encode LOS glycosyl transferases they have been named lgtA, lgtB, lgtC, lgtD, and lgtE. The DNA sequence analysis revealed that lgtA, lgtC, and lgtD contained poly-G tracts, which, in strain F62 were, respectively, 17, 10, and 11 bp. Thus, three of the LOS biosynthetic enzymes are potentially susceptible to premature termination by reading frame changes. It is likely that these structural features are responsible for the high-frequency genetic variation of gonococcal LOS.

Two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate.

We report that two N-acetylglucosaminyltransferases catalyze the biosynthesis of heparan sulfate in Chinese hamster ovary cells. The first enzyme initiates heparan sulfate biosynthesis and can be measured by the transfer of GlcNAc from UDP-GlcNAc to GlcUA beta 1-3Gal beta 1-O-naphthalenemethanol. The second enzyme catalyzes the polymerization of heparan sulfate and can be measured by the transfer of GlcNAc from UDP-GlcNAc to the nonreducing terminal GlcUA present in oligosaccharide fragments prepared from the Escherichia coli K5 capsular polysaccharide, N-acetylheparosan. Kinetic characterization of the initiating GlcNAc-transferase (alpha-GlcNAc-TI) indicates an apparent Km for UDP-GlcNAc of 36 +/- 4 microM. The apparent Km for UDP-GlcNAc of the polymerizing GlcNAc-transferase (alpha-GlcNAc-TII) is 230 +/- 30 microM. Both enzymes have broad pH optima and require a divalent cation for activity. alpha-GlcNAc-TI can use both Mn2+ and Ca2+, while alpha-GlcNAc-TII will use only Mn2+. Chinese hamster ovary cells deficient in the synthesis of heparan sulfate and lacking alpha-GlcNAc-TII activity and S49 Thy 1-a lymphoma cells deficient in alpha GlcNAc addition to phosphatidylinositol have wild-type alpha-GlcNAc-TI activity. Thus, distinct alpha-GlcNAc-transferases catalyze the initiation and polymerization of heparan sulfate.

Evidence for glycosphingolipid modification of the type 1 IFN receptor.

P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1). Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity. Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites. In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected. Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes. Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha. Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted. IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment. By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2. These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.

Epithelial glucosphingolipid expression as a determinant of bacterial adherence and cytokine production.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is a structural analog of ceramide that inhibits glucosylation of this molecule and thus of glucosphingolipid (GSL) expression by living cells. In this study, we used PDMP to slow the synthesis of the globoseries of GSLs (globo-GSLs) (derived from the precursor Gal alpha 1-4Gal beta 1-4Glc-ceramide) by cultured human kidney and large intestinal epithelial cells. The aim was to deplete the cells of receptors for P-fimbriated Escherichia coli and to examine the effects on the bacterially induced cytokine response. The mammalian cells (A498, HT-29, and Caco2) were cultured in the presence of PDMP in order to deplete them of GSLs. The cells were then subjected to GSL analysis or used to test bacterial adherence and cytokine production. The globo-GSLs were identified by thin-layer chromatography. Bacterial adherence was quantitated by microscopy, and interleukin-6 secretion was quantitated by the B9 bioassay. The interaction of bacteria with the globo-GSLs was studied by using E. coli strains and recombinant clones expressing P fimbriae. E. coli strains expressing type 1 fimbriae binding to mannose-containing glycoproteins were used as controls. PDMP treatment was found to reduce the content of the globo-GSLs in mammalian cells and the adherence of P-fimbriated E. coli to these cells. In contrast, PDMP treatment had no effect on the adherence of type 1-fimbriated E. coli or their activation of cytokine production by A498 cells. P-fimbriated E. coli elicited an interleukin-6 response in the A498 cells; this response was reduced after treatment with PDMP. The results emphasize the role of GSLs as receptors for P-fimbriated E. coli and for the cytokine response elicited by attaching bacteria.

Characterization of the specificity of binding of Moluccella laevis lectin to glycosphingolipids.

The specificity of Moluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal alpha-linked N-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc alpha 3GalNAc beta 3Gal alpha 4Glc beta 1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc alpha 3(Fuc alpha 2)Gal beta. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal alpha-linked galactose. When considering the use of the M. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance.

Human tumor-associated Le(a)-Le(x) hybrid carbohydrate antigen IV3(Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc)III3FucnLc4 defined by monoclonal antibody 43-9F: enzymatic synthesis, structural characterization, and comparative reactivity with various antibodies.

Two Le(a)-cross-reacting monoclonal antibodies (mAbs) were previously established which define complex tumor-associated carbohydrate antigens. The first mAb, 43-9F, was raised against human squamous cell lung carcinoma and shows preferential reactivity with various human cancers over normal cells. Its tumor cell binding activity is best inhibited by a milk oligosaccharide characterized as Le(a)-Le(x) [Mårtensson, S., et al. (1988) Cancer Res. 48, 2125], Gal beta 1-->3[Fuc alpha 1-->4] GlcNAc beta 1-->3Gal beta 1-->4]Fuc alpha 1-->3]-GlcNAc beta 1-->3Gal beta 1-->4Glc (1). The second mAb, ST-421, was raised against human gastric cancer xenograft in nude mice and found to have strong tumor growth-suppressing activity in nude mice. The epitope recognized by ST-421 was chemically identified as Le(a)-Le(a), Gal beta 1-->3 [Fuc alpha 1-->4]GlcNAc beta 1-->3Gal beta 1-->3-[Fuc alpha 1-->4] GlcNAc beta 1-->3Gal beta 1-->4Glc (2) [Stroud, M.R., et al. (1991) J. Biol. Chem. 266, 8439]. Both 43-9F and ST-421 cross-react with Le(a). Identification of the 43-9F antigen as structure 1 (Le(a)-Le(x)) is tentative since it was not based on isolation and chemical characterization of antigen from tumor cells or tissues. We therefore synthesized structure 1 starting from sialyl-nor-hexaosylceramide (VI3NeuAcnLc6), with sequential enzymatic hydrolysis by sialidase and beta-galactosidase followed by addition of beta 1-->3Gal with beta 1-->3 galactosyltransferase. This yielded the hybrid type 1/type 2 chain core structure IV3-(Gal beta 1-->3GlcNAc)nLc4, which was fucosylated with alpha 1-->3/4 fucosyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and Characterization of a Rat Testis Glycosphingolipid Based on Gangliotetraosylceramide and Having a Blood Group B Determinant Extended with β3-Linked N-Acetylgalactosamine1

Monoclonal antibody 14.2, directed against the GalNAc beta 3Gal alpha 3Gal beta epitope, was used to monitor the isolation of a novel glycosphingolipid from rat testes. Structural analysis was performed by mass spectrometry, proton NMR spectroscopy, gas chromatography-mass spectrometry after degradation, and exoglycosidase digestion. It was concluded that the structure was [formula: see text] This is the first report on an extended blood group B determinant.

Specific stimulation of Graves' disease thyrocytes by the natural anti-Gal antibody from normal and autologous serum.

Anti-Gal is a natural Ab abundantly produced in humans. It interacts specifically with the carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is expressed in large amounts on thyrocytes of nonprimate mammals, but not of humans. We have previously found that binding of anti-Gal to alpha-galactosyl epitopes on porcine thyrocytes results in stimulatory effects similar to those exerted by thyroid-stimulating hormone (thyrotropin). In the present study, we tested the hypothesis that anti-Gal may contribute to Graves' disease (GD) pathogenesis by stimulation of the thyrocytes of patients with this autoimmune disorder. Anti-Gal binding and stimulatory effects were assessed in primary thyrocyte cultures. Anti-Gal specifically bound to GD thyrocytes and induced an increase in cAMP synthesis, 125I uptake, and DNA synthesis in these cells. Furthermore, the stimulatory effects of autologous sera on GD thyrocytes were greatly reduced after specific depletion of anti-Gal from these sera. No binding and no stimulatory effects of anti-Gal were observed, however, with normal human thyrocytes and with thyrocytes from thyrotoxic patients who lack thyroid-stimulating Igs or thyrotropin binding inhibiting Igs. These in vitro stimulatory effects of anti-Gal on GD thyrocytes suggest that this natural Ab may contribute to the in vivo continuous stimulation of thyrocytes in GD patients. The possibility that anti-Gal may stimulate GD thyrocytes via interaction with aberrantly expressed alpha-galactosyl epitopes on the thyroid-stimulating hormone receptor is discussed.

Isolation, structural determination, and calcium-binding properties of the major glycoprotein present in Bufo japonicus japonicus egg jelly.

Although the previous studies showed that the jelly coat is essential in anuran fertilization under natural conditions, identification and structural studies of the macromolecules that play functional roles have remained to be elucidated. In the present study we isolated acidic glycoproteins (JGP) from the solubilized egg jelly of Bufo japonicus japonicus, and showed that they were the major non-dialyzable macromolecular components of the jelly coat. JGP was a typical mucin-type glycoprotein, and it showed high degree of polydispersity in molecular masses ranging over 100-4000 kDa, but both amino acid and carbohydrate compositions were practically identical among fractions, suggesting that JGP was composed of a repeating glycoprotein unit. Four types of short O-glycan chains were isolated from JGP by reductive beta-elimination and their structures were determined as: Gal beta 1-->3[NeuAc alpha 2-->6]GalNAcol (= N-acetylgalactosaminitol), Fuc alpha 1-->2Gal beta 1-->3 [NeuAc alpha 2-->6]GalNAcol, Fuc alpha 1-->2Gal beta 1-->3[GlcNAc beta 1-->6]GalNAcol, and Fuc alpha 1-->2Gal beta 1-->3-GalNAcol. These carbohydrate units (about 80% of the mass of JGP) were linked to nearly all the serine and threonine residues which accounted for 55% of total amino acid residues. The Ca(2+)-binding property of JGP was studied by equilibrium dialysis. The high Ca(2+)-binding capacity of JGP was abolished by its desialylation of JGP and was highly dependent on the JGP concentration. When the low JGP concentrations as in the hydrated Bufo jelly were used, a 50% increment of both n (the number of binding sites) and Kd (the dissociation constant of JGP-Ca2+) values was observed. This property of JGP is suited to retaining Ca2+ and keeping its concentration at that just necessary for fertilizing sperm.

Monoclonal antibody specific for alpha 2-->8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry.

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing alpha 2-->8-linked polysialic acid (-->8Neu5Gc alpha 2-->)n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (-->8KDN alpha 2-->)n-->6(KDN alpha 2-->3Gal beta 1-->3G alpha lNA-c alpha 1-->3) GalNAc alpha 1-->residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochemical characterization of oligosaccharide side chains of the glycoproteins of rat zona pellucida: an ultrastructural study.

The zona pellucida (ZP), an extracellular matrix which surrounds mammalian oocytes, is formed by different glycoproteins. Several studies have revealed that carbohydrate residues present in glycoproteins of ZP play a key role in the sperm-egg recognition. However, the origin and the biochemical composition of ZP remain to be completely resolved. ZP glycoproteins from rat ovarian follicles were investigated at light and electron microscopic level by the application of lectins conjugated to peroxidase, digoxigenin, and colloidal gold in combination with enzyme and chemical treatment. A quantitative analysis was also performed. ZP shows reactivity to WGA, DSA, LFA, AAA, RCA I, and MAA. SBA and PNA showed a variable reactivity ranging from negative to strongly positive. A uniform pattern of binding throughout ZP was observed with DSA, Con A, AAA, MAA, and LFA. However, labeling by RCA I and SBA was higher in the outer ZP while PNA and WGA showed a higher binding in the inner ZP. Lectin reactivity was detected in cortical granules, endoplasmic reticulum, Golgi apparatus, vesicles, and multivesicular bodies of oocytes. ZP contained the terminal disaccharides Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, and GalNAc beta 1,3Gal and the trisaccharides Neu5Ac alpha 2, 3Gal beta 1,4GlcNAc, Neu5Ac-Gal beta 1,3GalNAc, and Neu5Ac-GalNAc beta 1,3Gal sequences. The occurrence of Fucose residues alpha 1,6 linked to the inner core region of N-linked glycoproteins of ZP was demonstrated by the use of several fucose-specific lectins. Methylation-saponification treatment in combination with lectin cytochemistry reveals that Gal, GalNAc, and polyllactosamine residues of rat ZP glycoproteins contain sulphated groups. The reactivity observed in ooplasmic vesicles was similar to that of ZP, thus suggesting that the oocyte is the site of synthesis of ZP glycoproteins.

Characterization of porcine kidney neutral glycosphingolipids: identification of a carbohydrate antigen recognized by human natural antibodies.

We have investigated the glycosphingolipids of pig kidney with a special interest in identifying compounds which may be involved in the rejection of tissue in xenotransplantation. Nine neutral glycosphingolipids have been characterized in porcine kidney and structurally characterized by a combination of techniques including 1H-NMR, permethylation analysis and thin-layer chromatography (TLC) immunostaining with carbohydrate sequence-specific monoclonal antibodies. The major components are members of the globo family and represent the human pk and P antigens. Three other compounds were found to contain a neolacto core structure; the major neolacto compound carries a nonreducing terminal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc) recognized by the naturally-occurring human antibody, anti-Gal, and a second neolacto compound carries the blood group A trisaccharide (GalNAc alpha 1-3(Fuc alpha 1-2)Gal). These results are discussed with respect to tissue transplantation.

Structural characterization of intermediates in the blosynthetic pathway of neolacto glycosphingolipids: differential expression in human leukaemia cells

The biosynthesis of neolacto glycosphingolipids is thought to proceed via reactions catalysed by the two enzymes beta 1-3-N-acetylglucosaminyltransferase (beta 1,3GlcNAcT) and beta 1-4 galactosyltransferase (beta 1,4GalT). In general, only the products of the latter enzyme have been isolated from tissues and structurally characterized. Among the GlcNAc beta 1-3-R glycosphingolipids, only lactotrioasylceramide (Lc3Cer, the initial product in the biosynthesis of neolacto glycosphingolipids) has been isolated and structurally characterized. Longer-chain glycosphingolipids with a terminal GlcNAc-beta 1-3-R structure are considered to be intermediates in the synthesis of complex neolacto glycosphingolipids. We have detected a series of GlcNAc beta 1-3-R glycosphingolipids in extracts obtained from human leukocytes isolated from patients with leukaemia using a monoclonal antibody (TE5) which specifically recognizes these compounds. The structures of three of these compounds purified from chronic myelocytic leukaemia (CML) cells have been determined using a combination of enzymatic, immunostaining and chemical methods. The compounds were found to have the following structures: GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer (Lc3Cer) GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer (nLc5Cer) GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer (nLc7Cer) A longer-chain compound, apparently nLc9Cer, was also detected. TLC immunostaining analysis of glycosphingolipids isolated from cells obtained from patients with various leukaemias demonstrated that GlcNAc beta 1-3-R glycosphingolipids have a distribution that depends on the stage of differentiation and lineage of the cell population.(ABSTRACT TRUNCATED AT 250 WORDS)

Bi-antennary oligo-(N-acetyllactosamino)glycans of I-type are galactosylated preferentially at the GlcNAc?1-6Gal linked arms by ?1,3-galactosyltransferase of bovine thymus

alpha 1,3-Galactosylation of radiolabelled bi-antennary acceptors Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal-R (R = 1-OH, beta 1-4GlcNAc or beta 1-4Glc) with bovine thymus alpha 1,3-galactosyltransferase was studied. At all stages of the reactions the three acceptors reacted faster at the 1-->6 linked arm than at the 1-->3 linked branch. Hence, in addition to the doubly alpha 1,3-galactosylated products, practically pure Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal-R could be obtained from the three acceptors in reactions that had proceeded to near completion. The isomeric mono-alpha 1,3-galactosylated products were identified by using exoglycosidases to remove the branches unprotected by alpha 1,3-galactoses and by subsequently identifying the resulting linear glycans chromatographically.

Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements. The anchoring effect of the micelle allowed the detection of conformational fluctuations of the headgroup on the time scale of a few hundred picoseconds. NMR experiments performed on the GD1a/DPC micelles in H2O at low temperatures permitted the observation of hydroxyl proton resonances, contributing valuable conformational information.

Differential host-dependent expression of alpha-galactosyl epitopes on viral glycoproteins: a study of eastern equine encephalitis virus as a model.

The carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-galactosyl) is abundantly expressed on cells of non-primate mammals, prosimians and New World monkeys, where it is synthesized by the enzyme alpha 1,3-galactosyltransferase (alpha 1,3GT). Old World monkeys, apes and humans lack alpha 1,3GT and hence do not synthesize alpha-galactosyl epitopes. Instead, these species produce a natural antibody, anti-Gal, which interacts specifically with alpha-galactosyl epitopes and which constitutes up to 1% of circulating immunoglobulins in humans. We have used eastern equine encephalitis (EEE) virus as a model to examine the differential expression of alpha-galactosyl epitopes on the glycoproteins of virus propagated in cells that either produce or lack alpha 1,3GT. As predicted, virus propagated in Vero cells (derived from the African green monkey, an Old World monkey) did not express alpha-galactosyl epitopes. In contrast, virus propagated in mouse 3T3 cells (EEE3T3) expressed approximately 80 alpha-galactosyl epitopes per virion on both the E1 and the E2 envelope glycoproteins. Thus, expression of the alpha-galactosyl epitope on virions paralleled that on host cells. The binding of anti-Gal antibody to these epitopes on EEE3T3 virions partially neutralized virus infectivity, raising the possibility that anti-Gal production in hosts may influence the initial infectious stage of viruses expressing alpha-galactosyl epitopes.

Oligosaccharides derived from bovine articular cartilage keratan sulfates after keratanase II digestion: implications for keratan sulfate structural fingerprinting.

Keratan sulfate chains were isolated from bovine articular cartilage (6-8-year-old animals) and digested with keratanase II, an endo-beta-N-acetylglucosaminidase [Nakazawa, K., Ito, M., Yamagata, T., & Suzuki, S. (1989) in Keratan Sulphate: Chemistry, Biology and Chemical Pathology (Greiling, H., & Scott, J. E., Eds.) pp 99-110, The Biochemical Society, London]. Twenty-five borohydride-reduced oligosaccharides were purified chromatographically and characterized by one- and two-dimensional NMR spectroscopy. From the structures of these oligosaccharides the following conclusions can be drawn about the mode of action of keratanase II: (1) The enzyme cleaves the beta (1-->3)-glycosidic bond between 6-O-sulfated N-acetyl-glucosamine and galactose, the major products being mono- and disulfated disaccharides. (2) Larger oligosaccharides containing keratanase II susceptible bonds are produced which are resistant to further degradation, e.g., tetrasaccharides from the sulfated poly(N-acetyllactosamine) repeat sequence, fucose-containing penta- and hexasaccharides, and hexa- and heptasaccharides from the linkage region. (3) The enzyme cleaves the beta (1-->3)-glycosidic bond of a fucosylated 6-O-sulfated N-acetylglucosamine. (4) Sialic acid-containing capping fragments are always recovered as pentasaccharides, despite the presence of an apparently susceptible bond. Two new elements of skeletal keratan sulfate structure, namely, the highly sulfated cap NeuAc alpha 2-3Gal(6S) beta 1-4GlcNAc (6S) beta 1-3Gal(6S) beta 1-4GlcNAc (6S)-ol and the difucosylated sequence Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc(6S)-ol, have been identified. A structural model for articular cartilage keratan sulfate is proposed. The potential of the enzyme keratanase II for the structural fingerprinting of subnanogram quantities both of keratan sulfates and of sulfated oligosaccharide selectin ligands is discussed.

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.

Structural analysis of the N-glycans from human immunoglobulin A1: comparison of normal human serum immunoglobulin A1 with that isolated from patients with rheumatoid arthritis

The primary structures of the N-linked oligosaccharides from normal human serum IgA1 were determined by a combination of sequential exoglycosidase digestion, Bio-Gel P-4 chromatography, anion-exchange chromatography and one-dimensional n.m.r. spectroscopy. Three major N-linked disialylated biantennary-complex-type structures were found (55%). The remaining N-linked oligosaccharides consisted of at least nine further structures, some of which (7%) were of the triantennary type and included disialylated triantennary oligosaccharides with outer-arm fucose substitution [Fuc alpha 1-3(4)]. Compared with IgG, the N-glycan structures on IgA are more completely processed: the outer arms have a higher proportion of galactose and sialic acid, and only trace levels of incompletely galactosylated oligosaccharides, commonly found on IgG, were detected. Analysis of the sialylated O-glycans revealed that 64% were [NeuAc2 alpha 3(6)]2Gal beta 3GalNAc and 9% were [NeuAc2 alpha 3(6)]-Gal beta 4GlcNAc beta 6[NeuAc2 alpha 3(6)Gal beta 3]GalNAc, and 27% were monosialylated. The N-linked glycosylation of both serum IgA1 and IgG isolated from a group of six normal individuals was compared with that from ten patients with rheumatoid arthritis (RA). In contrast with the hypogalactosylation found in IgG from diseased sera, there was no evidence of an equivalent decrease in the galactosylation of the IgA1 oligosaccharides. In addition, the N-glycosylation of IgA1 was remarkably consistent within the group of normal individuals. These data suggest that incomplete galactosylation of N-linked glycans and its augmentation in RA does not extend to IgA1 and that the RA-associated galactosyltransferase deficiency may be restricted to cells producing gamma-chain.