Alpha 2-3Gal Beta 1-4GlcNAc Research Articles

Glycoconjugate histochemistry of the seminal vesicles of the Japanese miniature (Shiba) goat.

The present study localizes and characterizes complex glycoconjugates in the seminal vesicles of the Japanese Shiba goat, using several carbohydrate histochemical procedures, including lectin techniques at light and electron microscopic levels. Glandular epithelial cells and luminal secretions were shown to contain neutral and acidic glycoconjugates with various saccharide residues, such as alpha-D-Man, alpha-D-Glc, alpha-L-Fuc, beta-D-Gal, GalNAc, GlcNAc, and NeuAc (sialic acid). The terminal oligosaccharide chains of sialoglycoconjugates present in the seminal vesicles were NeuAc alpha 2-3Gal beta 1-3GalNAc and NeuAc alpha 2-3Gal beta 1-4GlcNAc. In addition, in lysosomes of the glandular epithelial cells alpha-D-Man, alpha-D-Glc, GlcNAc and NeuAc (sialic acid) residues could be detected, the secretory vesicles contained alpha-L-Fuc, and the endoplasmic reticulum exhibited alpha-D-Man and alpha-D-Glc residues. The complex glycoconjugates with various sugar residues found in the seminal vesicles of the goat may be involved in various fertilization-related events.

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's lymphoma and germinal centre B cells.

A new monoclonal antibody (TU-1) directed against the Gal alpha 1-4Gal beta 1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer(Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Gal beta 1-4Glc beta 1-1'Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer), galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), sulfatide (HSO3-3Gal beta 1-1'Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Gal alpha 1-4Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes.

A human antibody binds to alpha-galactose receptors and mimics the effects of Clostridium difficile toxin A in rat colon

BACKGROUND & AIMS: Nearly all human sera contain an immunoglobulin G antibody (antigalactose) that binds the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc expressed on cells from most mammals but not humans. Because the Clostridium difficile toxin A receptor in rodents contains this trisaccharide, the aim of this study was to examine whether antigalactose could mimic the enterotoxic effects of toxin A and bind to receptors containing this trisaccharide. METHODS: Fluid secretion, [3H]-mannitol permeability, and release of rat mast cell protease II and prostaglandin E2 were measured after luminal exposure of rat colon to either purified human anti-galactose, control immunoglobulin G, toxin A, or buffer. RESULTS: Toxin A (5 micrograms) and antigalactose (250 micrograms) but not control immunoglobulin (250 micrograms) stimulated colonic fluid secretion and caused increased mannitol permeability and rat mast cell protease II release. Antigalactose and toxin A and, to a lesser degree, control immunoglobulin G also stimulated release of prostaglandin E2, but only toxin A produced acute inflammation of rat colonic mucosa. Antigalactose and toxin A bound specifically to a single class of colonic brush border receptors with dissociation constants of 10(-6) mol/L and 5.4 x 10(-8) mol/L, respectively. CONCLUSIONS: Fluid secretion, increased permeability, and mast cell activation occur in rat colon when toxin A or human antigalactose immunoglobulin G bind to receptors bearing the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc. (Gastroenterology 1996 Jun;110(6):1704-12)

Characterization of Porcine Platelet Glycoproteins Recognized by Human Natural “Anti-Gal” Antibodies

Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain.

Localization of the Putative Sialic Acid-binding Site on the Immunoglobulin Superfamily Cell-surface Molecule CD22

B-lymphocyte antigen CD22 is a member of the recently described sialoadhesin family of immunoglobulin-like cell-surface glycoproteins that bind glycoconjugates terminating in sialic acid. One prominent ligand for CD22 is the highly glycosylated leukocyte surface protein CD45. Using surface plasmon resonance spectroscopy, we characterized the interaction of recombinant mouse CD22 with native CD45 purified from rat thymus (CD45-thy). By in situ desialylation and resialylation of immobilized CD45-thy, we show that mouse CD22 binds to the sialoglycoconjugate NeuGc alpha 2-6Gal beta 1-4GlcNAc carried on CD45-thy N-glycans. Previous studies have shown that the sialic acid-binding site lies within the two membrane-distal domains of CD22 (domains 1 and 2), which are V-set and C2-set immunoglobulin superfamily domains, respectively. To further localize the binding site, we have made 42 single amino acid substitutions throughout both domains. All 12 mutations that abrogated binding to CD45-thy without disrupting antibody binding were of residues within the GFCC'C" beta-sheet of domain 1. These residues are predicted to form a contiguous binding site centered around an arginine residue in the F strand that is conserved in all members of the sialoadhesin family. Our results provide further evidence that immunoglobulin superfamily cell adhesion molecules use the GFCC'C" beta-sheet of membrane-distal V-set domains to bind structurally diverse ligands, suggesting that this surface is favored for cell-cell recognition.

The reducing end of alpha Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera.

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-alpha-Gal gave weak inhibition, Gal alpha 1-3Gal substantially inhibited the reaction (400muM), and Gal alpha 1-3Gal beta 1-4GLcNAc was ten times more efficient (30 muM). The modification from alpha to beta anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-alphaGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal alpha 1-3Gal beta 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.

Specificity of xenoreactive anti-Gal alpha 1-3Gal IgM for alpha-galactosyl ligands.

The transplantation of organs from lower animals such as pigs into humans is prevented by a severe rejection reaction initiated by complement fixing xenoreactive natural antibodies. Most anti-pig xenoreactive natural antibodies in humans are thought to recognize Gal alpha 1-3Gal beta 1-4GlcNAc and are also thought to recognize, albeit less avidly, Gal alpha 1-6Glc. Gal alpha 1-6Glc has been used as a ligand for purification of 'anti-Gal alpha 1-3Gal antibodies' and as a therapeutic or reagent to prevent the binding of these antibodies to porcine organs or cells. We tested the specificity of anti-Gal alpha 1-3Gal IgM for Gal alpha 1-6Glc and related saccharides. Based on inhibition of binding of xenoreactive anti-Gal alpha 1-3Gal IgM to porcine cells by soluble saccharides, anti-Gal alpha 1-3Gal IgM in a human serum was found to consist of a mixture of antibodies which have a similar affinity for Gal alpha 1-3Gal but varying affinities for Gal alpha 1-6Glc and other structures. Twenty to 40% of the anti-Gal alpha 1-3Gal IgM from the population tested did not recognize Gal alpha 1-6Glc. The binding of anti-Gal alpha 1-3Gal IgM to Gal alpha 1-6Glc varied widely from individual to individual, some samples lacking almost entirely anti-Gal alpha 1-3Gal IgM which bound to Gal alpha 1-6Glc.

Isolation and characterization of gangliosides from Theileria sergenti.

The gangliosides of Theileria sergenti piroplasms were isolated and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated on TLC. G-1, G-2, G-3, and G-4 ganglioside showed the same mobility as GM3, sialosylparagloboside (SPG), i-active ganglioside, and I-active ganglioside on the TLC plate, respectively. In order to characterize the molecular species of gangliosides from T. sergenti, G-1, G-2, G-3, and G-4 gangliosides were purified and tested by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside had reactivity to anti-GM3 monoclonal antibody. G-2 gave reaction with monoclonal antibody to SPG containing N-glycolylneuraminic acid (NeuGc). G-3 showed reactivity to the anti-i-active ganglioside (NeuGc) monoclonal antibody. G-4 was recognized by the monoclonal antibody which reacts with I-active ganglioside (NeuGc). In addition, sialic acid moiety of the gangliosides from T. sergenti piroplasms was also analyzed. N-acetylneuraminic acid-containing gangliosides were hardly detectable in T. sergenti piroplasms. Gangliosides from T. sergenti (G-1, G-2, G-3, and G-4) carried only NeuGc as their sialic acid moiety. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuGc) [NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer], SPG (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], i-active ganglioside (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], and I-active ganglioside(NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3 (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6) Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], respectively.

Analysis of neutral glycosphingolipids of Theileria sergenti piroplasms.

Neutral glycosphingolipids (GSLs) were isolated from a lipid extract of Theileria sergenti piroplasms and analyzed by thin-layer chromatography (TLC) and liposome immune lysis assay (LILA); two predominant GSLs, designated as N-1 and N-2 were separated on TLC. N-1 GSL showed the same mobility as lactosylceramide (LacCer) on the TLC plate. On the other hand, the mobility of N-2 GSL on the TLC plate was identical to that of galactosylparagloboside. In order to characterize the molecular species of neutral GSLs from T. sergenti, N-1 and N-2 GSLs were tested by LILA with antibodies against LacCer and galactosylparagloboside, respectively, N-1 GSL had reactivity to anti-LacCer antibody and N-2 reacted with the antibody to galactosylparagloboside. These results suggest that N-1 and N-2 GSLs are LacCer (Gal beta 1-4Glc beta 1-1Cer) and galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), respectively.

Purification, properties and possible gene assignment of an ?1,3-fucosyltransferase expressed in human liver

alpha 1,3-Fucosyltransferase solubilized from human liver has been purified 40,000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with alpha 1,3-fucosyltransferase activity and had a specific activity of approximately 5-6 mumol min-1 mg-1 and an M(r) approximately 44,000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal beta 1-4GlcNAc, NeuAc alpha 2-3Gal beta 1-4GlcNAc and Fuc alpha 1-2Gal beta 1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc alpha 1-2Gal beta 1-4Glc and the Type 1 compound Gal beta 1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc alpha 2-6Gal beta 1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated alpha 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with alpha 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with the Fuc-TVI cDNA, suggests a provisional identification of Fuc-TVI as the major alpha 1,3-fucosyltransferase gene expressed in human liver.

Molecular Cloning, Expression, Chromosomal Assignment, and Tissue-specific Expression of a Murine α-(1,3)-Fucosyltransferase Locus Corresponding to the Human ELAM-1 Ligand Fucosyl Transferase

Terminal Fuc alpha 1-3GlcNAc moieties are displayed by mammalian cell surface glycoconjugates in a tissue-specific manner. These oligosaccharides participate in selectin-dependent leukocyte adhesion and have been implicated in adhesive events during murine embryogenesis. Other functions for these molecules remain to be defined, as do the tissue-specific expression patterns of the corresponding alpha-(1-3)-fucosyltransferase (alpha 1-3FT) genes. This report characterizes a murine alpha 1-3FT that shares 77% amino acid sequence identity with human ELAM ligand fucosyltransferase (ELFT, also termed Fuc-TIV). The corresponding gene maps to mouse chromosome 9 in a region of homology with the Fuc-TIV locus on human chromosome 11q. In vitro, the murine alpha 1-3FT can efficiently fucosylate the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc (apparent Km of 0.71 mM) to form an unusual tetrasaccharide (Gal alpha 1-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) described in periimplantation mouse tissues. The enzyme can also form the Lewis x determinant from Gal beta 1-4GlcNAc (Km = 2.05 mM), and the sialyl Lewis x determinant from NeuNAc alpha 2-3Gal beta 1-4GlcNAc (Km = 1.78mM). However, it does not yield sialyl Lewis x determinants when expressed in a mammalian cell line that maintains sialyl Lewis x precursors. Transcripts from this gene accumulate to low levels in hematopoietic organs, but are unexpectedly abundant in epithelia that line the stomach, small intestine, colon, and epididymus. Epithelial cell-specific expression of this gene suggests function(s) in addition to, and distinct from, its proposed role in selectin ligand synthesis.

Structural Analysis of the Oligosaccharides Derived from Glycodelin, a Human Glycoprotein with Potent Immunosuppressive and Contraceptive Activities

Glycodelin, also known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities. In this paper we report the first characterization of glycodelin-derived oligosaccharides. Using strategies based upon fast atom bombardment and electrospray mass spectrometry we have established that glycodelin is glycosylated at Asn-28 and Asn-63. The Asn-28 site carries high mannose, hybrid and complex-type structures, whereas the second site is exclusively occupied by complex-type glycans. The major non-reducing epitopes in the complex-type glycans are: Gal beta 1-4GlcNAc (lacNAc), GalNAc beta 1-4GlcNAc (lacdiNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacdiNAc), Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewisx), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (lacdiNAc analogue of Lewisx). It is possible that the oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae may manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell associated receptor. Oligosaccharides with fucosylated lacdiNAc antennae have previously been shown to potently block selectin-mediated adhesions and may perform the same function in glycodelin. The potent inhibitory effect of glycodelin on initial human sperm-zona pellucida binding is consistent with our previous suggestion that this cell adhesion event requires a selectin-like adhesion process. This result also raises the possibility that a convergence between immune and gamete recognition processes may have occurred in the types of carbohydrate ligands recognized in the human.

Anti‐Gal activity In diabetic patients transplanted with fetal porcine Islet cell clusters

The natural anti-Gal antibody seems to create a major obstacle for discordant xenotransplantation in humans. Anti-Gal, which is produced in large amounts in humans (1% of circulating IgG), interacts specifically with the carbohydrate structure Gal alpha 1-3Gal beta 1-4Glc-NAc-R (termed the alpha-galactosyl epitope). This epitope is present in large amounts on porcine cells, as well as on cells of other nonprimate mammals (1 x 10(6) to 35 x 10(6) epitopes/cell). The interaction of anti-Gal with alpha-galactosyl epitopes on the xenograft was found to mediate the immune destruction of discordant xenografts. In the present study, the human immune response to alpha-galactosyl epitopes on xenografts was assessed by measuring changes in anti-Gal titers and affinity in sera of diabetic patients transplanted with fetal porcine islet cell clusters. The activity of this antibody was assessed by a hemagglutination assay with RBC, by ELISA with mouse laminin as a solid-phase antigen, and by equilibrium dialysis with the radiolabeled free haptenic form of the alpha-galactosyl epitope, i.e. [3H]Gal alpha 1-3Gal beta 1-4GlcNAc. All assays revealed a marked increase in anti-Gal activity after transplantation. The increase in anti-Gal titers ranged between 8- and 64-fold. A similar increase was observed in the binding of free alpha-galactosyl epitopes to anti-Gal, as assayed in equilibrium dialysis. Immunoglobulin concentration did not increase after transplantation, suggesting that the observed increase in anti-Gal activity is the result of a specific immune response against alpha-galactosyl epitopes on the xenograft. The elevation in anti-Gal activity was observed in all three immunoglobulin classes and the highest activity was found within the IgG class. Analysis of IgG binding to fixed porcine endothelial cells suggested that most of the observed increased activity against these cells in transplanted patients may be attributed to the elevation in anti-Gal activity.

Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection.

Transplants performed between phylogenetically disparate species are subject to hyperacute rejection initiated by binding of xenoreactive natural Abs to endothelium in the donor organ. Binding of these Abs activates complement, leading to tissue injury and destruction of the graft. Human xenoreactive natural Abs recognize Gal alpha 1-3Gal beta 1-4GlcNAc (galactose alpha 1-3galactose beta 1-4-N-acetylglucosame); however, the relative importance of this Ag in graft rejection has not been proved. The present study was conducted to test the potential importance of alpha-galactosyl (alpha-Gal) determinants in the pathogenesis of hyperacute rejection. To this end, hearts (n = 3) from New World monkeys (Saimiri scureus, squirrel monkey), which can synthesize Gal alpha 1-3Gal, were transplanted heterotopically into Old World monkeys (Papio species, baboon), which do not synthesize Gal alpha 1-3Gal determinants and which have circulating anti-alpha Gal Abs. The xenografts were rejected in 51 to 56 min (mean +/- SD = 53.3 +/- 2.5), results similar to those observed in porcine grafts transplanted into baboons. Histologic analysis of the hearts revealed thrombosis and intraparenchymal hemorrhage and immune deposits consisting of IgM, Clq, C3, C4, C5b, and the membrane attack complex, but not properdin or factor B of the recipient deposited on graft endothelium. Sera obtained from baboons after perfusion of squirrel monkey kidneys revealed depletion of alpha-Gal-specific Abs and anti-pig endothelial cell Abs. These findings provide strong evidence that the Abs that accumulate in New World monkey organs during perfusion with baboon blood are the same Abs that would accumulate in a porcine organ transplanted into a primate and suggest that hyperacute rejection is not necessarily a reflection of phylogenetic distance but that the expression of terminal alpha-Gal residues provides an adequate target to initiate that process.

Enzymic Synthesis of Octadecameric Saccharides of Multiply Branched Blood Group I-Type, Carrying Four Distal .alpha.1,3-Galactose or .beta.1,3-GlcNAc Residues

Radiolabeled oligosaccharide constructs were prepared to evaluate carbohydrate determinants involved in gamete adhesion in mice. The octasaccharide primer GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (1) was incubated with UDP-GlcNAc and beta 1,6-GlcNAc-transferase of hog gastric microsomes, producing the tetraantennary decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (2). The decasaccharide was then incubated with UDP-Gal and beta 1,4-galactosyltransferase from bovine milk, yielding the tetradecasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (3). Incubation of the tetradecasaccharide 3 with UDP-Gal and alpha 1,3-galactosyltransferase from bovine thymus gave the octadecameric glycan Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (4).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of Human Plasma Sialoglycoproteins by the B Cell-specific Lectin CD22: SELECTIVE RECOGNITION OF IMMUNOGLOBULIN M AND HAPTOGLOBIN

CD22 is a cell-surface receptor of resting mature B cells that recognizes sialic acid (Sia) in the natural structure Sia alpha 2-6Gal beta 1-4GlcNAc (Powell, L. D., Jain, R. K., Matta, K. L., Sabesan, S., and Varki, A. (1995) J. Biol. Chem. 270, 7523-7532). Human umbilical vein endothelial cells (HEC) treated with inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) display increases in cell-surface CD22 ligands, caused by increased expression of the enzyme beta-galactoside alpha 2,6-sialyltransferase (Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) J. Biol. Chem. 269, 10637-10643; Hanasaki, K., Varki, A., and Powell, L. D. (1995) J. Biol Chem. 270, 7533-7542). Thus, CD22 could direct potential interactions between mature B cells and endothelial cells during inflammatory states. However, this would have to occur in the presence of blood plasma, which contains many sialoglycoproteins known to carry alpha 2-6-linked sialic acids. We show here that human plasma can indeed inhibit Sia-dependent binding of a recombinant soluble chimeric form of human CD22 (CD22Rg) to TNF-alpha activated HEC. Affinity adsorption of individual human plasma samples with immobilized CD22Rg showed that, of the numerous alpha 2-6-sialic acid containing glycoproteins in plasma, only three polypeptides with apparent molecular mass (under reducing conditions) of 74, 44, and 25 kDa bound, and were specifically eluted with alpha 2-6-sialyllactose. NH2-terminal amino acid sequencing of these high affinity CD22 ligands revealed that they are subunits of immunoglobulin M (IgM) and haptoglobin. Purified human IgM from pooled human plasma can be quantitatively bound by CD22Rg, and binding is blocked by alpha 2-6-sialyllactose, but not by alpha 2-3-sialyllactose. Pretreatment by sialidase or by mild periodate oxidation of sialic acid side chains abolishes these interactions. IgM at physiological concentrations also inhibits CD22Rg binding to TNF-alpha-activated HEC in a manner dependent not only upon its sialylation but also requiring its intact multimeric structure. These data show that CD22 is capable of highly selective recognition of certain multimeric plasma sialoglycoproteins that carry alpha 2-6-linked sialic acids. Notably, the two proteins that are selectively recognized are known to be involved in immune and inflammatory responses. Haptoglobin synthesis by the liver is markedly increased during the "acute phase response" to systemic inflammation, while IgM is the major product resulting from activation of resting CD22-positive B cells.

Characterization of Sialyloligosaccharide Binding by Recombinant Soluble and Native Cell-associated CD22: EVIDENCE FOR A MINIMAL STRUCTURAL RECOGNITION MOTIF AND THE POTENTIAL IMPORTANCE OF MULTISITE BINDING

CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing alpha 2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Sia alpha 2-6Gal beta 1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A. (1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 microM) for Neu5Ac alpha 2-6Gal beta 1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the alpha 2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(alpha 2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.

?1,3-Fucosylation of branched blood group I-type oligo-(N-acetyllactosamino)glycans by human milk transferases is restricted to distalN-acetyllactosamine units: The resulting isomers are separated by WGA-agarose chromatography

A partially purified preparation of alpha 1,3-fucosyltransferase(s) from human milk was used to [14C]fucosylate oligosaccharides containing Gal beta 1-4GlcNAc units. Substitution of N-acetyllactosamine at position 3' with a beta-linked N-acetyl-glucosamine enhanced the reactivity of the acceptor, whereas similar substitution at position 6' was inhibitory. Thus, the trisaccharide GlcNAc beta 1-6Gal beta 1-4GlcNAc (5), the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (11) and the triply branched decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (26) gave remarkably poor yields of alpha 1,3-fucosylated products in comparison to GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). beta 1,4-Galactosyl derivatives of 5 and 11, however, gave good yields of alpha 1,3-fucosylated products, but the fucosylation was restricted to the distal N-acetyllactosamine units of Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc (16), Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (18) and also in Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (22). Immobilized wheat germ agglutinin (WGA), possessing high affinity for 16 [1], revealed no affinity for the fucosylated derivative Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6Gal beta 1-4GlcNAc (17). The isomeric heptasaccharides Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (19) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4GlcNAc (20) were readily separated from each other on WGA agarose, and so were the isomeric nonasaccharides Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (23) and Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4GlcNAc (24).

Characterization of neutral glycosphingolipids in rat lens

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folch's partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

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.