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A Varki

Publications and source records attributed to A Varki.

At least 109 records · Page 6Linked to original sources
PubMed1993

CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin.

The B lymphocyte cell surface receptor CD22 is an adhesion molecule that can mediate binding to several leukocyte subsets. The first CD22 ligand to be identified was the receptor-linked phosphotyrosine phosphatase CD45, but several lines of evidence suggest that CD22 may interact with multiple counter receptors on adjacent lymphocytes. In the present work, we show that in addition to CD45, a soluble CD22-immunoglobulin fusion protein (CD22Rg) recognizes several other distinct lymphocyte sialoglycoproteins. CD22-mediated adhesion is dependent upon the presence of sialic acids on ligands. CD22Rg is observed to bind specifically to a 115-kDa sialoglycoprotein in COS cells transfected with an alpha-2,6-sialyltransferase cDNA, but not in COS cells transfected with unrelated cDNA clones, indicating that at least some CD22-mediated interactions require presentation of sialic acid in an alpha-2,6 linkage by CD22 ligands. In all cases, truncation of the side chain of sialic acids by mild periodate oxidation abolishes recognition by CD22Rg. Direct binding of CD22Rg to lymphoid cells also requires sialic acids and their side chains. Taken together, these observations indicate that CD22 is a sialic acid-binding lectin and may define a novel functional subset of immunoglobulin superfamily adhesion molecules.

Animals↗
PubMed1993

Natural ligands of the B cell adhesion molecule CD22 beta carry N-linked oligosaccharides with alpha-2,6-linked sialic acids that are required for recognition.

CD22 beta is a glycoprotein found on the surface of B cells during restricted stages of development. It is believed to play a role in cell-cell interactions and B cell activation. The accompanying paper (Sgroi, D., Varki, A., Braesch-Andersen, S., and Stamenkovic, I. (1993) J. Biol. Chem. 268, 7011-7018) shows that CD22 beta recognizes multiple glycoproteins on the surfaces of T and B cells and that sialylation of these ligands is essential for binding. To identify the structure(s) of the sialylated oligosaccharide(s) recognized by CD22 beta, [3H]glucosamine-labeled glycoproteins were purified from Daudi cells by adsorption onto a CD22 beta recombinant immunoglobulin (CD22 beta Rg) chimera attached to protein A-Sepharose (PAS), and the N-linked oligosaccharides were released by peptide N-glycosidase F. These released oligosaccharides failed to bind to CD22 beta Rg-PAS under the conditions used initially to adsorb the glycoproteins, but their elution from a column of CD22 beta Rg-PAS was significantly retarded. Populations of oligosaccharides with different affinities could be identified by their order of elution. Specific sialidases were used to determine the content of alpha-2,3- and alpha-2,6-linked sialic acid in these different populations and their contribution to binding. Multiantennary oligosaccharides with one alpha-2,6-linked residue bound marginally, and those with two or more bound more tightly. alpha-2,3-Linked sialic acid residues were without effect. Binding did not require divalent cations and was abrogated by mild periodate oxidation of the outer side chain of sialic acid. No marked differences in size or fucose content were found between the populations of high and low affinity oligosaccharides. However, the low affinity population could be partially converted into higher affinity by treatment with beta-galactoside alpha-2,6 sialyltransferase and CMP-sialic acid. Thus, CD22 beta is a mammalian lectin that can recognize specific N-linked oligosaccharide structures containing alpha-2,6-linked sialic acids.

Animals↗
PubMed1993

Enhanced interaction of L-selectin with the high endothelial venule ligand via selectively oxidized sialic acids.

The selectins are adhesion receptors that play key roles in leukocyte trafficking. Each has an N-terminal C-type lectin domain that binds to specific carbohydrates in a calcium-dependent manner. L-selectin recognizes sulfated, sialylated ligands on lymph node high endothelial venules. This recognition is abolished by strong periodate oxidation under conditions that destroy oligosaccharides. In contrast, mild periodate oxidation, which selectively oxidizes the side chain of sialic acid residues without affecting the underlying oligosaccharide, markedly enhances this interaction. The enhancement is calcium dependent, indicating that lectin recognition is maintained. Reduction of the sialic acid aldehydes generated by mild periodate to alcohol groups abolishes this effect. Covalent cross-linking of the oxidized ligand to L-selectin can be demonstrated, suggesting Schiff base formation between lysine residues of the selectin and the newly formed aldehydes. Such selectively oxidized sialylated ligands could be used to probe the lectin domains of the selectins and to identify lysine residues near the binding site. Also, this approach could be used to design drugs for disrupting leukocyte-endothelial interactions leading to pathological inflammation.

Animals↗
PubMed1993

Alpha- and beta-xylosides alter glycolipid synthesis in human melanoma and Chinese hamster ovary cells.

beta-D-Xylosides are often used to competitively inhibit proteoglycan synthesis by serving as primers for free glycosaminoglycan (GAG) chain assembly. Quite unexpectedly, we found that when human melanoma cells and Chinese hamster ovary cells are labeled with [3H] galactose in the presence of 4-methyl umbelliferyl beta-D-xyloside (Xyl beta 4MU), a large portion of the labeled acceptor does not consist of the expected GAG chains, but of the novel GM3 ganglioside-like structure: Sia-alpha 2,3-[3H]Gal beta 1, 4Xyl beta 4MU. Moreover, formation of this derivative is associated with an inhibition of glycosphingolipid synthesis by up to 78% without affecting synthesis of other [3H]Gal-labeled glycoconjugates. Inhibition occurs rapidly and equally for all glycolipid species and is partially abrogated by brefeldin A. Inhibition requires the addition of a single galactose residue to the xyloside within the lumen of the Golgi apparatus. This addition appears to be carried out by galactosyl transferase I that normally synthesizes the core region of GAG chains. Although alpha-xyloside does not inhibit proteoglycan synthesis, it is galactosylated, but not sialylated, and is nearly as effective as a beta-xyloside at inhibiting glycolipid biosynthesis. Similar results were obtained for human macrophage U937, and differentiated or undifferentiated PC12 cells. However, in neuroblastoma cell line MR23, no low molecular weight xyloside products were made and glycolipid synthesis was not inhibited. These results suggest that some of the previously documented effects of beta-xylosides might result, in part, from their inhibition of glycolipid synthesis. The mechanism of inhibition is not a direct competition for glycolipid synthesizing enzymes; rather, it is an unexplained result of formation of Gal beta 1,4Xyl-1 (alpha or beta)4MU.

Animals↗
PubMed1993

Biological roles of oligosaccharides: all of the theories are correct.

Many different theories have been advanced concerning the biological roles of the oligosaccharide units of individual classes of glycoconjugates. Analysis of the evidence indicates that while all of these theories are correct, exceptions to each can also be found. The biological roles of oligosaccharides appear to span the spectrum from those that are trivial, to those that are crucial for the development, growth, function or survival of an organism. Some general principles emerge. First, it is difficult to predict a priori the functions a given oligosaccharide on a given glycoconjugate might be mediating, or their relative importance to the organism. Second, the same oligosaccharide sequence may mediate different functions at different locations within the same organism, or at different times in its ontogeny or life cycle. Third, the more specific and crucial biological roles of oligosaccharides are often mediated by unusual oligosaccharide sequences, unusual presentations of common terminal sequences, or by further modifications of the sugars themselves. However, such oligosaccharide sequences are also more likely to be targets for recognition by pathogenic toxins and microorganisms. As such, they are subject to more intra- and inter-species variation because of ongoing host-pathogen interactions during evolution. In the final analysis, the only common features of the varied functions of oligosaccharides are that they either mediate 'specific recognition' events or that they provide 'modulation' of biological processes. In so doing, they generate much of the functional diversity required for the development and differentiation of complex organisms, and for their interactions with other organisms in the environment.

Glycoconjugates↗
PubMed1992

The readers NIH.

Explore the source record for details and available documents.

Financing, Government↗
PubMed1992

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.

Acetyl Coenzyme A↗
PubMed1992

Of pride, prejudice, and discrimination. Why generalizations can be unfair to the individual.

The categorization of individuals into groups can serve some useful purposes. If the individuals being grouped are heterogeneous, however, the categories used are likely to be flawed. Thus, any resulting generalizations are also flawed, especially when applied to a given individual in a group. The most serious attendant dangers can be misplaced pride, misguided prejudice, and unfair discrimination. The categorization of physicians according to their original medical training and the generalizations that result are examined here. The principles advanced could also be applied to many other forms of categorization.

Foreign Medical Graduates↗
PubMed1992

The spectrum of incomplete N-linked oligosaccharides synthesized by endothelial cells in the presence of brefeldin A.

Previous studies in many cell lines have shown that Brefeldin A (BFA) inhibits the forward movement of newly synthesized glycoconjugates by fusing the cis-, medial-, and trans-Golgi compartments with the rough endoplasmic reticulum. Studies on the oligosaccharide processing of individual glycoproteins have yielded confusing and incomplete results regarding the location of the block. Assuming that all glycoproteins with N-linked oligosaccharides follow the same endoplasmic reticulum to the Golgi pathway, a more complete picture on the location and nature of the block can be determined by analyzing N-linked oligosaccharides synthesized in the presence of BFA. In bovine pulmonary artery endothelial cells, BFA (0.1 microgram/ml) reversibly inhibits the secretion of greater than 95% of Tran35S and [3H]Man-labeled glycoproteins without affecting protein synthesis or N-linked glycosylation. In addition, BFA inhibits the synthesis and secretion of 35SO4-labeled oligosaccharides. Initial oligosaccharide trimming is uninhibited, but further processing is affected since the majority (65%) of the chains terminate only in beta-GlcNAc residues. Concomitantly, the proportion of [3H]Man-labeled N-linked anionic oligosaccharides is reduced from 60 to 20%, and the great majority of the charge is due to one sialic acid. The rate-limiting step for sialylation appears to be the branch selective addition of beta-Gal residues. The remaining charge is due to sulfate esters (0.6%) which normally account for greater than 10% of the anionic substituents. BFA also reduces the amount of phosphorylated chains by 80% and greatly diminishes further phosphodiester processing since the majority of these oligosaccharides (60%) contain a Man-6-PO4 residue in an acid-sensitive diester linkage. The addition of all polylactosamine chains, outer-branch fucose and terminal alpha-Gal residues are completely inhibited by BFA. Secretion, fucosylation, and sialylation are completely restored when BFA is removed, but the other modification steps are only partially restored. Our results indicate that addition of sulfate esters, terminal alpha-Gal residues, polylactosamine chains, outer-branch fucose residues, some initial phosphorylation, and most phosphodiester processing may occur beyond a compartment where some beta-Gal and sialic acid residues can be added. Essentially, all of the effects on oligosaccharide processing are partially or completely reversible.

Amidohydrolases↗
PubMed1992

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1992

Selectins and other mammalian sialic acid-binding lectins.

Several recently discovered mammalian cell adhesion proteins recognize and bind to sialic acid-containing ligands. Reports concerning the molecular specificities of these interactions have been intriguing but somewhat confusing, partly because of pitfalls in methodology or interpretation. Nevertheless, these protein-carbohydrate recognition phenomena are important in the normal biology of blood cells and in the pathophysiology of many diseases.

Animals↗
PubMed1992

Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.

Antibodies, Monoclonal↗
PubMed1991

Developmental abnormalities in transgenic mice expressing a sialic acid-specific 9-O-acetylesterase.

9-O-acetylation of sialic acids is tissue specific and developmentally regulated. We have selectively destroyed these O-acetyl groups during murine embryogenesis by expressing the 9-O-acetyl-sialic acid-specific esterase of influenza C. DNA constructs driven by the metallothionein promoter arrested development at the 2-cell stage and gave a markedly decreased yield of live mice. A similar construct driven by the phenylethanolamine-N-methyltransferase promoter did not cause this block, but gave transgenic mice with selective expression of esterase in the retina and the adrenal gland. These organs showed variable abnormalities in organization, while all other tissues examined appeared normal. The ganglioside 9-O-acetyl-GD3 was selectively destroyed in target tissues. Thus, 9-O-acetylated sialic acids may play an role in murine development at the 2-cell stage and in certain differentiated tissues.

Acetylation↗
PubMed1991

GMP-140 binds to a glycoprotein receptor on human neutrophils: evidence for a lectin-like interaction.

GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism.

Antibodies, Monoclonal↗
PubMed1991

The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells.

Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1-3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium.

Amino Sugars↗
PubMed1991

Radioactive tracer techniques in the sequencing of glycoprotein oligosaccharides.

Complete sequencing of the oligosaccharide units of glycoproteins can be performed by conventional physical techniques if nanomole quantities of pure molecules are available. However, isolation of sufficient quantities of a glycoprotein may not be technically feasible (e.g., the analysis of biosynthetic intermediates, or rare molecules). Alternatively, partial structural analyses may answer the biological question at hand. In both instances, radioactive sugars can be used to metabolically label the oligosaccharide units of a glycoprotein, permitting substantial structural characterization. Several aspects of this approach are discussed in this overview, including selection of the labeled precursor, maximization of uptake and incorporation, determinants of the specificity of labeling, and general principles for the release and structural analysis of labeled oligosaccharides. Particular advantages include simplicity, ease of use without sophisticated instrumentation, and the fact that purification to radiometric homogeneity is sufficient. Radioactive tracer techniques cannot replace conventional approaches to sequencing oligosaccharides. However, they do provide a rapid, relatively simple approach to obtaining considerable information from limited amounts of material. For molecules such as short-lived biosynthetic intermediates, there is no substitute for these techniques. This approach has been responsible for the initial identification and characterization of many novel oligosaccharides of biological interest.

Animals↗