Showing papers on "Sialic acid published in 1991"

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

Abstract: The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the hig...

Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages

Abstract: Macrophage subpopulations in the mouse express a lectin-like receptor, sialoadhesin (originally named sheep erythrocyte receptor, SER), which selectively recognizes sialoglycoconjugates and is likely to be involved in cellular interactions of stromal macrophages in haematopoietic and lymphoid tissues. In this report we describe the purification and ligand specificity of sialoadhesin isolated from mouse spleen. Purified sialoadhesin, a glycoprotein of 185 kd apparent Mr, agglutinated sheep or human erythrocytes at nanomolar concentrations in a sialic acid-dependent manner. Low angle shadowing and electron microscopy showed that sialoadhesin consisted of a globular head region of approximately 9 nm and an extended tail of approximately 35 nm. To investigate the specificity for sialic acid, we studied the interaction of sialoadhesin with derivatized human erythrocytes, glycoproteins, and glycolipids. In conclusion, sialoadhesin specifically recognizes the oligosaccharide sequence Neu5Ac alpha 2----3Gal beta 1----3GalNAc in either sialoglycoproteins or gangliosides. These findings imply that specific sialoglycoconjugates carrying this structure may be involved in cellular interactions between stromal macrophages and subpopulations of haematopoietic cells and lymphocytes.

Structural requirements for the carbohydrate ligand of E-selectin.

Abstract: The acute inflammatory response requires that circulating leukocytes adhere to, and then migrate through, the vascular wall at the site of injury or infection. Several receptors have been implicated in this adhesion and migration process, including the selectins, a family of carbohydrate-binding proteins. The ligand for one of these proteins, E-selectin (LECAM-2, ELAM-1) has been described by several groups to contain a polylactosamine structure bearing a terminal sialic acid residue and at least one fucose residue. We report here a more detailed investigation into the minimum structural requirements for carbohydrate recognition by E-selectin. Using both direct binding and inhibition studies we demonstrate that the sialyl Lewisx tetrasaccharides Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, and Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc are the smallest oligosaccharides recognized by the lectin. In addition, an oligosaccharide containing the sialyl Lewisa epitope is also recognized, but less avidly. We propose a structural model of functional groups necessary for recognition by E-selectin, based on these data and additional experiments on modifications of sialic acid and the reducing terminal saccharide.

GMP-140 Binds to a Glycoprotein Receptor on Human Neutrophils: Evidence for a Lectin-like Interaction

Abstract: 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.

Serum sialic acid concentration and cardiovascular mortality.

Abstract: OBJECTIVE--To determine whether serum sialic acid concentration may be used to predict short and long term cardiovascular mortality. DESIGN--Prospective study on all men and women who had their serum sialic acid concentration measured as part of a general health survey in 1964 or in 1965. All were followed up for an average of 20.5 years. SETTING--Geographical part of the county of Varmland, Sweden. SUBJECTS--Residents in the area participating in a health check up in 1964-5 (27,065 men and 28,037 women), of whom 372 men (169 with incomplete data and 203 lost to follow up) and 345 women (143 and 202 respectively) were excluded; thus 26,693 men and 27,692 women entered the study. The study sample was restricted to subjects aged 40-74 during any of the 20 years9 follow up. MAIN OUTCOME MEASURES--Serum sialic acid concentration, serum cholesterol concentration, diastolic blood pressure, body mass index at the general health survey visit; cardiovascular and non-cardiovascular deaths during three periods of follow up (0-6 years, 7-13 years, and 14-20 years), according to the Swedish mortality register, in subjects aged 45-74. RESULTS--Mean serum sialic acid concentration (mg/100 ml) was 68.8 (SD 8.0) for men and 69.2 (8.0) for women; the average concentration increasing with age in both sexes. A total of 5639 (21%) men and 3307 (12%) women died during the follow up period, in whom death in 3052 (54%) men and 1368 (41%) women was from cardiovascular causes. During short (0-6 years), medium (7-13 years), and long (14-20 years) term follow up the relative risk of death from cardiovascular disease increased with increasing serum sialic acid concentration. The relative risk (95% confidence interval) associated with the highest quartile of sialic acid concentration compared with the lowest quartile was 2.38 (2.01 to 2.83) in men and 2.62 (1.93 to 3.57) in women. Similar results were found for deaths from non-cardiovascular disease with relative risks of 1.50 (1.34 to 2.68) in men and 1.89 (1.57 to 2.28) in women, but these relative risks were significantly lower than those for deaths from cardiovascular disease (p less than 0.001 and p less than 0.005 respectively). In multivariate analysis of total mortality and of cardiovascular mortality with sialic acid concentration, serum cholesterol concentration, diastolic blood pressure, and body mass index as independent variables the impact of sialic acid concentration was virtually the same as in univariate analysis. CONCLUSION--Serum sialic acid concentration is a strong predictor of cardiovascular mortality. A possible explanation of these findings is that the serum sialic acid concentration may reflect the existence or the activity of an atherosclerotic process, and this may warrant further investigation.

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

Abstract: 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.

Expression, structure, and function of the CD23 antigen.

Abstract: Publisher Summary The chapter explains that the structure of CD23 predicts both a lectin activity and a role in cellular adhesion. However, this view requires more experimental support, and, moreover, the binding to IgE does not require carbohydrate interactions. Interestingly, all the well-documented functions of CD23 (antigen presentation, parasite killing, and mediator release) are IgE dependent whereas most of the functions ascribed to sCD23 are clearly IgE independent. A hallmark of the CD23 molecule is that this transmembrane glycoprotein is continuously cleaved into soluble fragments, referred to as soluble CD23 (sCD23) that are released in the extracellular fluid. Most intriguingly, the two forms of CD23 may have different and sometimes antagonistic functions. In humans, some sCD23 fragments retain the ability to bind to IgE and have therefore been named IgE-binding factors (IgE–BFs). Human CD23 is a single-chain 45-kDa glycoprotein; it contains one chain of N-linked carbohydrates of the complex type, several O-linked carbohydrates, and sialic acid residues. Immunoprecipitation studies using either anti-CD23 Mab orIgE reveal the presence of three components with masses of 60–95, 45, and 37 kDa. The high-molecular-weight component is because of the formation of aggregates of the 45-kDa molecules after solubilization of the cells. Indeed, the molecules of 45 and 60–95 kDa display identical peptide maps and they share several antigenic determinants. This chapter discusses the functions of membrane CD23 and soluble CD23. In healthy individuals the serum level of sCD23 remains stable over a long period of time, indicating that it is not affected by environmental factors. CD23 is found at low levels on the majority of mature resting B cells, and freshly isolated PBMCs express only type a CD23 mRNA.

The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant.

Abstract: The S protein of bovine coronavirus (BCV) has been isolated from the viral membrane and purified by gradient centrifugation. Purified S protein was identified as a viral hemagglutinin. Inactivation of the cellular receptors by sialate 9-O-acetylesterase and generation of receptors by sialylation of erythrocytes with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicate that S protein recognizes 9-O-acetylated sialic acid as a receptor determinant as has been shown previously for intact virions. The second glycoprotein of BCV, HE, which has been thought previously to be responsible for the hemagglutinating activity of BCV, is a less efficient hemagglutinin; it agglutinates mouse and rat erythrocytes, but in contrast to S protein, it is unable to agglutinate chicken erythrocytes, which contain a lower level of Neu5,9Ac2 on their surface. S protein is proposed to be responsible for the primary attachment of virus to cell surface. S protein is proposed to be responsible for the primary attachement of virus to cell surface receptors. The potential of S protein as a probe for the detection of Neu5,9Ac2-containing glycoconjugates is demonstrated.

Expression of alpha 2,6-linked sialic acid residues in neoplastic but not in normal human colonic mucosa. A lectin-gold cytochemical study with Sambucus nigra and Maackia amurensis lectins.

Abstract: Increased sialylation of cell surface glycoconjugates has been demonstrated in malignant tumors and shown to be correlated with the invasive and metastatic growth of colon carcinoma cells. The authors have applied the Maackia amurensis lectin, which interacts with alpha 2,3-linked sialic acid, and the Sambucus nigra I lectin specific for alpha 2,6-linked sialic acid. In human colon, alpha 2,3-linked sialic acid was detectable in normal and transitional mucosa as well as in adenomas with different degrees of dysplasia and in carcinoma. In contrast, alpha 2,6-linked sialic acid as visualized with Sambucus nigra I lectin was found only in severe dysplasia and carcinoma. Thus expression of binding sites for Sambucus nigra I lectin was associated with the occurrence of histologic features of malignancy. It is concluded that malignant transformation in human colonic epithelium is accompanied by the de novo expression of an alpha 2,6 sialyl-transferase. These findings provide the basis for more detailed studies of the possible role of cell surface glycoconjugates bearing alpha 2,6-linked sialic acid in growth behavior of human colonic epithelial cells.

Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis.

Abstract: Monoclonal antibodies (MAb) 3F11 and 06B4 recognize epitopes that are conserved on gonococcal lipooligosaccharides (LOS), present on some meningococcal LOS, and conserved on human erythrocytes. LOS of some group B and C prototype meningococcal LOS strains (LOS serotypes L1 to L8) treated with neuraminidase showed increased expression of the 3F11 and 06B4 MAb-defined epitopes. Neuraminidase-treated LOS separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stained showed a shift in migration from a component with a mass of approximately 4.8 kDa to a component with a mass of between 4.5 and 4.6 kDa. The same strains grown in medium with excess CMP-N-acetylneuraminic acid had LOS that shifted in migration to a slightly higher component (mass, approximately 4.8 kDa). Chemical analysis of the neuraminidase-digested products from one LOS indicated it contained approximately 1.5% sialic acid. Covalent linkage between sialic acid and the LOS was confirmed by analysis of de-O-acylated and dephosphorylated LOS by liquid secondary ion mass spectrometry. Three studies show that some meningococci contain sialic acid in their LOS, that the sialic acid is cleaved and lost in conventional acetic acid hydrolysis, and that the sialic acid alters the expression of MAb-defined epitopes.

Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells

Abstract: We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).

Method and composition for synthesizing sialylated glycosyl compounds

Abstract: The present invention provides a method for synthesizing a sialylated glycosyl compound comprising reacting in the presence of each other a sialic acid, a glycosyl compound, a CMP-sialic acid regenerating system, a pyrophosphate scavenger and catalytic amounts of a CMP-sialic acid synthetase and a sialyl transferase having substrate specificity for the glycosyl compound. The present invention also provides a composition for sialylating glycosyl compounds comprising a sialic acid, CMP-sialic acid regenerating system, a pyrophosphate scavenger and a catalytic amount of CMP-sialic acid synthetase. The composition can further comprise an aqueous solvent having a suitable buffer and enzyme cofactors as well as a catalytic amount of a sialyl transferase having substrate specificity for the glycosyl compound. A phagemid-transformed E. coli. that overproduces CMP-sialic acid synthetase is also disclosed.

Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Gal beta 1-3GlcNAc) or type 2 (Gal beta 1-4GlcNAc) disaccharide units.

Abstract: Theadhesion ofPseudomonas aeruginosa totype1 (Gal,11-3GlcNAc) andtype2 (Galpll-4GlcNAc) disaccharide determinants wasstudied ina microtiter adhesion assayanda thin-layer chromatography bacterial overlay assay. Theoligosaccharides wereprepared fromhumanbreast milkandhumanurine and wereconjugated tohexadecylaniline toformneoglycolipids thatwereusedintheassays. Boththemucoidand thenonmucoid strains thatwerestudied recognized thedisaccharide determinants. Sialylation ofthe oligosaccharides didnotsuppress binding inthethin-layer chromatography assay, butt2-6-linked sialic acid blocked binding inthemicrotiter assay. Theuseofbovine serumalbumin instead ofgelatin asablocking agent against nonspecific binding completely suppressed binding inthethin-layer chromatography assay. Isogenic nonpiliated mutants ofnonmucoid strains constructed byinterrupting thepilin generetained their adhesive capacity forthedisaccharide units, indicating thatbinding tothedisaccharides wasmediated byanonpilus adhesin(s). Furthermore, twomonoclonal antibodies thatrecognize thetype2 disaccharide determinant (GaI01-4GlcNAc) partially inhibited adhesion ofapair ofpiliated andnonpiliated isogenic strains tomucin. Thisstudy suggests thatP.aeruginosa utilizes anonpilus adhesin(s) tobindtodisaccharide units commonly found inmucins, inaddition topili andalginate, twopreviously described adhesins. Receptors forPseudomonas aeruginosa havebeenfound onatleast twotypes ofglycoconjugates, namely, glycolipids andmucins. Theminimal structure recognized onglycolipids isclaimed tobeGalNAc1-4Gal sequence commonly found intheglycosphingolipid

Fusion properties of cells persistently infected with human parainfluenza virus type 3: participation of hemagglutinin-neuraminidase in membrane fusion.

Abstract: Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.

Quantitative determination of N-glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as a tumor-associated sialic acid by gas chromatography-mass spectrometry.

Abstract: N-Glycolylneuraminic acid (NeuGc) is distributed in most animals except humans and chickens. However, human and chicken cancerous tissues often synthesize this heterophilic sialic acid as a tumor-associated Hanganutziu-Deicher antigen [M. Naiki and H. Higashi, Adv. Exp. Med. Biol., 152: 445-456, 1982; H. Higashi et al., Cancer Res., 45: 3796-3802, 1985]. In this paper, NeuGc in human cancerous tissues and chicken Marek's disease lymphoma cell lines was determined quantitatively with gas chromatography-mass spectrometry analysis using mass fragmentography. The detectable limit of NeuGc was 40 pg (0.12 pmol) in each injection using 5 ng of trideuteriomethyl ester trideuteriomethyl glycoside of the sialic acid as an internal standard sample when a pair of ions at m/e 386 and 389 was chosen for ion monitoring. NeuGc was detected in ganglioside-rich fractions of various human cancerous tissues from 5 of 8 patients examined but was not detected in glycosphingolipids of normal human tissues. The contents of NeuGc in these cancerous tissues ranged from 0.02 to 0.5% of the total sialic acid content. NeuGc was also detected in freeze-dried samples of 5 different cell lines from chicken Marek's disease lymphomas but was not detected in a cell line from chicken lymphoid leukosis lymphoma and normal chicken skeletal muscle tissue. The contents of NeuGc in the positive cell lines ranged from 0.03 to 0.11% of the total sialic acid content. These results indicate that NeuGc can be synthesized in both humans and chickens in some cancers.

Sulfate contributes to the negative charge of podocalyxin, the major sialoglycoprotein of the glomerular filtration slits.

Abstract: Podocalyxin is the major sialoprotein of the rat glomerulus. Its function is to maintain the filtration slits of the glomerular epithelium open by virtue of its high net negative charge. We have used biosynthetic labeling and oligosaccharide analysis to characterize the anionic-charge-carrying moieties on this protein. Kidney slices from 2-day-old rats were biosynthetically labeled with [35S]Cys, [3H]Man, [3H]GlcN, and 35SO4, after which podocalyxin was immunoprecipitated and purified by SDS/PAGE. All these labels were incorporated into podocalyxin. Immunoprecipitates were subjected to digestion with specific glycosidases or digested with Pronase followed by chromatographic analysis of the released glycopeptides. Podocalyxin was susceptible to digestion with N-Glycanase and O-Glycanase, indicating the presence of both N- and O-linked oligosaccharides. Approximately 30% of the [3H]GlcN-labeled glycopeptides bound to Con A, confirming the presence of high mannose, hybrid, or biantennary N-linked structures; alkaline borohydride treatment confirmed the presence of O-linked oligosaccharides. Analysis of the 35SO4-labeled glycopeptides indicated that both the N- and O-linked structures were sulfated. We conclude that in newborn rat kidney (i) podocalyxin contains both O- and N-linked oligosaccharides [high mannose or hybrid type, biantennary, and complex (sialylated) type], (ii) podocalyxin is sulfated, and (iii) sulfate is located on both O-linked oligosaccharides and on glycopeptides carrying tri- or tetrantennary N-linked structures. These results indicate that the net negative charge of podocalyxin is most likely derived from sulfate as well as from sialic acid residues.

Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells.

Abstract: Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellular fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribution of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Aggregation responses of neutrophils as well as adhesion responses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-dependent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells.