Sialic Acid Residues Of Glycoproteins Research Articles

Inhaled corticosteroids increase siglec-5/14 expression in sputum cells of COPD patients.

Recent studies show that several Siglec receptors, such as Siglec-8 and Siglec-14, may be important therapeutic targets in asthma and COPD. Siglecs are a family of lectins belonging to the immunoglobulin superfamily and recognize sialic acid residues of glycoproteins. Most of Siglecs have intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIM), implicating them in the suppression of immunoreceptor signaling. Siglec-5/14 may be involved in the negative regulation of innate immune responses. The aim of this study was to analyze Siglec-5/14 expression in induced sputum cells of COPD patients in the following treatment combinations: (1) a long-acting beta2-agonist, formoterol; (2) formoterol combined with a long-acting antimuscarinic agent, tiotropium; and (3) formoterol combined with an inhaled corticosteroid or formoterol combined with tiotropium and with an inhaled corticosteroid. Siglec expression was assessed in sputum cells by flow cytometry using a specific monoclonal antibody. Double staining of cells indicated that Siglec-5/14 is expressed in monocyte/macrophages and neutrophils, but not in lymphocytes. Siglec-5/14 expression was significantly higher in patients receiving combined therapy including inhaled corticosteroids compared with patients taking only formoterol or formoterol + tiotropium. Our results suggest that inhaled corticosteroids may exert beneficial or negative effects, depending on the patients' phenotype, through increased immunosuppressive Siglec-5 or immunoactivatory Siglec-14 receptors, respectively.

Microglial CD33-Related Siglec-E Inhibits Neurotoxicity by Preventing the Phagocytosis-Associated Oxidative Burst

Sialic acid-binding Ig-like lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-E is a mouse CD33-related Siglec that preferentially binds to sialic acid residues of the cellular glycocalyx. Here, we demonstrate gene transcription and protein expression of Siglec-E by cultured mouse microglia. Siglec-E on microglia inhibited phagocytosis of neural debris and prevented the production of superoxide radicals induced by challenge with neural debris. Soluble extracellular Siglec-E receptor protein bound to the neural glycocalyx. Coculture of mouse microglia and neurons demonstrated a neuroprotective effect of microglial Siglec-E that was dependent on neuronal sialic acid residues. Increased neurotoxicity of microglia after knockdown of Siglece mRNA was neutralized by the reactive oxygen species scavenger Trolox. Data suggest that Siglec-E recognizes the intact neuronal glycocalyx and has neuroprotective function by preventing phagocytosis and the associated oxidative burst.

Influenza virus binds its host cell using multiple dynamic interactions

Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609-9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus-cell binding quantitatively at the molecular level.

Alleviation of Neurotoxicity by Microglial Human Siglec-11

Sialic acid-binding Ig superfamily lectins (Siglecs) are members of the Ig superfamily that recognize sialic acid residues of glycoproteins. Siglec-11 is a recently identified human-specific CD33-related Siglec that binds to alpha2,8-linked polysialic acids and is expressed on microglia, the brain resident innate immune cells. Polysialylated neuronal cell adhesion molecule (PSA-NCAM) is a putative ligand of Siglec-11. We observed gene transcription and protein expression of Siglec-11 splice variant 2 in human brain tissue samples by RT-PCR and Western blot analysis. Siglec-11 was detected on microglia in human brain tissue by immunohistochemistry. Human Siglec-11 splice variant 2 was ectopically expressed by a lentiviral vector system in cultured murine microglial cells. Stimulation of Siglec-11 by cross-linking suppressed the lipopolysaccharides (LPS)-induced gene transcription of the proinflammatory mediators interleukin-1beta and nitric oxide synthase-2 in microglia. Furthermore, phagocytosis of apoptotic neuronal material was reduced in Siglec-11 transduced microglia. Expression of PSA-NCAM was detected on microglia and neurons by immunohistochemistry and RT-PCR. Coculture of microglia transduced with Siglec-11 and neurons demonstrated neuroprotective function of Siglec-11. The neuroprotective effect of Siglec-11 was dependent on polysialic acid (PSA) residues on neurons, but independent on PSA on microglia. Thus, data demonstrate that human Siglec-11 ectopically expressed on murine microglia interacts with PSA on neurons, reduces LPS-induced gene transcription of proinflammatory mediators, impairs phagocytosis and alleviates microglial neurotoxicity.

New method for determining the sugar composition of glycoproteins, glycolipids, and oligosaccharides by high-performance liquid chromatography.

A new method is reported that can be performed within a single vessel to analyze the composition of aldose, hexosamine, and sialic acid residues of glycoproteins, glycolipids, and oligosaccharides. Glycoconjugates are treated with sialidase or subjected to mild acid hydrolysis, before being treated with N-acetylneuraminic acid aldolase to convert the free sialic acid residues to their corresponding N-acylmannosamines. The reaction mixture is then successively subjected to acid hydrolysis (in order to produce monosaccharides), N-acetylation, and conversion with p-aminobenzoic acid ethyl ester (ABEE). The ABEE-converted monosaccharides are simultaneously determined by reverse-phase high-performance liquid chromatography. Determination of the sugar compositions of bovine fetuin, II3NeuGc alpha-LacCer, and 3'-sialyllactose with this method was found to be highly accurate. Linearity of the peak area vs. the amount of bovine fetuin ranged from 1 to 50 micrograms in all ABEE-converted monosaccharides. With a slight modification to this method, sialic acid residues can be separately determined as NeuAc and NeuGc. This novel method and its modified version are used to demonstrate the sugar compositions of alpha 1-acid glycoproteins from several sources.

Effect of Crataeva nurvala on the biochemistry of the small intestinal tract of normal and stoneforming rats

Certain biochemical constituents were analysed in small intestinal tract tissues of rats fed a normal and a calculi-producing diet. Changes were seen in calculogenic rats with respect to the carbohydrate and sialic acid residues of glycoproteins along with some lipid fractions. Treatment with Crataeva nurvala bark decoction lowered levels of small intestinal (Na +,K +)-ATPases. The action of the decoction on the small intestinal tract seems to be mediated through (Na +,K +)-ATPases, which in turn may affect the transport of metabolites.

Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.

45] Properties of a sialic acid-specific lectin from the slug Limax flavus

Publisher Summary Lectins are a group of nonimmunoglobulin-like proteins that exhibit a high binding specificity toward carbohydrate residues of glycoproteins and glycolipids. They have found wide application in studies on glycoproteins because they interact with carbohydrates in a highly specific manner. In spite of the ubiquitous presence of lectins in nature, only a few have been characterized to be specific for sialic acid. It has been recently demonstrated that this slug contains a lectin that is highly specific for sialic acid residues of glycoproteins. This lectin, limax flavus agglutinin (LFA), has been recently purified to homogeneity. The chapter discusses the hemagglutination assay process and the purification procedure. The macromolecular properties of LFA have been presented in the chapter in a tabular form. There is a discussion about the use of LFA–Sepharose 4B in affinity chromatography.

A sialic acid-specific lectin from the slug Limax flavus

The slug, Limax flavus, contains a lectin that appears to be highly specific for sialic acid residues of glycoproteins. The carbohydrates which inhibited the hemagglutinating activity of the slug lectin and the concentration of the carbohydrate which gave a 50% inhibition are as follows: N-acetylneuraminic acid, 0.13 m m; N-glycolylneuraminic acid, 0.90 m m; d-glucosamine, 4.9 m m; d-galactosamine, 7.6 m m; N-acetyl- d-glucosamine, 23 m m; and N-acetyl- d-galactosamine, 24 m m. d-Galactose, d-glucose, d-mannose, α-methyl- d-glucoside, α-methyl- d-mannoside, l-arabinose, d-xylose, l-fucose, d-glucuronic acid, lactose, and sucrose were found to be ineffective as inhibitors of the hemagglutinating activity of the slug lectin. Hemagglutination by slug lectin was strongly inhibited by bovine submaxillary mucin and fetuin but not by sialic acid-free bovine submaxillary mucin or fetuin.

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. I. Observations in hepatocytes.

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of [3H]N-acetylmannosamine (8 mCi) and then sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the silver grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. Meanwhile, the proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h and over 55% at 4 and 24 h; two-thirds of these grains lay over the sinusoidal membrane, and the remainder were equally divided over the lateral and bile canalicular membranes. Many silver grains also appeared over lysosomes at the 4- and 24-h time intervals, accounting for 15-17% of the total. At 3 and 9 d after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling such that by 9 d only a very light reaction remained. Because our biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), the interpretation of these results is that sialic acid is incorporated into these molecules in the Golgi apparatus and that the latter then migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. It is possible that some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.

The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. II. Observations in tissues other than liver.

Biochemical evidence from the preceding paper indicated that [3H]N-acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.

Selective spin labelling of sialic acid residues of glycoproteins and glycolipids in erythrocyte membranes: A novel method to study cell surface interactions

FEBS LettersVolume 115, Issue 2 p. 185-188 Full-length articleFree Access Selective spin labelling of sialic acid residues of glycoproteins and glycolipids in erythrocyte membranes A novel method to study cell surface interactions J.B. Feix, J.B. Feix Department of Chemistry, University of Kentucky, Lexington, KY 40506, USASearch for more papers by this authorD.A. Butterfield, D.A. Butterfield Department of Chemistry, University of Kentucky, Lexington, KY 40506, USASearch for more papers by this author J.B. Feix, J.B. Feix Department of Chemistry, University of Kentucky, Lexington, KY 40506, USASearch for more papers by this authorD.A. Butterfield, D.A. Butterfield Department of Chemistry, University of Kentucky, Lexington, KY 40506, USASearch for more papers by this author First published: June 30, 1980 https://doi.org/10.1016/0014-5793(80)81164-1Citations: 33AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 J.E. Sadler, J.C. Paulson, R.L. Hill, J. Biol. Chem., 254, (1979), 2112– 2119. 2 R. Kornfield, S. Kornfield, J. Biol. Chem., 245, (1970), 2536– 2545. 3 R.W. Jeanloz, J.F. Codington, A. Rosenberg C.-L. Schengrund Biological Roles of Sialic Acid (1976), Plenum New York 221– 238. 4 J.P. Behr, J.M. Lehn, FEBS Lett., 31, (1973), 297– 300. 5 T.J. Greenwalt, E.A. Steane, Brit. J. Haematol., 25, (1973), 217– 226. 6 C.G. Gahmberg, K. Itaya, S.-I. Hakomori, E.D. Korn Methods in Membrane Biology 7, (1976), Plenum New York 179– 210. 7 E.L.M. Ochoa, A.B. Bangham, J. Neurochem., 26, (1976), 1193– 1198. 8 D.A. Butterfield, A.D. Roses, S.H. Appel, D.B. Chesnut, Arch. Biochem. Biophys., 177, (1976), 226– 234. 9 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 10 L. Warren, J. Biol. Chem., 234, (1959), 1971– 1975. 11 T.L. Steck, J. Yu, J. Supramol. Struct., 1, (1973), 220– 232. 12 J. Folch-Pi, M. Lees, G.H. Sloane-Stanley, J. Biol. Chem., 226, (1957), 497– 509. 13 T.L. Steck, G. Dawson, J. Biol. Chem., 249, (1974), 2135– 2142. 14 R.F. Borch, M.D. Bernstein, H.D. Durst, J. Am. Chem. Soc., 93, (1971), 2897– 2904. 15 P.L. Nordio, L.J. Berliner Spin Labelling. Theory and Applications (1976), Academic Press New York 29– 35. 16 T.H. Liao, P.M. Gallop, O.O. Blumenfield, J. Biol. Chem., 23, (1973), 8247– 8253. 17 O.O. Blumenfield, P.M. Gallop, T.H. Liao, Biochem. Biophys. Res. Commun., 48, (1972), 242– 251. 18 P.V. Tishler, C.J. Epstein, Anal. Biochem., 22, (1968), 89– 98. 19 L. Van Lenten, G. Ashwell, J. Biol. Chem., 246, (1971), 1889– 1894. 20 R.G. Spiro, J. Biol. Chem., 239, (1964), 567– 573. 21 C.G. Gahmberg, L.C. Anderson, J. Biol. Chem., 252, (1977), 5888– 5894. 22 G. Poggi, C.S. Johnson, J. Magn. Res., 3, (1970), 436– 445. 23 J.D. Aplin, M.A. Bernstein, C.F.A. Cullings, L.D. Hall, Carb. Res., 70, (1979), C9– C12. Citing Literature Volume115, Issue2June 30, 1980Pages 185-188 ReferencesRelatedInformation