Showing papers on "Sialic acid published in 1979"

Human alternative complement pathway: membrane-associated sialic acid regulates the competition between B and beta1 H for cell-bound C3b.

Abstract: The relative affinity of B and β1H for particle-bound C3b is influenced by the relative abundance of cell surface sialic acid moieties and determines whether or not the particle has the capacity to activate the human alternative complement pathway. The effect of membrane sialic acid on the binding of B and β1H to particle bound C3b was determined with radiolabeled proteins and a technique for separating bound from free ligand by centrifugation of the particles through an oil/aqueous interphase. The affinity constants at equilibrium and the effective number of C3b binding sites for B and β1H on the particles were determined by Scatchard analysis of the binding data. The sheep erythrocyte (E s ) served as a nonactivating particle, whereas the activating particles included E s from which 80% of the membrane sialic acid residues had been removed by sialidase or 80 to 85% had been converted to heptulosonic acid by NaIO 4 and NaBH 4 treatment and zymosan particles that lack sialic acid. The affinity constants at equilibrium for C3b on E s C3b were 1 × 10 7 M -1 for β1H and 2.1 × 10 6 M -1 for B, in the presence of 5 mM Mg ++ , indicating an approximately 5-fold greater affinity of β1H for the binding sites that were present in approximately equal numbers for the two ligands as determined by Scatchard analysis. The affinity of B at equilibrium for C3b bound to sialic acid-deficient sheep erythrocytes and to zymosan was the same as that for C3b on E s , and the calculated number of effective binding sites for B agreed with the number of sites estimated by the prior uptake of radiolabeled C3b. In contrast, the number of C3b binding sites with an affinity for β1H of approximately 10 7 M -1 on sialic acid-deficient E s C3b or ZyC3b was only ⅕ to ⅙ the number interacting with B. The presence of additional binding sites for β1H with an affinity so low that they were not revealed by the Scatchard analysis because of the dominant effect of the high affinity sites was determined in competitive binding studies between B and β1H for C3b on sialic acid-deficient surfaces. The affinity constant of β1H for these sialic acid independent C3b sites is approximately 7 × 10 5 M -1 , a K β1H at least 10-fold less than that of the sialic acid-dependent high affinity C3b sites. The relative inefficiency of β1H in competing with 125 I-B for uptake by C3b on the sialic acid-deficient erythrocytes correlated with the decreased capacity of β1H to decay-dissociate C3b,Bb from such particles. Thus a deficiency of sialic acid diminishes the number of high affinity C3b sites for β1H and creates an activating particle on which binding of B by C3b is favored relative to β1H and decay-dissociation of C3b,Bb by β1H is impaired.

Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen

Abstract: Fibronectin mediates the adhesion of cells to collagen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectin-mediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GD1a were the most effective inhibitors followed by GD1b greater than GM1 greater than GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GD1a modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin.

Form variation in Escherichia coli K1: determined by O-acetylation of the capsular polysaccharide.

Abstract: The chemical basis for the alternating antigenic change called form variation noted for the Escherichia coli K1-capsular polysaccharide has been shown by 13C nuclear magnetic resonance to be a result of random O-acetylation of C7 and C9 carbons of the alpha-2-8-linked sialic acid homopolymer. A serologic method (antiserum agar) was developed to identify and isolate the form variants. The O-acetyl positive and O-acetyl negative K1 polysaccharides had unique biochemical and immunologic properties. The O-acetyl-positive variants resisted neuraminidase hydrolysis in contrast to the susceptibility of the O-acetyl negative variant to this enzyme. In addition, O-acetylation altered the antigenicity of the O-acetyl polysaccharides. When injected as whole organisms, O-acetyl positive organisms produced anti-K1 -antibodies in rabbits specific for this polysaccharide variant. O-acetyl negative organisms were comparatively less immunogenic; however, antibodies induced by these organisms reacted with both K1 polysaccharide variants. Burros, injected with either variant, produced antibodies reactive with both K1 polysaccharides.

Three types of blood group I specificity among monoclonal anti-I autoantibodies revealed by analogues of a branched erythrocyte glycolipid.

Abstract: Blood group I activities of the purified glycosphingolipid lacto-N-iso-octaosyl ceramide (Fromula: see text) and 8 of its analogues have been evaluated with 11 anti-I sera including 5 anti-I sera previously tested. All but one of the antisera were inhibited by the lacto-N-iso-octaosyl structure. Three types of I-specificity could be distinguished although none of the anti-I sera was identical in its inhibition patterns with the nine glycophingolipid analogues. The anti-I sera Ma and Woj represent the first type and require an intact Galbeta1 leads to 4GlcNAcbeta1 leads to 6 chain, the anti-I sera Step, Gra, Ver, and Ful represent the second type which requires Galbeta1 leads to 4GlcNAcbeta1 leads to 3 chain with branching, and the anti-I sera Phi, Da, Sch, and Low belong to the third type which requires both branches to be intact. Anti-I antibodies varry in their ability to react with their antigenic determinants in the presence of external substitutions with alpha-linked galactose or sialic acid.

Glycosyltransferase and Glycosidase Activities in Ovarian Cancer Patients

Abstract: In order to elucidate the mechanism of appearance of abnormal glycoproteins in cancer, activities of glycoprotein glycosyltransferases and glycosidases were determined in the homogenates prepared from normal ovaries and ovarian epithelial adenocarcinoma. Significantly high activities (more than normal mean + 2 S.D.) of these enzymes were found as follows: galactosyltransferase and sialyltransferase in 100%; fucosyltransferase 1 (exogenous acceptor, fetuin minus sialic acid and galactose) in 86%; fucosyltransferase 2 (fetuin minus sialic acid, acceptor) in 45%; N-acetylglucosaminyltransferase 1 (ovalbumin acceptor) in 53%; N-acetylglucosaminyltransferase 2 (ribonuclease A as acceptor) in 10% of the samples analyzed. Among the glycosidases, substantially elevated activities above normal controls were found as follows: N-acetyl-β-d-glucosaminidase in 85%; N-acetyl-β-d-galactosaminidase in 63%; N-acetyl-β-d-galactosidase in 50%, and those of α-l-fucosidase in 35% of the tumors. In serum of these cancer patients, only levels of galactosyltransferase were consistently elevated compared to controls. Increases in serum levels for other transferases were as follows: fucosyl-1, 10%; fucosyl-2, 60%; sialyl-, 20%; N-acetylglucosaminyl-1, 90%; N-acetylglycosaminyl-2, in 80% of the serum samples from ovarian carcinoma patients. Galactosyltransferase thus appears to be an excellent marker for ovarian carcinoma.

Enzymatic Properties of Neuraminidases from Arthrobacter ureafaciens

Abstract: Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.

Inhibition of platelet aggregation by native and desialised alpha-1 acid glycoprotein

Abstract: The alpha-1 acid glycoprotein (orosomucoid; AAG) is a normal constituent of human plasma (650±215 µg ml−1) which increases in concentration as much as fivefold in association with acute inflammation and cancer, and thus is recognised as an acute phase protein1,2. AAG consists of a single polypeptide chain, has a molecular weight of 44,100, and contains ∼45% carbohydrate including 12% sialic acid; it is the most negatively charged of the plasma proteins3. Certain of the biological properties of AAG are related to its sialic acid content1,3; thus, clearance and immunogenicity of AAG are markedly increased on desialisation4,5. The biological functions of AAG are largely unknown. AAG has the ability to inhibit certain lymphocyte reactivities including blastogenesis in response to concanavalin A, phytohaemagglutinin and allogeneic cells6, and these inhibitory effects are enhanced in association with desialisation7. In view of these observations, a report that unphysiologically large (5–15 mg ml−1) amounts of AAG inhibit the platelet aggregation induced by ADP and adrenaline8, and evidence that a sialic acid-deficient species of AAG appears elevated in several chronic disease states9,10, we compared the effects of AAG and its desialised counterpart (AAG-D) on platelet aggregation. We report that desialisation of AAG is associated with increased expression of activity inhibitory to the platelet aggregation otherwise observed on stimulation with ADP, collagen or thrombin.

Structures of the Asparagine-Linked Sugar Chains of Human Chorionic Gonadotropin

Abstract: From the viewpoint of the physiological function of the carbohydrate moieties of glycoproteins, glycohormones and glycoenzymes are of current interest. These molecules have sugar components widely found in all glycoproteins.

Serum Sialic Acid and Sialyltransferase as Monitors of Tumor Burden in Malignant Melanoma Patients

Abstract: This study examines two associated tumor markers in malignant melanoma, sialic acid and sialyltransferase (EC 2.4.99.1). Both markers were measured in the same sera from 66 malignant melanoma patients, 20 rheumatoid arthritis patients, and age- and sex-matched normal controls. Bound sialic acid was measured by the thiobarbituric acid method, and sialyltransferase activity was measured by cytidine 5′-monophosphate- N -[4-14C]acetylneuraminic acid incorporation in desialated fetuin. Serum values for both markers were higher among rheumatoid arthritis patients than among controls ( p = < 0.0001). Melanoma patients were divided into three groups: Group I, 34 patients with no evidence of disease at the time of sampling; Group II, 13 patients with minimal tumor burden; and Group III, 19 patients with advanced metastatic disease. For sialic acid, there were significant differences between all group comparisons except the normals versus Group I. By contrast, for sialyltransferase only sera from Group III patients showed significant increased activity. Using the calculated upper limit of normal of 2.37 µmol/ml for sialic acid and 18.1 nmol/ml/hr for sialyltransferase, 2 (6%) Group I, 4 (31%) Group II, and 18 (95%) Group III patients had sialic acid elevations, while corresponding results for sialyltransferase were 0, 0, and 4 (42%). There was a direct relationship between sialic acid and sialyltransferase, especially for melanoma patients with established disease ( p = 0.00002). Of 24 patients with objective evidence of changing tumor burden 19 (79%) showed corresponding alterations in sialic acid concentration, while 14 (58%) showed such changes in sialyltransferase. Sialic acid was directly related to recurrence rate in Group I patients, while sialyltransferase was not. When used as monitors of tumor burden either to detect recurrent disease or to follow response to treatment, tumor markers need not be tumor specific. In our hands, sialic acid levels more closely reflect tumor burden than does sialyltransferase activity.

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Abstract: Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.

Immunodeterminant specificity of human immunity to type III group B streptococcus.

Abstract: The type III polysaccharides of group B Streptococcus in its native state chemically consists of glucose, galactose, glucosamine, and sialic acid. The core of this polysaccharide lacks sialic acid and precipitates with type III antiserum to give a partial identity with the precipitate between the native antigen and this serum. The core determinant is immunochemically similar to the capsular polysaccharide of type XIV Streptococcus pneumoniae, while the native type III group B streptococcal polysaccharide does not cross-react with type XIV pneumococcal antiserum. In human sera, it is antibody directed to the native antigen which correlates very highly with opsonic immunity (r = 0.94) while a poorer correlation exists between antibody to the core antigen and opsonins (r = 0.51 P less than 0.001). In natural infections, as association exists between low levels of maternal antibody to the native antigen and risk of disease in the infant. This association is not true for antibody to the core structure, where both infected infants and their mothers have much higher levels of antibody to the core than the native antigens. Infected infants are also more likely to respond to infection by developing antibody to the native antigen. Immunization of 12 adults with multivalent pneumococcal polysaccharide induced significantly better antibody response to the core antigen than to the native, and this vaccine induced opsonic activity in only one recipient. Immunization of adults with type III group B streptococcal antigens induced antibody to the native determinant which correlated with opsonic activity. Therefore, it would appear that native group B streptococcal polysaccharides will provide the best candidate antigens for immunization.

Glycoproteins and human cancer. 1. Circulating levels in cancer serum.

Abstract: Total protein and sialic acid levels were determined in the supernatant of serum treated with perchloric acid. Patients with either localized or advanced metastatic malignancy have significantly elevated mean serum values. The highest levels occur in patients with lung, GI, GYN cancer, lymphoma and malignant melanoma. Patients with leukemia and multiple myeloma have slightly elevated values, but they were not significantly different from normal. Patients following curative surgery have normal values while patients in clinical remission following chemotherapy have elevated mean serum protein and NANA levels. Elevated values also occur in patients with benign tumors and 12% of patients with nonmalignant disease. Tumor cells appear to shed macromolecules which contribute to the observed elevation of serum protein and sialic acid levels.

Inhibition of the lectin-induced mitogenic response of thymocytes by glycolipids.

Abstract: Exogenous gangliosides at concentrations found in serum inhibit the concanavalin A- (Con A) induced mitogenic response of mouse thymocytes. Of four gangliosides tested, the trisialoganglioside, GT1, was the most potent inhibitor. Ceramides, cerebrosides, and sialic acid were not inhibitory at any concentration tested. The inhibition by gangliosides was not due to interference with Con A binding as shown by direct binding studies with [3H]acetyl-Con A nor was it due to a nonspecific killing effect. Thymocytes exposed to a ganglioside concentration 5 times that required to inhibit mitogenesis were still capable of excluding trypan blue up to 44 hr after ganglioside addition. Furthermore, ganglioside inhibition could be reversed by washing the cells 4 hr after addition of the glycolipid. A productive interaction with Con A occurs in the presence of ganglioside as shown by a Con A-induced increase in carbohydrate metabolism. However, uridine and thymidine incorporation are inhibited by the presence of ganglioside. Complete inhibition could be achieved if the glycolipid were added as late as 24 to 28 hr after the Con A in a 48-hr mitogenic assay. The results are discussed in light of recent findings that elevated levels of gangliosides are found in in the sera of tumor-bearing animals, and it is suggested that gangliosides shed by tumor cells could be involved in the generalized immunosuppression observed in such animals.

Partial structure of a membrane glycopeptide from virus-transformed hamster cells.

Abstract: The predominant surface glycopeptide from a clone of baby hamster kidney cells transformed by Rous sarcoma virus (C13/B4), metabolically labeled with L-[14C]fucose, has been characterized for the first time. This glycopeptide represents 19% of the total radioactivity removed by trypsin from the cell surface of the transformed fibroblasts and is more abundant in the transformed cells than in the normal counterpart. Purification of the glycopeptide after digestion with Pronase was by successive chromatography on DEAE-cellulose and Sephadex G-50. The monosaccharide content of the glycopeptide was 42, 127, 138, 114, and 243 nmol of fucose, sialic acid, galatose, mannose, and glucosamine, respectively. A partial structure of the glycopeptide was proposed from the results of sequential enzymatic degradation coupled with gas-liquid chromatographic analysis of the resultant monosaccharides. All of the enzymes used were purified and pretested on natural substrates and found to remove terminal monosaccharides of the correct configuration, quantitatively. The purification and properties of an alpha-L-fucosidase from rat testes were described. All of the radioactivity in the glycopeptide, recovered as fucose, was present at the core and was removed by treatment with this alpha-L-fucosidase. The proposed structure is a triantennary, completely sialylated, complex glycopeptide containing a core region of beta-D-mannose, beta-D-N-acetylglucosamine, and alpha-L-fucose.

Thyroxine and Propylthiouracil Effects on Alpha- and Beta-adrenergic Receptor Number, Atpase Activities, and Sialic Acid Content of Rat Cardiac Membrane Vesicles

Abstract: Summary: Membrane vesicle preparations from the hearts of euthyroid, hyperthyroid, and hypothyroid rats were analyzed in an attempt to identify biochemical changes which might correlate with known functional changes occurring in these thyroid states. Several sarcolemmal constituents which have been shown to influence myocardial contractility were measured in membrane vesicle preparations from the hearts of animals in the three thyroid states. These constituents included the apparent number of alpha- and beta-adrenergic receptors (judged from specific binding of radiolabeled adrenergic antagonists) and Na-, K--ATPase activity. As a control for the recovery of sarcolemma in the preparations, the sialic acid content was measured in all preparations. The activity of K-, Ca2--ATPase, a sarcoplasmic reticulum enzyme which regulates intracellular ionized Ca2- concentration, was also measured. Membrane vesicles of the thyroxine-treated hyperthyroid rats showed a decrease (41%, p

Sialic-acid content of low-density lipoproteins controls their binding and uptake by cultured cells.

Abstract: The (high-affinity receptor)-mediated uptake of homologous low-density (low-rho) lipoproteins by cultured human arterial smooth muscle cells or human skin fibroblasts is controlled by the sialic acid content of low-rho lipoprotein particles This conclusion is derived from the following results 1 Gangliosides incubated with native low-rho lipoproteins associate with low-rho lipoprotein particles Low-rho lipoproteins modified by associated GLac1, GGtet1, and GGtet2b + GGtet3 gangliosides are internalized by arterial smooth muscle cells at a rate up to 80% lower than native low-rho lipoproteins or those preincubated with desialized gangliosides 2 The inhibitory effect of gangliosides is specific for high affinity uptake and not detectable on skin fibroblasts deficient in low-rho-lipoprotein receptor 3 Desialyzed low-rho lipoproteins are internalized by smooth muscle cells up to 100% faster than native low-rho lipoproteins, the enhancement of uptake corresponding to the degree of desialization