Removal Of Terminal Sialic Acid Research Articles

Altered glycosylation in Madin-Darby canine kidney (MDCK) cells after transformation by murine sarcoma virus

The changes in glycosylation of an immortalized epithelial cell line (MDCK) before and after progression towards a more malignant phenotype have been studied. The parental MDCK-3 cells were immortalized after long-term passage in vitro and have shown no tendency for spontaneous acquisition of malignancy-related phenotypes such as tumorigenicity, invasion and metastasis. They conserved morphological and functional characteristics of the epithelial tissue of origin. The ras-MDCK cells acquired the fully malignant phenotype after transformation with a Harvey murine sarcoma virus; they were immortalized, invasive in vitro and produced invasive and also metastatic tumors after subcutaneous injection into nude mice. Using immobilized lectins and gel chromatography, before and after liberation of O-linked glycans from the peptide moieties and also after removal of terminal sialic acid, we have found differences in the glycosylpeptides of both whole cells and cell surface trypsinates from ras-MDCK cultures as compared to the parental MDCK-3 cultures: (i) more sialic acid in the N-linked tri- and tetra-antennary structures; (ii) more fucosylation in the N-glycosylpeptides; (iii) more bi-antennary N-glycosylpeptides and less O-linked glycans; and (iv) a lower molecular weight of the O-linked glycans probably due to a decreased sialylation. It is concluded that alterations in sialylation and fucosylation of the cell surface exposed glycans accompanied progression of MDCK-3 cells towards a more malignant phenotype.

The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity.

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.

The lectin-like interaction between recombinant tumor necrosis factor and uromodulin.

The polypeptide of uromodulin, an immunosuppressive glycoprotein isolated from human urine, has been shown to be identical to that of Tamm-Horsfall glycoprotein and is synthesized exclusively in the kidney (Hession, C., Decker, J. M., Sherblom, A. P., Kumar, S. (1987) Science 237, 1479-1484). Uromodulin binds recombinant murine interleukin 1 alpha with high affinity, and this binding can be inhibited by addition of specific saccharides (Muchmore, A. V., and Decker, J. M. (1987) J. Immunol. 138, 2541-2546). We now report that uromodulin binds recombinant human tumor necrosis factor (rTNF) with high affinity. Both diacetylchitobiose and Man(alpha 1-6)(Man(alpha 1-3]-Man-O-ethyl are effective inhibitors of the binding, whereas a wide variety of other saccharides are not inhibitory. Although Tamm-Horsfall glycoprotein contains predominantly tetraantennary N-linked chains, the binding to rTNF is unaffected by removal of terminal sialic acid, galactose, and N-acetylhexosamine residues. Fractionation of a Pronase digest of uromodulin by gel filtration yields material that inhibits the binding of uromodulin to rTNF but is of lower molecular weight than the major oligosaccharide. Uromodulin does not inhibit the cytotoxic activity of rTNF as monitored by lysis of tumor cell targets but effectively protects mice from lethal challenge with lipopolysaccharide, an event that may involve lymphokine toxicity. We have previously shown that rTNF binds to sections of human kidney and is localized in the same region as uromodulin. Thus, rTNF interacts with uromodulin via carbohydrate chains that are less processed than the major tetraantennary chain, and this interaction may be critical in promoting clearance and/or reducing toxicity of TNF and other lymphokines.

Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro.

The interaction in vitro between rat peritoneal macrophages and homologous, sialidase-treated lymphocytes was investigated. Lymphocytes were isolated from blood, thymus, and spleen on a density gradient. Total sialic acids obtained by acid hydrolysis were 10 nmol/10(8) lymphocytes, composed of 29% N-acetyl-neuraminic acid and 71% N-glycoloylneuraminic acid. Sialidase treatment released maximally 33% of membrane sialic acids. Lymphocytes were bound to peritoneal macrophages to an extent which increased in parallel with the amount of sialic acids released, whereas binding of untreated lymphocytes was not significant. This interaction was inhibited by free galactose and substances containing terminal galactose residues. Asialoorosomucoid with its oligoantennary sugar chains proved to be a 10(5) times more potent inhibitor of the interaction than lactose. The addition of homologous serum had no influence on binding. Electron microscopy revealed that vital lymphocytes were tightly bound to macrophages and only damaged lymphocytes appeared to be phagocytozed. The experiments demonstrate that the interaction between rat peritoneal macrophages and sialidase-treated lymphocytes is mediated by a macrophage receptor specific for galactose. This sugar is demasked on the surface of lymphocytes after the removal of terminal sialic acids. The role of this mechanism in cell recognition, elimination and homing of lymphocytes is discussed.

Changes in (Na+ + K+) ATPase activity and the composition of surface carbohydrates in erythrocyte membranes in alcoholics.

(Na+ + K+)ATPase activity and sensitivity to the inhibitory effect of ethanol and noradrenaline in vitro as well as the concentrations of sialic acid, galactose, and hexosamine were determined in erythrocyte membranes in 20 healthy controls and in 20 alcoholic patients within 24 hr of withdrawal. Basal (Na+ + K+)ATPase activity, the sensitivity of the enzyme to inhibition by ethanol and noradrenaline added in vitro, and the concentrations of sialic acid and galactose were significantly reduced in the patients (p less than 0.0005). All of these abnormalities were significantly correlated to each other as well as to the estimated daily quantity of ethanol consumed. After enzymatic removal of terminal sialic acid and terminal and sialic acid-bound beta-galactose, the (Na+ + K+)ATPase differences between the patients and the controls were eliminated. The results showed that the previously reported reduced inhibition of (Na+ + K+)ATPase by ethanol in the presence of noradrenaline in brain membranes in chronically ethanol-fed animals is also found in erythrocyte membranes in alcoholic humans. Abnormal carbohydrate composition of glycolipids and/or glycoproteins of the membrane surface appeared to be related to the (Na+ + K+)ATPase changes, possibly due to interference either with K+ transport or surface electrostatics or, directly or indirectly, with the conformation of (Na+ + K+)ATPase.

Sialidase-enhanced lectin-like mechanism for Actinomyces viscosus and Actinomyces naeslundii hemagglutination.

Laboratory strains representing six numerical taxonomy clusters and fresh isolates of human Actinomyces viscosus and Actinomyces naeslundii were studied by standard flocculation slide tests for the ability to hemagglutinate erythrocytes (RBC) from various animal species. Human AB and horse RBC were agglutinated more frequently and rapidly than others; guinea pig RBC were agglutinated by only a few strains. Human AB RBC were selected for studies of hemagglutination mechanisms. Treatment of RBC with clostridial neuraminidase (NTRBC) greatly enhanced hemagglutination for almost all strains. In hapten inhibition experiments in which various concentrations of sugars were used, beta-galactosides were the most effective inhibitors of hemagglutination for both RBC and NTRBC; inhibition of NTRBC agglutination required higher concentrations. Soybean lectin agglutinated both RBC and NTRBC but not Actinomyces cells. NTRBC agglutinated at a 125-fold-lower concentration. Hemagglutination was sensitive to ethylenediaminetetraacetate for one strain tested. Hemagglutination reactions were reversible by addition of beta-galactosides. The ability of Actinomyces strains to "prime" RBC for hemagglutination by removing sialic acid to expose more penultimate beta-galactoside sites was studied by recycling Actinomyces-agglutinated RBC which were dispersed with a lactose solution and washed free of bacteria (primed RBC). Priming in this manner augmented subsequent hemagglutination by indicator Actinomyces strains and made the RBC more sensitive to agglutination by soybean lectin. The priming ability of Actinomyces strains generally correlated with the amount of sialic acid removed from primed RBC. Strains representing the numerical taxonomy clusters differed in both their hemagglutinating and priming activities. Cluster 5 strains (typical A. naeslundii) were good agglutinators of RBC, NTRBC, and primed RBC but were poor primers. Cluster 3 strains (atypical A. naeslundii) were the weakest hemagglutinators but could prime RBC adequately for subsequent agglutination by other strains. Together, these data indicate that Actinomyces hemagglutination proceeds via a two-step mechanism: (i) neuraminidase removal of terminal sialic acid and (ii) lectin-like binding to exposed beta-galactoside-associated sites on the RBC. Strains differ in the extent to which they can perform the two functions, and this specificity may relate to their taxonomic classification.

Alkaline phosphatase of mouse teratoma stem cells: Immunochemical and structural evidence for its identity as a somatic gene product

The immunochemical and structural characteristics of the alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from mouse teratoma stem cells derived from the OTT-6050 teratoma (ascitic and solid tumors and the F9 and PCC4 cell lines) have been compared to those of the alkaline phosphatases expressed in normal mouse placenta and several adult organs. Crossreactivity of the stem cell alkaline phosphatase with antisera reacting with placental, kidney, liver, and brain alkaline phosphatases indicated that the stem cell enzyme had common antigenic determinants. Structural studies utilizing two-dimensional electrophoresis of the (32)P-labeled alkaline phosphatase subunits showed that the stem cell, placental, and kidney alkaline phosphatases differed only in their sialic acid content and comigrated after removal of terminal sialic acid by neuraminidase digestion. Furthermore, one-dimensional peptide mapping of partial proteolysis fragments from (32)P-labeled enzymes demonstrated identical fragmentation patterns for the stem cell and somatic enzymes. These immunochemical and structural data indicate that the stem cell alkaline phosphatase is the same core enzyme as that produced in the mouse placenta and kidney, with different amounts of terminal sialic acid. The one mouse alkaline phosphatase examined that differed from the other enzymes was the intestinal alkaline phosphatase. This isoenzyme was not immunochemically crossreactive with the other alkaline phosphatases, did not comigrate in two-dimensional electrophoresis after neuraminidase digestion, and did not give identical peptide maps after partial proteolysis.

Aglycosylantibody. Effects of exoglycosidase treatments on autochthonous antibody survival time in the circulation.

Rabbit anti-hapten antibodies were purified by affinity chromatography and characterized immunochemical for in vivo studies of their blood clearance rate and organ distribution after treatment with various glycosidases. Following sequential removal of sialic acid, galactose, and N-acetylglucosamine with the appropriate cellulose-immobilized exoglycosidases, the antibody populations were recharacterized, radiolabeled, and introduced intravenously into the original animals. Using double radioiodine lables it was possible to demonstrate alterations in purified antibody survival times in the circulation and altered organ distribution after glycolytic cleavage. Removal of terminal sialic acid resulted in rapid blood clearance and enhanced localization of asialoantibody in the liver. Subsequent removal of penultimate galactose residues returned both antibody survival time in the circulation and organ distribution to near normal. Removal of subpenultimate N-acetylglucosamine moieties resulted in aglycosylantibody survival values which were intermediate between asialo- and asialoagalactoantibodies. Removal of the three saccharide also increased kidney localization. The results are evaluated based on current concepts of the biological roles of protein-linked carbohydrate and plasma glycoprotein survival time in the circulation.