Turnover of Free Sialic Acid, CMP-Sialic Acid, and Bound Sialic Acid In Rat Brain

Abstract: Adult male rats were injected intraventricularly with N-[³H]acetylmannosamine. After different time intervals the rats were killed and free sialic acid, CMP-sialic acid, lipid- and protein-bound sialic acid were isolated from brain and the specific radioactivities determined. Maximal specific radioactivity was reached after approximately 4 h for CMP-sialic acid, after 10–12 h for free sialic acid and after approximately 42 h for lipid-and protein-bound sialic acid. After some days the specific radioactivities of all four pools were the same and decreased equally, with a calculated turnover rate of approximately 3.5 weeks. The conclusion was that this phenomenon was the result of reutilisation of sialic acid and/or precursors. Therefore, the calculated turnover is not the turnover of bound sialic acid, but merely the rate of leakage of sialic acid and/or precursors out of the brain, so that no real turnover can be measured by this method. The first few hours after injection the specific radioactivity of CMP-sialic acid rose above that of free sialic acid. It is supposed that a compartmentalization exists of free sialic acid. The newly synthesized sialic acid molecules are not secreted into the cytoplasmic pool but are preferentially used for the synthesis of CMP-sialic acid. The results and conclusions are discussed in view of the general problems concerning turnover measurements of glycoconjugates.     

The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.     

Author index for volume 85

A colorimetric assay of sialic acid has been developed in which bound sialic acid is oxidized by periodic acid so as to quantitatively release formaldehyde. In the second step of the reaction formaldehyde reacts with methyl-3-benzothiazolinone-2-hydrazone, giving a colored compound. This method does not require a prior hydrolysis and presents the advantage of great accuracy.     

Usefulness and potential pitfalls of sialic acid determination in sera of patients with ovarian tumors

Increasing evidence in the literature indicates that serum sialic acid is increased in cancer patients suggesting a possible usefulness of its determination as a tumor marker. However there are many discrepancies in the data reported, probably due to methodological errors, mainly in lipid bound sialic measurement. In this paper we illustrate the results obtained when we applied a method worked out in our laboratory for the determination of total and fractionated sialic acid (lipid and protein bound) to the analysis of sera from patients with ovarian tumors and the preliminary data on the follow up of selected cases. The potential pitfalls in using this relatively new tumor marker will be critically evaluated.     

Specific Staining of Sialic Acid Components on Sodium Dodecyl Sulfate Polyacrylamide Gel

Specific staining of sialic acid components after sodium dodecyl sulfate polyacrylamide gel electrophoresis can be carried out as follows: 1) extract glycoprotein of erythrocyte membranes or serum by the phenol-saline method, 2) electrophorese the extract on 5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate at constant current, 3) treat the gel with chilled 0.04 M HIO₄ for 45 minutes, 4) replace the periodic acid solution with one containing resorcinol 0.6 g, cone. HCl 50 ml, 0.1 M CuSO₄ 0.5 ml and H₂O 50 ml, 5) warm the container in boiling water until blue violet sialic acid bands become clear, 6) replace the staining solution with a mixture of equal parts water and concentrated HCl and observe at once.     

Glycosidically bound sialic acid levels as a predictive marker of postoperative adjuvant therapy in gastric cancer

Summary A group of 293 gastric cancer patients were examined to see if the preoperative value of glycosidically bound sialic acid is a predictor of prognosis and effectiveness of postoperative adjuvant therapy. All patients had gastrectomies and were histologically confirmed to have primary adenocarcinoma of the stomach. Some patients then received either postoperative adjuvant chemotherapy or immunochemotherapy. Patients with sialic acid levels less than 74.5 mg/dl survived significantly longer than those with sialic acid levels of 74.5 mg/dl or of 85.3 mg/dl and over. No significant differences in survival were found among patients treated by gastrectomy alone, gastrectomy plus chemotherapy and gastrectomy plus immunochemotherapy. However, patients with abnormally elevated levels of sialic acid survived significantly longer when they were treated with immunochemotherapy after gastrectomy than those treated by gastrectomy alone or with chemotherapy after gastrectomy. By using Cox's multivariate regression model, pTNM stages, postoperative adjuvant therapy (chemotherapy and immunochemotherapy) and preoperative serum levels of sialic acid were examined as prognostic variables. Postoperative therapy was a significant prognostic variable in patients with abnormally elevated levels of sialic acid. The preoperative serum level of sialic acid is a promising predictive marker of the response to postoperative adjuvant immunochemotherapy.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase)

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the sialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens$\text{N-}\text{acetylneuraminate}$ glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspension medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca²⁺ in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that a restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

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.     

Sialic acid metabolism in rat liver: effect of carbon tetrachloride

Sialic acid metabolism was investigated in the livers of control rats and of rats treated with a single oral dose (1.5 ml/kg body weight) of carbon tetrachloride. The main change observed during the necrotic stage of CCl4 poisoning (18 h after treatment) was a highly significant reduction in sialyltransferase activity. Slight reciprocal changes in neuraminidase activities, i.e., a small decrease in cytosolic neuraminidase and a small increase in the membrane bound enzyme were also observed. At 72 h after CCl4 treatment, during the stage of liver regeneration, the main change was a marked elevation in membrane-bound neuraminidase (two fold above control values). Moderate increases in the specific activities of CMP-N-acetylneuraminic acid synthetase and sialyltransferase were also observed. A considerable decrease in the sialic acid content of the isolated smooth endoplasmic reticulum (one half of control values) was detected at 72 h after CCl4 administration. The sialic acid content of the rough endoplasmic reticulum, on the other hand, remained at control levels.     

Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase).

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the dialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens N-acetylneuraminate glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspenion medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca (2+) in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

Chemical determination of cationized ferritin bound to synaptosomes

Surface charge of synaptosomes was studied with cationized ferritin (CF) binding at pH between 4 and 7. The characteristic of binding resembles that of the electrophoretic mobility and suggests an electrostatic interaction between CF and membranes. When sialic acid was partially removed from synaptosomes, a reduction in binding was detected. This suggests that sialic acid is one determinant of surface charge of synaptosomes. The present method differs from other CF studies in that the amount of bound CF was measured chemically instead of by electron microscopy. This method could be useful in other cell systems.     

A simple and reliable method for the detection of sialylation in complex/hybrid-type carbohydrate chains of glycoproteins by mixed lectins

A practical and convenient method for discriminating between the presence and the absence of sialic acid in carbohydrate chains of glycoproteins was devised using paramagnetic beads and two lectins, Sambucus sieboldiana lectin (SSA) and Ricinus communis agglutinin (RCA120). The glycoproteins of transferrin or thyroglobulin were firstly captured to paramagnetic beads that were previously coated with corresponding antibody, and then the lectins of RCA120-biotin and SSA-FITC were bound to the glycoproteins on the paramagnetic beads. Finally, the fluorescence intensity of the beads was measured to determine the ratios of lectins RCA120-biotin/Cy5-streptavidin to SSA-FITC. The mixed lectins method showed bigger difference of the ratios between the presence and the absence of sialic acid, indicating higher discrimination efficiency than the ordinary non-mixed lectins method. Furthermore, statistical analysis by two-side t-test indicated that the mixed lectins method was more highly reliable than the ordinary non-mixed lectins method in discriminating between the presence and the absence of sialic acid. The reaction with the two lectins can be performed in a single tube and readily automated taking advantage of the use of paramagnetic beads.     

Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ([1-14C]N-acetylmannosamine) and a radioactive precursor of ceramide ([3,3-3H2]serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.     

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.     

Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.     

Sialic acid metabolism in sialuria fibroblasts

Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid (N-acetylneuraminic acid, NeuAc). Fibroblasts cultured from the three known cases of sialuria contained 70-200-fold increases in soluble sialic acid, but normal concentrations of bound sialic acid. The sialic acid appeared in the cytosolic fraction of the cells on differential centrifugation, and was susceptible to borohydride reduction, suggesting that accumulated sialic acid was in the form of NeuAc and not CMP-NeuAc. In biochemical studies, CMP-NeuAc (50 microM) inhibited the UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase of normal fibroblasts by 84-100%, but inhibited the epimerase from sialuria cells by only 19-31%. Feeding sialuria cells up to 5 mM D-glucosamine for 72 h increased free sialic acid content 20-60%, but normal cells were unaffected by this treatment. Cytidine feeding (5 mM, 72 h) reduced the NeuAc content of sialuria cells, initially 112, 104, and 266 nmol/mg protein, by 63-71 nmol/mg protein; CMP-NeuAc concentrations, initially 4, 2, and 5 nmol/mg protein, increased by 14-33 nmol/mg protein. Consequently, the total cellular content of soluble sialic acid (NeuAc + CMP-NeuAc) was lowered 14-46% by cytidine feeding. The inheritance pattern of sialuria has not been determined. However, cells from both parents of one sialuria patient contained normal concentrations of free sialic acid, and the parental epimerase activity also responded normally to CMP-NeuAc. We conclude that the basic biochemical defect in all known cases of sialuria is a failure of CMP-NeuAc to feedback-inhibit UDP-GlcNAc 2-epimerase and cytidine feeding can lower the intracellular soluble sialic acid concentration of sialuria cells.     

Sialyltransferase, sialic acid and sialoglycoconjugates in metastatic tumor and human liver tissue

1. The specific activities of sialytransferase in metastatic tumor sites were 5-37% of those of uninvolved non-cancerous tissue. The non-cancerous host tissues had a slightly lower average sialyltransferase activity than that of non-pathological control livers (986 vs 1194 dpm/min/mg protein). 2. The levels of total and bound sialic acid are increased 1.4 to 7.2-fold in homogenates, supernatants and resuspended pellets of metastatic tumor sites compared to non-cancerous and non-pathological control livers. For all these tissues, 24-29% of the bound sialic acid is found on soluble components (in the 16,300 g supernatant). 3. Soluble sialoglycoconjugates from most metastatic tumor sites give gel filtration profiles different from those of non-cancerous and non-pathological control livers.     

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.     

Roles of Exopolysaccharides and Lipopolysaccharides in the Adsorption of the Siphovirus Phage NM8 to Rhizobium meliloti M11S Cells

The exopolysaccharides produced by Rhizobium meliloti M11S inhibited nonspecifically the adsorption of phage NM8 by coating the cells. But lipopolysaccharides (LPS) had a specific inhibitory effect. Only the polysaccharide moiety of LPS, composed of glucose, glucosamine, galactose, 3-deoxy-D-manno-octulosonic acid (KDO), and large amounts of sialic acid, inhibited phage adsorption; neither the lipid A moiety nor a cellular glucan was involved. Rhizobium strains lacking sialic acids did not bind phage NM8. Inhibition of phage binding by lectin specific for N-acetylneuraminic acid demonstrated that phage NM8 bound to sialic acids. Preincubation of the phage with monosaccharides showed that inactivation of phage was very stereospecific for N-acetylneuraminic acid. Phage adsorption was also strongly inhibited by N-acetylglucosamine, which is not present in the LPS. Therefore, the receptor for phage NM8 appears to be a saccharide site, probably involving the acetyl groups of sialic acids. Received: 8 March 1996 / Accepted: 29 June 1996