O-acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-O-acetylated sialic acids

We have previously described the preparation and use of 9-O-[acetyl-3H]acetyl-N-acetylneuraminic acid to identify sialic acid O-acetylesterases in tissues and cells (Higa, H. H., Diaz, S., and Varki, A. (1987) Biochem. Biophys. Res. Commun. 144, 1099-1108). All tissues of the adult rat showed these activities, with the exception of plasma. Rat liver contained two major sialic acid esterases: a cytosolic nonglycosylated enzyme and a membrane-associated glycosylated enzyme. The two enzymes were found in similar proportions and specific activities in a buffer extract of rat liver acetone powder. By using the latter as a source, the two enzymes were separated, and the glycosylated enzyme was purified to apparent homogeneity by multiple steps, including ConA-Sepharose affinity chromatography and Procion Red-agarose chromatography (yield, 13%; fold purification, approximately 3000). The homogeneous enzyme is a 61.5-kDa disulfide-linked heterodimeric protein, whose serine active site can be labeled with [3H]diisopropyl fluorophosphate. Upon reduction, two subunits of 36 kDa and 30 kDa are generated, and the 30-kDa subunit carries the [3H]diisopropyl fluorophosphate label. The protein has N-linked oligosaccharides that are cleaved by Peptide N-glycosidase F. These chains are cleaved to a much lesser extent by endo-beta-N-acetylglycosaminidase H, indicating that they are mainly complex-type glycans. The enzyme activity has a broad pH optimum range between 6 and 7.5, has no divalent cation requirements, is unaffected by reduction, and is inhibited by the serine active site inhibitors, diisopropyl fluorophosphate (DFP) and diethyl-p-nitrophenyl phosphate (Paraoxon). Kinetic studies with various substrates show that the enzyme is specific for sialic acids and selectively cleaves acetyl groups in the 9-position. It shows little activity against a variety of other natural compounds bearing O-acetyl esters. It appears to deacetylate di-O-acetyl- and tri-O-acetyl-N-acetylneuraminic acids by first cleaving the O-acetyl ester at the 9-position. The 7- and 8-O-acetyl esters then undergo spontaneous migration to the 9-position, where they can be cleaved, resulting in the production of N-acetylneuraminic acid. In view of its interesting substrate specificity, complex N-linked glycan structure, and neutral pH optimum, it is suggested that this enzyme is involved in the regulation of O-acetylation in membrane-bound sialic acids.     

O-acetylation and de-O-acetylation of sialic acids. 7- and 9-o-acetylation of alpha 2,6-linked sialic acids on endogenous N-linked glycans in rat liver Golgi vesicles

We have previously shown that radioactivity from [acetyl-3H]AcCoA is concentrated into isolated intact rat liver Golgi vesicles. The incorporated radioactivity occurred in acid-soluble and acid-insoluble components, and the acid-insoluble fraction included O-acetylated sialic acids (Varki, A., and Diaz, S. (1985) J. Biol. Chem. 260, 6600-6608). Nearly all of the protein-associated radioactivity was found to be in sialic acids alpha 2-6-linked to N-linked oligosaccharides on endogenous glycoproteins. Incubation of the vesicles with CMP-[3H]sialic acid resulted in labeling of a very similar group of glycoproteins. The 3H-O-acetyl groups were found at both the 7- and the 9-positions of N-acetylneuraminic acid residues at the end of the labeling reaction. Although 7-O-acetyl groups can undergo migration to the 9-position under physiological conditions, kinetic studies using O-acetyl-14C-labeled internal and O-acetyl-3H-labeled external standards indicate that during the labeling, release, and purification, negligible migration occurred. Studies with mild periodate oxidation provided further confirmation that O-acetyl esters are added directly to both the 7- and the 9-positions of the sialic acids in this system. The acid-soluble, low molecular weight component is released from the vesicles by increasing concentrations of saponin, and its exit parallels that of CMP-[14C]sialic acid taken up during the incubation. The vesicles themselves are impermeant to free acetate. However, even after short incubations, this saponin-releasable radioactivity was almost exclusively in [3H] acetate and not in [3H]acetyl-CoA. The apparent Km for accumulation of the [3H]acetate is almost identical with that for the generation of the acid-insoluble O-acetylated sialic acids. Most of this accumulation of free acetate is also blocked by coenzyme A-SH. Only a small portion arises from the action of an endogenous esterase on the 3H-O-acetylated sialic acids. Taken together, the results indicate that accumulation of free [3H]acetate occurs within the lumen of the vesicles in parallel with O-acetylation of sialic acids and is probably a product of abortive acetylation. It is not known if this reaction occurs in vivo. Permeabilization of Golgi vesicles to low molecular weight molecules with saponin does not alter the rate of acetylation substantially. Furthermore, double label studies suggest that the intact acetyl-CoA molecule does not gain access to the lumen of the vesicles. These results indicate that the acetylation reaction may have a different mechanism from previously described Golgi glycosylation reactions, wherein specific transporters concentrate sugar nucleotides for use by luminally oriented transferases.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

O-acetylation and de-O-acetylation of sialic acids. Sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues

We and others have recently described 9-O-acetyl-sialic acid esterase (9-O-Ac-SA esterase) activities that appear to be specific for removal of O-acetyl esters from the 9-position of naturally occurring sialic acids. We have now examined a variety of species for such enzymes and found them in vertebrates and higher invertebrates, but not in plants or in lower invertebrates. This evolutionary distribution correlates well with that of the sialic acids themselves. All of the 9-O-Ac-SA esterase activities tested were inhibited by diisopropyl fluorophosphate (DFP) in a dose-dependent fashion. This indicates that each of these enzymes has a serine active site similar to the well known serine esterases and serine proteases. Methyl esterification of the carboxyl group of 9-O-acetyl-N-acetylneuraminic acid significantly reduced the activity of all of the 9-O-Ac-SA esterases against the O-acetyl group. This indicates that each of these enzymes may recognize the negatively charged carboxyl group of the sialic acid. Enzymes that recognize anionic substrates frequently have an essential arginine residue (Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886). We therefore studied the effects of the arginine-specific modifying reagents 2,3-butanedione and phenylglyoxal on 9-O-Ac-SA esterase activities from influenza C virus, human erythrocytes, rat liver, starfish gonads, and sea bass brain. All of these enzymes were inhibited in a dose-dependent fashion by both reagents, under conditions previously known to avoid nonspecific modification. In contrast, the typical serine proteases trypsin and kallikrein and the serine esterase acetylcholinesterase were not significantly affected, even by the highest concentrations of these reagents used. These data indicate that five 9-O-Ac-SA esterase activities from evolutionarily distinct origins all have serine active sites and essential arginine residues. We postulate that the arginine residue is involved in substrate recognition via the negatively charged carboxyl group of the sialic acids. Thus, these 9-O-Ac-SA esterase activities may be members of a previously undescribed class of serine esterase.     

Induction of sialic acid 9-O-acetylation by diverse gene products: implications for the expression cloning of sialic acid O- acetyltransferases

Sialic acids can be modified by O-acetyl esters at the 7- and/or 9- position, altering recognition by antibodies, lectins and viruses. 9(7)- O-acetylation is mediated by a sialic acid-specific O- acetyltransferase, which has proven difficult to purify. Two groups have recently isolated cDNAs possibly encoding this enzyme, by expression cloning of human melanoma libraries in COS cells expressing the substrate ganglioside GD3. Pursuing a similar approach, we have isolated additional clones that can induce 9-O-acetylation. One clone present in a melanoma library encodes a fusion protein between a bacterial tetracycline resistance gene repressor and a sequence reported to be part of the P3 plasmid. Expression of the open reading frame is necessary for inducing 9-O-acetylation, indicating that this is not a reaction to the introduction of bacterial DNA. Another clone from a rat liver cDNA library induced 9-O-acetylation on COS cells expressing alpha2-6-linked sialic acids, and encodes an open reading frame identical to the Vitamin D binding protein. However, truncation at the 5' end eliminates the amino-terminal hydrophobic signal sequence, predicting cytosolic hyperexpression of a truncated protein. Thus, diverse types of cDNAs can indirectly induce sialic acid 9-O- acetylation in the COS cell system, raising the possibility that the real enzyme may be composed of multiple subunits which would not be amenable to expression cloning. Importantly, the cDNAs we isolated are highly specific in their ability to induce 9-O-acetylation either on alpha2-6-linked sialic acids of glycoproteins (truncated vitamin D binding protein) or on the alpha2-8-linked sialic acids of gangliosides (Tetrfusion protein). These data confirm our prior suggestion that a family of O-acetyltransferases with distinctive substrate specificities exists in mammalian systems.      

Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus

An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the cDNA encoding a murine sialic acid-specific 9-O-acetylesterase and RNA expression in cells of hematopoietic and non-hematopoietic origin.

We describe the isolation of a cDNA encoding a murine sialic acid-specific 9-O-acetylesterase as well as its expression pattern in cells of both hematopoietic and non-hematopoietic origin. This enzyme catalyzes the removal of O-acetyl ester groups from position 9 of the parent sialic acid N-acetylneuraminic acid. The cDNA is 2105 nt in length and encodes a protein of 541 amino acids with a predicted molecular weight of 61 kDa, not including oligosaccharides linked to eight potential N-glycosylation sites. The cDNA encoding the acetylesterase displays a widespread distribution in various cell lines and tissues. Expression studies of B lineage cell lines and primary fetal liver cells revealed a developmentally regulated expression pattern in cells of hematopoietic origin. Given the importance of 9-O-acetylation of sialic acids, the cloning of the cDNA encoding a sialic acid-specific 9-O-acetylesterase will be helpful in understanding further the regulation of this post-translational modification and the biological consequences thereof.     

O-acetylation and de-O-acetylation of sialic acids in human colorectal carcinoma.

A decrease in the level of O-acetylated sialic acids observed in colorectal carcinoma may lead to an increase in the expression of sialyl Lewis(X), a tumor-associated antigen, which is related to progression of colorectal cancer to metastasis. The underlying mechanism for this reduction is, however, not fully understood. Two enzymes are thought to be primarily responsible for the turnover of O-acetyl ester groups on sialic acids; sialate-O-acetyltransferase (OAT) and sialate-O-acetylesterase (OAE). We have previously reported the characterization of OAT activity from normal colon mucosa, which efficiently O-acetylates CMP-Neu5Ac exclusively in the Golgi apparatus prior to the action of sialyltransferase. In this report we describe the identification of a lysosomal and a cytosolic OAE activity in human colonic mucosa that specifically hydrolyses 9-O-acetyl groups on sialic acid. Utilizing matched resection margin and cancer tissue from colorectal carcinoma patients we provide strong evidence suggesting that the level of O-acetylated sialic acids present in normal and diseased human colon may be dependent on the relative activities of OAT to lysosomal OAE. Furthermore, we show that the level of free cytosolic Neu5,9Ac2 in human colon is regulated by the relative activity of the cytosolic OAE.     

Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Application to sialic acid assay in analytical de-O-acetylation.

With dimethyl sulphoxide instead of butanol in the thiobarbituric acid assay for sialic acid, a non-fading chromophore with lambdamax. = 549 nm was produced in a homogeneous solution, allowing dilution of the test mixture in case of high colour yield. This test adapted well to studies on alkaline de-O-acetylation. Bovine and rat submaxillary mucins, and rabbit Tamm-Horsfall urinary sialoproteins contain O-acetyl isomers of neuramine acid that are resistant to the thiobarbituric acid assay. Alkaline de-O-acetylation converted resistant O-acetylneuraminic acid into thiobarbituric acid-reactive sialic acid, and such conversion paralleled de-O-acetylation as measured by the ferric hydroxamate method. The colour increment was similar when the alkaline treatment of bovine submaxillary mucin either preceded or followed the acid hydrolysis. Only alkaline preptreatment was effective with rat submaxillary mucin. By selecting optimal conditions for alkaline de-O-acetylation, O-acetyl isomers can be accurately assessed by the thiobarbituric acid assay.     

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.     

A comparison of acetylation in vitro of microsomal, homogenate, and Golgi fractions of rat liver

The activity of acetyltransferase was detected in the microsomal fraction of rat liver by incubation with [3H]acetyl-CoA and by analyses using sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Endogenous membrane proteins of relatively high molecular weight were found to serve as substrates. Optimal conditions for assay of the enzyme were defined. A deacetylase activity was also detected, which was inhibited by 2 mM ethylenediaminetetraacetic acid. Further subfractionation disclosed that the acetyltransferase activity was most enriched in the Golgi fraction, in which its specific activity was some ninefold greater than in the total homogenate. The radioactive labelling of Golgi-associated proteins observed was relatively intense, exceeding that of histone and ribosomal proteins in the homogenate. Analysis of the acetylated Golgi fraction by two-dimensional electrophoresis revealed approximately 90 radioactive polypeptides. Various treatments demonstrated that a minimum of 80% of the incorporated radioactivity was present as derivatives of N-acetylneuraminic acid, principally N-acetyl-9-mono-O-acetylneuraminic acid (Neu5,9Ac2). The sialic acid O-acetyltransferase activity detected is thus probably identical to that reported by Varki and Diaz; the intense labelling of proteins reflects the ability of Golgi apparatus fractions to take up and concentrate acetyl-CoA. Protein-bound radioactive Neu5,9Ac2 was also detected in the medium of hepatocytes incubated with N-[3H]acetylmannosamine, demonstrating that these cells synthesize certain proteins containing acetylated sialic acids, some of which may be secreted. The data confirm that the Golgi apparatus is a major site of acetylation of protein-bound sialic acids in rat liver in vitro and provide new information showing that many glycoproteins undergo this particular type of modification.     

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.     

Characterization of a carbohydrate epitope defined by the monoclonal antibody H185: sialic acid O-acetylation on epithelial cell-surface mucins

Sialic acids comprise a large family of derivatives of neuraminic acid containing methyl, acetyl, sulfate, and phosphate among other groups, which confer specific physicochemical properties (e.g., hydrophobicity and resistance to hydrolases) to the molecules carrying them. Several years ago, a monoclonal antibody, designated H185, was developed, which binds to cell membranes of human corneal, conjunctival, laryngeal, and vaginal epithelia and whose distribution is altered on the ocular surface of patients with keratinizing disease. Recent findings using immunoprecipitation and immunodepletion techniques have demonstrated that, in human corneal epithelial cells, the H185 antigen is carried by the membrane-associated mucin MUC16. In this study, we show that the H185 epitope on human corneal cells and in tear fluid is an O-acetylated sialic acid epitope that can be selectively hydrolyzed in an enzyme-concentration-dependent manner by sialidase from Arthrobacter ureafaciens and to a lesser extent by sialidases from Newcastle disease virus, Clostridium perfringens, and Streptococcus pneumoniae. Binding of the H185 antibody was impaired by treatment of tear fluid with a recombinant 9-O-acetylesterase from influenza C virus. Two O-acetyl derivatives, Neu5,7Ac(2) and Neu5,9Ac(2), were identified in human tear fluid by fluorometric high-performance liquid chromatography (HPLC) and electrospray mass spectrometry (MS). Immunoprecipitation of the H185 epitope from human corneal epithelial cells revealed that Neu5,9Ac(2) was the major derivative on the mucin isolate. These results indicate that exposed wet-surfaced epithelia are decorated with O-acetyl sialic acid derivatives on membrane-associated mucins and suggest that O-acetylation on cell surfaces may protect against pathogen infection by preventing degradation of membrane-associated mucins.     

Role of linkage specific 9-O-acetylated sialoglycoconjugates in activation of the alternate complement pathway in mammalian erythrocytes

Substitution of the -OH group at C-9 of sialic acid by an O-acetyl ester has been suggested to modify various biological phenomena that are regulated by sialic acids. Amongst them, enhancement of erythrocyte lysis by 9-O-acetylated sialic acid determinants through modulation of the alternate pathway of complement has been extensively studied on murine erythrocytes [1]. A variable expression of linkage specific 9-O-acetylated sialoglycoconjugates as defined by the lectinogenic epitope of Achatinin-H namely 9-O-acetylated sialic acid 26Gal NAc was identified on rabbit, guinea pig, hamster, rat, mouse and human erythrocytes. This differential expression of linkage specific 9-O-acetylated sialoglycoconjugates strongly correlated with the susceptibility of mammalian erythrocytes to lysis by the alternate pathway of complement. Additionally, low levels of antibodies directed against O-acetylated sialic acids in these mammalian species suggested that these constitutively present determinants have low immunogenicity. Taken together, our results indicate that complement mediated hemolysis depends not simply upon the extent of surface 9-O-acetylated sialic acids present but more importantly upon the specific linkage.     

Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase.

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.     

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.     

The transport and utilization of acetyl coenzyme A by rat liver Golgi vesicles. O-acetylated sialic acids are a major product

When intact rat liver Golgi vesicles were incubated with [acetyl-3H]acetyl coenzyme A, radioactivity was incorporated into the vesicles in a manner dependent upon temperature, time, protein, and acetyl-CoA concentration. The vesicles concentrated the label 121-fold relative to the medium within 20 min, suggesting an active transport mechanism operating in intact vesicles, and incorporated more than 50% of this label into acid-insoluble materials. This was supported by the finding that incorporation was markedly reduced by Triton X-100 at levels above its critical micellar concentration. While the intravesicular low molecular weight fraction was predominantly free acetate, acetate ions themselves were not permeant to the vesicles. Double-label experiments suggested that the transport process involved the entire acetyl-CoA molecule. This was further supported by the fact that coenzyme ASH, palmitoyl-CoA and butyryl-CoA were markedly inhibitory. Incorporation was optimal at 22 degrees C at pH 7.0, and was moderately stimulated by ATP. However, compounds known to abolish proton gradients or to inhibit the Golgi proton pump had no effect. The apparent Km for the utilization process was 0.61 microM with a Vmax of 21.3 pmol/mg of protein/min. Oligomycin and 4,4'-diisothiocyanostilbene-2,2'disulfonic acid were inhibitory, whereas CMP-NeuAc, UDP-GlcNAc, adenosine 3'-phosphate, 5'-phosphosulfate, atractylosides, tunicamycin, 2'5'-ADP, and 3',5'-ADP were not, showing that this transport process is distinct from other nucleotide transporters previously described in rat liver Golgi. 75-85% of the radioactivity incorporated was shown to be in O-acetylated sialic acids, by neuraminidase release, purification, and high pressure liquid chromatography. The majority of the neuraminidase-resistant radioactivity was released by alkaline hydroxylamine as [3H]acetylhydroxamate, but a significant fraction was resistant to this treatment. The nature of the non-sialic acid radioactivity remains unknown. The existence of this transport mechanism provides yet another level at which the O-acetylation of sialic acids could be regulated.     

Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group-specific transport system for acidic monosaccharides

Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.     

Synthesis of N-acetylneuraminic acid and of CMP-N-acetylneuraminic acid in the rat liver cell.

Adult male rats, under starving and normal conditions, were injected intravenously with N-acetyl[3H]mannosamine and after various time intervals the specific radioactivities of free N-acetylneuraminic acid (NeuAc) and CMP-N-acetylneuraminic acid were determined in the liver. The specific radioactivity of free NeuAc was high even within 20s after injection; the maximum was reached between 7 and 10 min. The specific radioactivity of CMP-NeuAc showed a lag phase of approx. 1 min. Thereafter it increased quickly and rose above the specific radioactivity of free NeuAc, reaching a maximum about 20 min after injection. These results point to a channelling of the newly synthesized NeuAc molecules into a special compartment, from which they are preferentially used by the enzyme CMP-sialic acid synthetase. It is suggested that the cytosolic enzyme N-acetylneuraminic acid 9-phosphate phosphatase is working in concert with the nuclear localized enzyme CMP-N-acetylneuraminic acid synthetase. Incorporation of radioactive sialic acid into sialoglycoproteins in liver occurred 2 min after injection, and after 10 min bound radioactivity began to appear in the circulation, indicating a transport time of 8 min of sialoglycoproteins from the point of attachment of sialic acid to the point of excretion.