Sialic Acid Activation

Glycobiology vol. 1 no. 5 pp. 441-447, 1991

MINI REVIEW Sialic acid activation

Edward L.Kean a few instances has the sugar nucleotide actually been measured in animal tissues. Harms et al. (1973) determined the con- Department of Ophthalmology and the Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA centration of CMP-NeuAc in rat liver to be 40.8 nmol/g tissue. The pool size of CMP-NeuAc in Chinese hamster ovary cells Cytidine 5-monophosphosialic acid (CMP-sialic acid) is the was measured by Briles et al. (1977) (1.6 nmol/mg cell activated form of sialic acid which is required for the bio- protein). Corfield et al. (1976) reported the presence of CMP- synthesis of sialic acid-containing complex carbohydrates. NeuAc and CMP-9-O-Ac-NeuAc in bovine submandibular Its discovery over 30 years ago by the laboratory of Dr Saul glands, and provided a partial identification. Carey and Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 Roseman was a landmark in research dealing with the bio- Hirschberg (1979) isolated CMP-NeuAc from mouse liver and synthesis of these compounds. A review is presented of the determined its concentration (37 nmol/g). salient features concerning this molecule: its discovery, The enzymatic synthesis of CMP-NeuAc was reported by chemistry, biosynthesis, subcellular location, enzymatic Roseman (1962) using a preparation from hog submaxillary cleavage, transport and molecular biology. This report does glands, and by Warren and Blacklow (1962a, b) using a pre- not deal with its utilization by the sialyltransferases. paration from Neisseria meningitidis. CMP-sialic acid is synthesized by the following reaction: CTP + sialic acid -• Key words: cytidine 5'-monophosphosialic acid/CMP-sialic CMP-sialic acid + PPi. Unlike the synthesis of other sugar acid/synthetase/hydrolase/subcellular location nucleotides which involves a reaction between a nucleotide triphosphate and a sugar-1-phosphate, the biosynthesis of CMP- sialic acid is unique in using the non-phosphorylated sugar donor (a reaction which also occurs in the formation of CMP- KDO). Subsequently, several laboratories described the syn- Properties of CMP-NeuAc and CMP-NeuAc synthetase thesis of CMP-NeuAc, among which are reports by Shoyab et al. (1964) using a preparation from sheep brain; Kean and In 1959, Comb, Shimizu and Roseman reported the discovery Roseman (1966a, b) using preparations from hog submaxillary of a new sugar nucleotide, CMP-N-acetylneuraminic acid gland and E.coli; van den Eijnden et al. (1972) with a calf brain (CMP-NeuAc)1. The compound was isolated from the cells of preparation; Hultsch et al. (1972), from rat liver; Schauer Escherichia coli K-235, the organism that produces a homo- et al. (1980) on a frog liver preparation. The properties of the polymer of sialic acid, colominic acid. Now, some 30 years compounds synthesized by these sources were similar in terms later, the gene governing its synthesis has been cloned. The of composition, chemical and enzymatic reactivity and stability, intervening years saw the wide demonstration that CMP-sialic and chromatographic properties such as great sensitivity to acid was the activated form of sialic acid required for the bio- acid; relatively stable at neutral or alkaline pH; resistance to the synthesis of sialic acid-containing complex carbohydrates. The actions of 5' nucleotidase, phosphodiesterase, phosphatase, discovery was a key one in furthering our understanding of the neuraminaldolase and neuraminidases; under acid conditions biosynthesis of this class of compounds. Although but a one- they underwent typical colorimetric reactions for sialic acid; page letter to the Editor, this initial report revealed many of the resistance to reduction with NaBHj. In general, the enzymes properties of this unique compound. This preliminary com- synthesizing CMP-NeuAc from all sources examined also had munication was followed by an extensive characterization of similar properties, as summarized in Table I. Unlike the CMP-NeuAc from E.coli K-235 (Comb et al., 1966). The most enzymes from N.meningitidis and E.coli K-235, the prepara- unique characteristic of the compound was the presence of one tions from the animal tissues (submaxillary gland, sheep brain, instead of two phosphate residues as is found in other sugar retina, rat liver) also functioned with N-glycolylneuraminic nucleotides, a property shared with only one other molecule of acid (NeuGc) as substrate. Also, unlike the enzyme from this type, CMP-3-deoxy-D-manno-octulosonate (CMP-KDO; N. meningitidis, the reaction carried out by the hog submaxillary Ghalambor and Heath, 1966), a compound involved in bacterial enzyme was shown to be reversible (Kean and Roseman, cell wall biosynthesis. 1966a, b); the ratio of the forward reaction to the reverse While the biosynthesis of CMP-NeuAc has been readily reaction was estimated to be - 1000:1. Reversibility could not demonstrated in a variety of animal tissues (see below), in only be detected with the enzyme from sheep brain (Shoyab et al., 1964). (This property was not reported in the other tissues.) The possibility that carboxyl activation might be involved in the 'The nomenclature in common use in this field is followed in which the term 'sialic acid' is the family name of the group of nine carbon amino sugar formation of CMP-NeuAc by the mammalian enzyme was acids having an acyl group on the amino nitrogen. Individual sialic acids ruled out in view of the lack of hydroxamate formation during are derivatives of neuraminic acid, such as AZ-acetylneuraminic acid or NeuAc, the reaction (Kean and Roseman, 1966a). whose systematic name is 5-acetamido-3,5-dideoxy-D-glycero-r>-galacto- The anomeric configuration about carbon atom 2 of the sialic nonulosonic acid. acid portion of CMP-NeuAc has been controversial. On the This review is dedicated to Dr Saul Roseman as part of a symposium in his honour at the 1 lth International Symposium on Glycoconjugates, held on June basis of its chemical properties and optical rotation, and 30-July 5, 1991, in Toronto, Canada. assuming that the Hudson isorotation rules applied, the linkage

TaMe I. Comparison of properties of CMP-sialic acid synthetases

Warren and Blacklow Kean and Roseman Shoyab van den Eijnden Kean Van Dijk Schauer (1962a, b) (1966a, b) et al. (1964) et al. (1972) (1970) et al. (1973) et al. (1980)

Form of enzyme Soluble Soluble Soluble Soluble Nuclear Nuclear Soluble Tissue Neisseria meningitidis Hog submaxillary Sheep brain Calf brain Hog retina, Kidney cortex Frog liver rat liver

Km (NeuAc) 0.25 mM 0.8 mM 06 mM 0 90 mM 0.72 mM 1.6 mM 1.6 mM

Km (NeuGc) Inactive 2.3 mM 2.3 mM - 1.40 mM - 2 3 mM

Km (CTP) 0.56 mM 060 mM 1.8 mM 0.50 mM 0.48 mM 1.6 mM 0 6 mM pH optimum 8.5 90 8.0 9.0 8.5 8.5-9.0 9.0 Fold purified 205^03 264-528 39 4.9 - - 250 Sulphydryl requirement Required Stabilizing Stimulatory Present Stabilizing Stabilizing Present Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 Reversibility No Yes No - - - - Stability Freeze/thaw Crude, stable Crude, stable - Stable Unstable Unstable Purified, unstable Purified, unstable Purified, stable 4°C Stable (2 weeks) Stable (3 weeks) Stable Stable Stable (1 week) Stable, 1 week (2 weeks) Metal; optimal Mg; (25 mM) Mg; (20 mM) Mg; (30 mM) Mg; (40 mM) Mg; (20 mM) Mg; (3.0 mM) Mg or Ca; concentration (50 mM)

was suggested to be (3 by Comb et al. (1966). This assignment 1971). The nuclear location of the sialic acid-activating enzyme received support by the study of Haverkamp et al. (1979) who was firmly established by studies using highly purified nuclei examined the anomeric configuration by means of I4C NMR from hog retina, rat liver, kidney, spleen and brain (Kean, spectroscopy. Stone and Kolodny (1971), however, have con- 1970, 1972a). When corrected for the yield of nuclei, from cluded from circular dichroism studies of CMP-NeuAc that the 54 to 90% of the total activity could be ascribed to the nuclei ketosidic linkage is a. Indirect support for the /3 configuration from these several tissues. The kinetic properties and stability may be obtained from the investigations of Yu and Ledeen of the nuclear enzyme were similar to those of the soluble form (1969), who examined the stereochemistry of the two anomeric described previously, as seen in Table I. methyl ketosides of NeuAc. The configuration to which the With the possibility that the nuclear location was due to a-D assignment was made was susceptible to the action of artifactual redistribution of cellular constituents as a result of neuraminidase, while the /3 ketoside was resistant. CMP-NeuAc homogenization, the nuclear location of CMP-NeuAc syn- has been observed to be resistant to the action of neuraminidase, thetase was examined further by employing a procedure to consistent with the /3 assignment. Since the configuration of fractionate the cell that did not involve homogenization. Harvey sialic acid in glycoconjugates occurs only with the a con- (1956) demonstrated that unfertilized sea urchin eggs could be figuration, a single inversion of configuration would, therefore, segmented into nucleate and non-nucleate portions merely by accompany the transfer of sialic acid from the sugar nucleotide centrifugation. Fractions (halves and quarters of the eggs) thus to the acceptor. enriched with nuclei can be obtained by this mild procedure. The distribution of CMP-sialic acid synthetase was followed in the nucleate and anucleate segments, and was observed to accompany the nuclear-enriched fractions (Kean and Bruner, Subcellular location; nuclear CMP-NeuAc synthetase 1971), an observation consistent with the nuclear location of the CMP-NeuAc synthetase examined in each of the reports enzyme. described above was in the form of a solubilized enzyme, The nuclear location of the enzyme was supported by reports suggesting that the enzyme was cytoplasmic in location. How- from several laboratories. CMP-NeuAc synthetase was also ever, subcellular fractionation studies performed on the retina found in high concentration in the nuclear fraction of homo- of the hog revealed high enzymatic activity associated with the genates of rat brain, and remained associated with membrane crude nuclear fraction and also with partially purified nuclei fragments after lysis of the nuclei (Gielen et al., 1971). Van (Kean, 1969). Consistent with these early observations, when Dijk et al. (1973) examined the distribution of the CMP-NeuAc the sialic acid-activating enzyme was measured in various synthetase in calf kidney and found high activity in the nuclei regions of the hog lens, activity was found only in the capsule- purified from several regions of the kidney. The association of epithelial region, the only site in the ocular lens where the enzyme with nuclear membranes was demonstrated by the nucleated cells are found. In retrospect, the nuclear location of retention of most (72%) of the activity by the residual pellet the enzyme was presaged from the results of a tissue survey after treatment with Triton X-100, although the enzyme could carried out previously in which rat erythrocytes were shown to be readily solubilized with 0.1 M salt solutions. The latter ob- be inactive (Kean and Roseman, 1966a). Subsequently, CMP- servation was consistent with that obtained with nuclei purified NeuAc synthetase was found in bovine leucocytes (Gielen from rat liver (Kean, 1970). The ready solubilization of the et al., 1970) and in tonsillar lymphocytes (Kean and Bruner, nuclear activity by KC1 and phosphate buffers strongly suggests

442 Sialk acid activation that the soluble form of the enzyme is brought about by con- showing that the ratios of the specific activities of NeuAc and ditions of isolation. Van den Eijnden (1973) observed that calf CMP-NeuAc were lower in the nucleus than in the whole liver, brain nuclei treated with TX-100, resulting in the removal of contrary to the expectation that the converse would hold if the nuclear membranes and contaminating cytoplasmic material, initial site of synthesis were the nucleus. The suggestion was still retained most of the CMP-NeuAc synthetase activity. The made that the synthetase leaves the nucleus and functions enzyme was thus concluded to be a true nuclear component elsewhere. and not to be present due to artifactual adsorption from the The nuclear location of the activating enzyme has been cytoplasm. observed in every nucleated cell type which has been examined. The subcellular localization of CMP-NeuAc synthetase was The significance of this subcellular locus in the economy of the again examined by Coates et al. (1980). Confirming previous cell, however, remains obscure. observations, the enzyme was shown to be localized in purified nuclei from rat liver. In addition, treatment with TX-100 and CMP-sialic acid hydrolase deoxycholate, which removed inner and outer nuclear membranes and nuclear pores, failed to remove the enzyme. Thus, In addition to the synthesis of CMP-sialic acid, factors that evidence was again provided that the enzyme in the nucleus was regulate its breakdown could modulate the availability of this not due to absorption from the cytoplasm. The nuclear location

compound for use in the biosynthesis of glycoproteins and Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 was given additional support by the observation by Coates et al. gangliosides. The enzymatic cleavage of CMP-NeuAc was first (1980) of the retention of -85% of the activity by nuclei described by Shoyab and Bachhawat (1967a, b) who sub- isolated by non-aqueous techniques from L 929 and BHK cells. sequently partially purified the CMP-NeuAc-degrading enzyme In addition, the soluble fraction which contained the remaining from sheep liver (Shoyab and Bachhawat, 1969). On the basis activity also contained DNA, suggesting that its presence could of subcellular distribution studies, Shoyab and Bachhawat have been due to nuclear contamination. These findings are (1969) concluded that the hydrolase was located in the nuclear strong evidence supporting the almost exclusive nuclear loca- and mitochondrial fractions. A unique type of metabolic control tion of CMP-NeuAc synthetase. Consistent with these in vitro might have been required if both the synthesis and degradation investigations, in vivo studies using enucleated L-cells (cyto- of this sugar nucleotide occurred on the same subcellular plasts) showed 5-fold lower relative ability to synthesize CMP- organelle, the nucleus. When the subcellular location of the NeuAc than did nucleated cells (Coates etal., 1980). An CMP-NeuAc hydrolase was examined in rat liver, however, it examination by van Rinsum et al. (1983) of the subcellular was shown to be an enzyme of the plasma membrane (Kean, localization in rat liver of CMP-NeuAc synthetase and of the 1972b; Kean and Bighouse, 1974); no activity was detectable enzymes involved in the formation of NeuAc clearly demon- in purified nuclei. In addition to differences in subcellular strated that the latter were soluble enzymes with < 2 % located location with the synthetase, many of the properties of the in the nucleus, and affirmed that essentially all of the synthetase synthetase and hydrolase are in sharp contrast to one another, was localized in the nucleus. as seen in Table n. Thus, while CTP is an obligatory substrate By studying the time course of the synthesis of NeuAc and for the synthetase, it inhibits the hydrolase; exogenous sulph- CMP-NeuAc in rat liver in vivo, Ferwerda et al. (1983) con- hydryl compounds stabilize the synthetase, but inhibit the cluded that there was a special pool of newly synthesized hydrolase; the synthetase has an absolute requirement for a NeuAc channelled to the synthetase. However, while the divalent cation, a characteristic which could not be demon- nuclear location of CMP-NeuAc synthetase itself was con- strated for the hydrolase from rat liver; while the synthetase of firmed by Ferwerda et al. (1986), its function at the nucleus hog submaxillary gland catalysed a reversible reaction, the was questioned. This was based primarily on calculations hydrolase reaction was irreversible. The hydrolase was also

Table II. Comparison of properties of CMP-NeuAc synthetase and CMP-NeuAc hydrolase

CMP-NeuAc synthetase CMP-NeuAc hydrolase Kean (1970) Kean and Bighouse (1974) Shoyab and Bachhawat (1969) Van Dijk et al (1976)

Tissue Rat liver, bovine retina Rat liver Sheep liver Calf kidney Subcellular location Nucleus Plasma membrane Nucleus and mitochondria Plasma membrane pH optimum 9.0 9.0 9.0 8.9 Metal requirement Mg (20 mM) None Mn (stimulatory) Required Effect of exogenous SH Stabilizing Inhibitory Inhibitory Partial inhibition Effect of CTP Obligatory substrate Inhibitory Inhibitory Partial inhibition Effect of UDP-GlcNAc - Inhibitory - Slight inhibition Tissue distribution Wide Limited (live and kidney) Wide - Substrate #„ CTP, 0.48 mM; NeuAc, 0.72 mM CMP-NeuAc, 0.4 mM CMP-NeuAc, 0 09 mM CMP-NeuAc, 0.47 mM Stability Frozen/thawed Unstable Stable Stable Stable 4°C Stable 3 weeks Stable a few days Stable Stable Reversibility Reversibility Irreversible - -

443 E.L.Kean

detected in calf kidney by Van Dijk et al. (1976) who con- and is widely distributed in the animal kingdom. Although it is firmed the plasma membrane localization of the enzyme. With not present in normal adult human tissue, it is present in fetal the exception of a stimulation by divalent metal ions of the calf human tissues and in certain human cancers. Both the means of kidney enzyme, the properties of the latter were almost identical the biosynthesis of the glycolyl derivative and its activation to those described for rat liver enzyme by Kean and Bighouse have remained unresolved until recently. Reactivity toward (1974). After confirming the plasma membrane location of the both NeuAc and NeuGc was observed with the soluble enzymes hydrolase in plasma membranes of rat liver, Van Dijk et al. from hog submaxillary gland (Kean and Roseman, 1966a, b), (1977) examined the regional distribution of the enzyme in the sheep brain (Shoyab etal., 1964), and frog liver (Schauer plasma membrane. They concluded that CMP-NeuAc hydro- et al., 1980), and the nuclear enzymes of rat liver (Kean, 1970) lase was localized at the sinusoidal and lateral parts of the cell and hog retina (Kean, 1969), as well as the enzyme from the membrane of the hepatocyte. Since activity was detected with unfertilized sea urchin egg (Kean and Bruner, 1971). The intact cells, it was suggested that the hydrolase was located on formation of the glycolyl and acetyl derivatives by other the outside of the cellular membrane, although a rigorous invertebrate species has also been reported (Warren, 1963). examination of the topography of the hydrolase in the plasma These observations suggested that a single enzyme catalysed the membrane remains to be performed. Carey et al. (1980) synthesis of both sugar nucleotides. The bacterial enzymes, observed the hydrolysis of CMP-NeuAc within microsomal however, could not use NeuGc as a substrate (Warren and vesicles. This latter hydrolase apparently did not have the same Blacklow, 1962; Kean and Roseman, 1966a, b). In contrast to Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 properties as the plasma membrane CMP-NeuAc hydrolase. previous suggestions that the glycolyl group was formed from Lepers et al. (1990) have also observed the degradation of free NeuAc or glycosidically bound NeuAc, or via the forma- CMP-sialic acid to free sialic acids by rat and mouse liver Golgi tion of vV-glycolylmannosamine, Shaw and Schauer (1988) vesicles, again indicative of the action of a CMP-sialic acid demonstrated that the conversion to the glycolyl group occurred hydrolase activity. Muilerman et al. (1984) were able to isolate by the action of a soluble hydroxylase from porcine sub- two sugar nucleotide hydrolases from rat liver, only one of mandibular glands acting on CMP-NeuAc and producing CMP- which had activity for CMP-NeuAc and which appeared to be NeuGc. Using murine myeloma cell lines, Muchmore et al. the same as the hydrolase described previously by Kean and (1989) presented several lines of evidence demonstrating that Bighouse (1974). Both of these enzymes, however, were the formation of the glycolyl derivative takes place primarily regarded to be members of the family of non-specific by hydroxylase action on CMP-NeuAc. Similar results were nucleotide-sugar pyrophosphatase/phosphodiesterases. A wide obtained using preparations from other tissues which express variety of sugar nucleotides and nucleotide phosphates were the glycolyl derivative. Thus, the formation of CMP-NeuGc observed to inhibit the action of the hydrolase (Kean and occurs in animal tissues by the hydroxylase reaction and can Bighouse, 1974; Van Dijk etai, 1976). It is not clear yet also be formed by direct reaction of the glycolyl derivative with whether the CMP-NeuAc hydrolase of the plasma membrane is the synthetase. Bouhours and Bouhours (1989) observed that an enzyme distinct from other nucleotide pyrophosphatases. the expression of the glycolyl derivative in GM3 ganglioside in rat small intestine was controlled by a single autosomal dominant gene, and its formation was perfectly correlated Metabolic regulation with the activity of CMP-NeuAc hydroxylase. Interestingly, using mutants devoid of hydroxylase activity, Bouhours and The earliest indication of a regulatory influence by CMP- Bouhours (1989) also presented evidence for the existence of NeuAc on sialic acid metabolism was the observation by another hydroxylase accounting for the production of 20-30% Kornfeld et al. (1964) that CMP-NeuAc inhibited the activity of NeuGc in intestinal glycoproteins that did not use CMP- of UDP-N-acetylglucosamine (UDP-GlcNAc) 2 epimerase, an NeuAc as substrate. enzyme which leads to the formation of 7v-acetylmannosamine, a key intermediate in the biosynthesis of the sialic acids. The net effect would be an inhibition of sialic acid biosynthesis. O-Acetylated sialic acids Also, as a result of the inhibition, UDP-GlcNAc would accumulate. Kean and Bighouse (1974) observed that UDP- In addition to the formation of the JV-glycolyl derivative, GlcNAc inhibits the destruction of CMP-NeuAc by the hydro- O-acetylated compounds are a major class of sialic acid lase, an effect which could thus reinforce the feedback in- derivatives. Kean and Roseman (1966a, b) observed that the hibition of the formation of N-acetylmannosamine. Recently, it enzyme purified from the hog submaxillary gland was inactive has been demonstrated that the metabolic lesion in sialurea is a with 4-O-Ac-NeuAc and 7-O-Ac-NeuAc as substrates. The loss of feedback control by CMP-NeuAc of the GlcNAc-2- enzyme purified from frog liver by Schauer et al. (1980) also epimerase, resulting in an overproduction of sialic acid did not react with NeuAc O-acetylated at C-4 or C-9. However, (Weiss et al. 1989; Sepalla et al., 1991). Another aspect of the the formation of the CMP-derivatives of 4-O-Ac-NeuAc and complex system of regulation concerning sialylation may be the 9-<9-Ac-NeuAc was reported by Schauer and Wember (1973) geographic separation within the cell of nuclear CMP-NeuAc by extracts of submandibular glands from several species. The synthetase and plasma membrane CMP-NeuAc hydrolase, two 4-0 diacetyl derivative was also active with the enzyme from mutually exclusive enzymatic activities. N.meningitidis (Warren and Blacklow, 1962). In contrast, Higa and Paulson (1985) were unable to demonstrate the formation of CMP-4-OAc-NeuAc with preparations from bovine and Activation of other sialic acids equine submaxillary glands. A predominant product obtained when using this substrate was CMP-NeuAc, due most likely to N-dycolylneuraminic acid O-deacetylation by the action of an O-acetyltransferase in the AM31ycolylneuraminic acid (NeuGQ is one of the most preparation. Similarly, a calf brain enzyme from which esterase commonly occurring modifications of Af-acetylneuraminic acid activity had been removed was active in the formation of

444 Sialk acid activation

CMP-9-t>-Ac-NeuAc, but not the 4-0-Ac derivative. Higa and formation and, in addition, the latter required the presence of Paulson (1985) concluded that the presence of 4-0-Ac NeuAc the lipid component which was shown to be undecaprenol on glycoproteins and mucins was due to O-acetyltransferase phosphate (Troy et al., 1975). This would constitute a lipid- activity on glycosidically bound sialic acid, and did not involve activated sialic acid, functioning as a more immediate precursor activation at the sugar nucleotide level. Higa and Paulson in sialyl polymer biosynthesis than the sugar nucleotide. Sialyl- (1985) also observed that the use of Mn2"1" instead of Mg2+ phosphorylundecaprenol, although not as yet completely charac- resulted in a shift of the pH optimum of the synthetase from terized, has thus been proposed an an intermediate in the pH 9.5 to 7.0, a condition which would minimize hydrolysis of synthesis of the high mol. wt sialyl polymers. It may function 0-acetyl groups. as a direct donor of sialic acid residues, or as a site for polymer formation by accepting additional sialic acid groups producing Transport of CMP-sialk acid higher oligosialylundecaprenols. The latter intermediate would then serve as the donor of polysialic acid to endogenous While evidence from several laboratories has demonstrated that acceptors, catalysed by polysialyltransferase, resulting in the CMP-NeuAc synthetase is located in the nucleus of the cell, as formation of the final sialylpolymers (Troy et al., 1975). Thus described above, the sugar nucleotide must be made available far, lipid activation of the sialic acids, either in this manner or to the sialyltransferases of the Golgi for utilization. -What is the as dolichol intermediates, has not been reported in animal Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 direct source of the sugar nucleotide and how is it delivered to systems. the Golgi? Carey et al. (1980) demonstrated that CMP-NeuAc can penetrate mouse liver microsomal vesicles, is present in Molecular biology the lumen, and suggested that the process involves a carrier- mediated transport system. The accumulation of CMP-NeuAc CMP-NeuAc synthetase was purified to homogeneity from an by microsomes of the hen oviduct was also observed by O18:K1 strain of E.coli RS218 by Vann et al. (1987). Many of Hanover and Lennarz (1982). Further analysis (Sommers and its properties were examined and shown to be similar to those Hirschberg, 1982) demonstrated that the CMP-NeuAc transport of the enzymes described previously, such as pH optimum, was solely into Golgi-derived vesicles and not into smooth metal requirement, catalytic activity. The gene encoding the or rough endoplasmic reticulum vesicles. The process was enzyme was localized and isolated. The mol. wt of the poly- inhibited by proteolysis of the Golgi vesicles, suggesting the peptide (50 000 daltons) encoded by the CMP-NeuAc gene was existence of translocation carrier proteins that face the cyto- identical to that obtained for the enzyme purified from E.coli plasm. Capasso and Hirschberg (1984a) observed that 5'-CMP by ammonium sulphate fractionation, absorption and affinity and 5'-UMP were the most effective inhibitors of the trans- chromatography. Previously, based on analysis by gel filtration location of CMP-NeuAc into Golgi vesicles. Evidence was and SDS-PAGE, Schauer et al. (1980) estimated the mol. wt obtained for a coupled exchange in the Golgi between CMP- for the synthetase from frog liver to be 163 000 daltons, with NeuAc and CMP (Capasso and Hirschberg, 1984b). Added no indication of the presence of subunits. The complete nucleo- information concerning the important role played by the trans- tide sequence of the gene encoding CMP-NeuAc synthetase location of CMP-NeuAc was the demonstration that mutants cloned from the E. coli genome was determined by Zapata et al. with decreased sialylation of glycoproteins and gangliosides (1989). Consistent with the functional similarities of CMP- were also deficient in CMP-NeuAc translocation (Deutscher NeuAc synthetase and CMP-KDO synthetase, several regions et al., 1984). Uptake studies of CMP-NeuAc and CMP-NeuGc of homology were observed in their amino acid sequences. by rat liver Golgi vesicles revealed that there was no significant Evidence was obtained suggesting that the sialic acid-activating distinction by the CMP-sialic acid transporter for either sugar enzyme from the bacterial system may also exist as dimers or nucleotide, and their mutual inhibition of uptake indicated that higher aggregates, thus suggesting differences in tertiary or the same carrier molecule was involved (Lepers et al., 1989, quaternary structure as compared to the enzyme from animal 1990). The immediate source of CMP-NeuAc in the cell for tissues (Schauer et al., 1980) in which no subunit composition delivery to the Golgi, however, is unclear. Is it secreted into the was indicated. Advantage was taken of the ability to clone the cytoplasm after synthesis by the nuclear enzyme and then CMP-NeuAc gene for the production of CMP-NeuAc in large- transported into the Golgi or are other processes involved? scale amounts (Shames et al., 1991). Gene constructs were Thus, Igarashi et al. (1985) have observed interaxonal transport engineered such that the CMP-NeuAc synthetase gene was of CMP-NeuAc. The findings of Ferwerda et al. (1983) would overexpressed to the extent of 8-10% of the soluble E.coli suggest that there may also be translocation of the synthetase protein. After a single purification step by column chroma- from the nucleus to the cytoplasm or to the Golgi. CMP-NeuAc tography, an enzyme of >95% homogeneity was obtained. synthetase has been detected in the Golgi (Creek et al., 1980). Using this preparation, multi-gram amounts of CMP-NeuAc The low yield in the Golgi (1% of total cellular activity) were synthesized in high yield and high purity. This same makes it difficult to conclude whether this is a native site or its methodology was applied to the production of various NeuAc presence is due to the redistribution of nuclear contents as a analogues. This is in contrast to the 'large scale' preparations result of homogenization. Analysis for DNA argued against previously carried out, as for example in the studies on CMP- the latter. NeuAc hydrolase (Kean and Bighouse, 1974), in which usually -350 jimol (-200 mg) of radioactive and non-radioactive Lipid-activated sialic acid CMP-NeuAc were prepared. The possible involvement of a lipid-linked sialic acid as an Concluding remarks intermediate in the biosynthesis of sialic acid-containing polymers in E.coli was revealed by Troy et al. (1975) and In that CMP-sialic acid was shown to be a unique, activated Vijay and Troy (1975). The incorporation of NeuAc from form of sialic acid, absolutely required for the biosynthesis of CMP-NeuAc into a lipoidal component preceded sialyl polymer the sialic acid-containing complex carbohydrates, its discovery 445 E.L.Kean

Briles.E.B., Li,E. and Kornfeld.S. (1977) Isolation of wheat germ agglutinin- resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. J. Biol. Chem., 252, 1107-1116. Plasma CapassoJ.M. and Htrschberg.C.B. (1984a) Effect of nucleotides on trans- Membrane location of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles. Biochim. Biophys. Ada, 777, 133-139. CapassoJ. and Hirschberg.C.B. (1984b) Mechanisms of glycosylation and sulfation in the Golgi apparatus: Evidence for nucleotide sugar/nucleoside Sial^C Acid monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in + CTP—-••CMP-sialic the Golgi apparatus membrane. Proc. Natl. Acad. Sci. USA, 81, 7051-7055 1 "* acid. Carey,D.J. arid Hirschberg.C.B. (1979) Metabolism of A'-acetylneuraminic acid Nucleus in mammals: Isolation and characterization of CMP-A'-acetylneuraminic acid. Biochemistry, 10, 2086-2092. Carey,D.J., Sommers.L.W. and Hirschberg.C.B. (1980) CMP-W-acetylneuraminic acid: Isolation from and penetration into mouse liver microsomes. Cell, 19, 597-605. Coates.SW., Gumey.T.Jr, Sommers,L.W., Yeh,M. and Hirschberg.C.B. Scheme 1. (1980) Subcellular localization of sugar nucleotide synthetases. J Biol. Chem., 255,9225-9229. Comb,D.G., Shimizu.F. and Roseman,S. (1959) Isolation of cytidine-5'-mono- Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 by Roseman's laboratory was thus a fundamental one for phospho-iV-acetylneuraminic acid. /. Am. Chem. Soc., 81, 5513. research in this field that has spanned many years and involved Comb.D.G., Watson,D.R. and Roseman,S. (1966) The sialic acids: EX. Isola- tion of cytidine 5'-monophospho-A'-acetylneuraminic acid from Escherichia many laboratories worldwide. Research concerning the forma- coh K-235 J. Biol. Chem., TAX, 5637-5642. tion of CMP-sialic acid, its turnover and the logistics of its Corfield.A.P., Ferreira do Amaral.C, Wember.M. and Schauer.R. (1976) The delivery in the cell has provided surprises and has left several metabolism of O-acyl-A'-neuraminic acids. Eur. J. Biochem., 68, 597-610 as yet unanswered questions. One of the most enigmatic Creek,K.E., Weisman.D. and Morrt.D.J. (1980) The subcellular distribution characteristics of the CMP-sialic acid-activating enzyme is its of cytidine 5'-monophosphosialic acid synthetase in rat liver. Eur. J. Cell unique location in the nucleus. Equally puzzling is the plasma Biol, 23, 157-165 Deutscher.S.L., Nuwayhih.N., Stanley,P., Briles.E.I.B. and Hirschberg.C.B. membrane location of the cleaving enzyme, CMP-NeuAc (1984) Translocation across Golgi vesicle membranes: A CHO glycosylation hydrolase, an enzyme about which much remains to be known. mutant deficient in CMP-sialic acid transport. Cell, 39, 295-299 Both of these loci are separate from the site of the utilization Ferwerda.W., Blok.C.M. and Van Rinsum.J. (1983) Synthesis of A'-acetyl- of CMP-sialic acid, the Golgi. Other than speculation, the neuraminic acid and of CMP-A'-acetylneuraminic acid in the rat liver cell. reason for this unique compartmentalization within the cell of Biochem J., 216, 87-92. Ferwerda.W., Blok.C.M. and Van RinsunU. (1986) CMP-A'-acefylneuramiruc the enzymes concerned with the formation, utilization and acid: is it synthesized in the nucleus? Glycoconjugate J., 3, 153—161. turnover of CMP-sialic acid, most of which were discovered Ghalambor,M.A. and Heath,E.C. (1966) The biosynthesis of cell wall lipopoly- over 20 years ago, remains unknown. The relationships saccharide in Escherichia coli. IV. Purification and properties of cytidine between nuclear CMP-sialic acid synthetase, the hydrolase, and monophosphate 3-deoxy-D-mannooctulosonate synthetase. J. Biol. Chem., delivery to the Golgi are depicted in Scheme 1. 241, 3216-3221. Gielen.W., Schaper.R. and Pink.H. (1970) Neuraminidase und cytidinmono- Although this review is concerned with the activation of the phosphat-A'-acerylneuraminat-synthetase in rinderleukozyten. Hoppe-Seyler's 7, sialic acids, attention must be drawn to the pioneering research PhysicA. Chem., 351, 768-770. on the sialyltransferases by Roseman's laboratory which flowed Gielen.W., Schaper,R. and Pink.H. (1971) Die subzellulare anordnung und die akitivat der cytkiinmonopriospnat-A'-acetylneuraminat-synthetase im jungen from the discovery of CMP-NeuAc. While a vast literature on rattengehim. Hoppe-Seyler's Z Physiol. Chem., 352, 1291-1296. the siaJyltransferases exists, the work by Roseman, Carlson, HanoverJ.A. and Lennarz.W.J. (1982) Transmembrane assembly of N-linked Jourdian, McGuire, Kaufman, Basu and Bartholomew, sum- glycoproteins. Studies on the topology of saccharide-lipid synthesis. J. Biol. marized in Methods in Enzymology (1966), as well as that of Chem., 257, 2787-2794. Aminoff, Dodyk and Roseman (1963), laid the foundation for Harms.E., Kreisel.W., Morns.H.P. and Reutter.W. (1973) Biosynthesis of A'-acetylneuraminic acid in Morris hepatomas. Eur. J. Biochem., 32, 254-262. our understanding of these reactions, and for research in that Harvey,E.B. (1956) The American Arbaaa, and Other Sea Urchins. Princeton area which followed. University Press, Princeton, NJ. HaverkampJ., Spoormaker,T., Dorland.L , VliegenthartJ.F.G. and Schauer.R. (1979) Determination of the £-anomeric configuration of cytidine 5'-mono- Acknowledgements phospho-A'-acetylneuraminic acid by 13C NMR spectroscopy. / Am. Chem. Soc., 101, 4851—4S53. This work was supported in part by US Public Health Service Research Grants Higa.H.H. and PaulsonJ.C. (1985) Sialylation of glycoprotein oligosaccharides EY 00393 and EY 03685, and the Ohio Lions Eye Research Foundation. with A'-aeetyl-, A'-glycolyl-, and A'-O-dlacetylneuraminic acids. /. Biol. Chem., 260, 8838-8849. Hultsch.E., Reutter.W. and Decker.K. (1972) Conversion of A'-acetylglucos- Abbreviations amine to CMP-A'-acetylneuraminic acid in a cell-free system of rat liver. Biochim. Biophys. Acla, 237, 132-140. CMP-KDO, CMP-3-deoxy-D-manno-otulosonate; CMP-NeuAc, CMP-A'-acetyl- Igarashi.M., Komiya,Y and Kurokawa.M. (1985) CMP-sialic acid, the sole neuraminic acid; CMP-sialic acid, cytidine 5'-monophosphosialic acid; NeuGc, sialosyl donor, is intra-axonally transported. FEBS Lett., 192, 239-242. A'-glycolylneuraminic and; UDP-GlcNAc, UDP-A'-acetylglucosamine Kean.E.L. (1969) Sialic acid activating enzyme in ocular tissue. Exp. Eye Res.. 8, 44-54. Kean,E.L. (1970) Nuclear cytidine 5'-monophosphosialic acid synthetase J. Biol. References Chem., 245, 2301-2308. Kean.E.L. (1972a) CMP-sialic acid synthetase of nuclei. Methods Enzymol.. Aminoff.D , Dodyk,F. and Roseman,S. (1963) Enzymatic synthesis of XXVm, 413—421. colorrunic acid. /. Biol. Chem., 238, PCI I77-PC1178. Kean.E.L. (1972b) CMP-sialic acid hydrolase. Methods Enzymol.. XXVm. BouhoursJ.-F. and Bouhours.D. (1989) Hydroxylation of CMP-NeuAc 983-990. controls the expression of A'-glycolylneuraminic acid in GM3 ganglioside of Kean.E.L. and Bighouse.K.J. (1974) Cyodine 5'-monophosphosialic acid hydro- the small intestine of inbred rats. J. Biol. Chem., 264, 16992-16999. lase; subcellular location and properties. J Biol. Chem.. 249. 7813-7823.

446 Sialic acid activation

Kean.E.L. and Bruner,W.E. (1971) Cytidine 5'-monophosphosialic acid syn- Van Dijk.W.. Maier.H and van den Eijnden.D.H. (1977) CMP-A'-acetyl- thetase: Activity and localization in the nucleate fragments of the unfertilized neuraminic acid hydrolase, an ectoenzyme distributed unevenly over the sea urchin egg. Exp. CeU Res., 69, 384-392. hepatocyte surface. Biochim. Biophys. Acta, 466, 187-197. Kean.E.L. and Roseman.S. (1966a) The sialic acids. X. Purification and Vann.W.F, Silver.R.P, Abeijon.C, Chang.K., Aaronson.W., Sutton.A., properties of cytidine 5'-monophosphosialic acid synthetasc J. Bioi. Chem., Finn.C.W., Linder.W. and Kotsatos.M. (1987) Purification, properties, and 241, 5643-5650. genetic location of Escherichia coli cytidine 5'-monophosphate A'-acetyl- Kean.E.L. and Roseman.S. (1966b) CMP-sialic acid synthetase Methods neuraminic acid synthetase. J. Biol. Chem., 262, 17556-17562. Enzymol., Vm, 208-215 Van RinsumJ., Van Dijk.W., HooghwinkelJ.M. and Ferwerda.W. (1983) Kornfdd.S., Komfeld.R., Neufdd.E F. and O'Bnen.P.J. (1964) The feedback Subcellular localization and tissue distribution of sialic acid precursor forming control of sugar nucleotide biosynthesis in liver Proc. Nail. Acad. Sri. USA. enzymes. Biochem. J., 210, 21-28. 52, 371-379. Vijay,I.K. and Troy.F.A. (1975) Properties of membrane-associated sialyl- Lepers.A., Shaw,L., Cacan.R., Schauer,R., MontreuilJ. and Verbert.A. (1989) transferase of Escherichia coli. J. Biol. Chem., 250 164-170. Transport of CMP-A'-glycolylneuraminic acid into mouse liver Golgi vesicles. Warren,L (1963) The distribution of sialic acid in nature. Comp Biochem. FEBS Lai., 250, 245-250. Physiol, 10, 153-171. Lepers.A., Shaw,L., Schneckenburger.P., Cacan.R., Verbert,A. and Schauer.R. Warren,L and Blacklow.R.S. (1962a) Biosynthesis of A'-acetylneuraminic acid (1990) A study on the regulation of A'-glycorylneuraminic acid biosynthesis and and cytidine-5'-monophospho-A'-acetylneuraminic acid in Neisseria meningiti- utilization in rat and mouse liver. Eur. J. Biochem., 193, 715-723. dis. Biochem. Biophys. Res. Common., 7, 433—438. Muchmore.E.A., Milewski.M., Varki.A and Diaz.S. (1989) Biosynthesis of Warren,L. and Blacklow.R.S. (1962b) The biosynthesis of cytidine 5'-mono- A'-glycolylneuraminic acid. J. Biol. Chem., 264, 20216-20223. phospho-A'-acetylneuraminic acid by an enzyme from Neisseria meningitidis. Downloaded from https://academic.oup.com/glycob/article/1/5/441/621435 by guest on 01 October 2021 Muilerman.H.G., Lasthuis,A.-M., HooghwinkelJ.M. and Van Dijk,W. (1984) J. Biol. Chem., 237, 3527-3534. On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in Weiss.O., Tietze.E, Gahl.W.A., Sepalla.R. and Ashwell.G. (1989) Identification rat liver. Biochem. J., 220, 95-103. of the metabolic defect in sialurea. J Biol. Chem., 264, 17635-17636. Roseman.S. (1962) Enzymatic synthesis of cytidine 5'-monophospho-sialic acids. Yu.R.K. and Ledeen.R. (1969) Configuration of the ketosidic bond of sialic acid Proc. Nail. Acad. Sa. USA, 48, 437-441. J. Biol. Chem., 244, 1306-1313 Roseman.S., Carlson.D.M., Jourdian.G.W.. McGuire.E.J.. Kaufman, B.. Zapata,G., Vann.W.F., Aaronson.W., Lewis,M.S. and Moos.M. (1989) Basu.S. and Bartholomew.B (1966) Animal sialic acid transferases (Sialyl- Sequence of the cloned Escherichia coli Kl CMP-A'-acetylneuraminic acid transferases). Methods Enzymol., VIII, 354-372. synthetase gene. J. Biol. Chem., 264, 14769-14774. Schauer,R. and Wember.M. (1973) Studies on the substrate specificity of acetylneuraminate cytidyltransferase and sialyltransferase of submandibular Received on June 20. 1991; accepted on August 15. 1991 glands from cow, pig, and horse. Hoppe-Sexler's Z Physiol. Chem., 354. 1405-1414. Schauer.R., Haverkamp.J. and Ehrtich.K. (1980) Isolation and characterization of acetylneuraminate cytidyltransferase from frog liver Hoppe-Seyler's Z Physiol. Chem., 361, 641-648. Sepalla.R., Tietze.F., Kransewich.D , Weiss.P . Ashwell.G . Barsh.G . Thomas.G H , Packman,S and Gahl.W.A. (1991) Sialic acid metabolism in sialurea fibroblasts. J. Biol. Chem.. 266, 7456-7461. Shames,S.L., Simon,E.S., Chnstopher.C.W., Schmid.W., Whitesides.G.M. and Yang.L-L. (1991) CMP-A'-acetylneuraminic acid synthetase of Eschenchia coir, high level expression, purification and use in the enzymatic synthesis of CMP-A'-acetylneuraminic acid and CMP-neuraminic acid derivatives. aycobiology, 1, 187-191. Shaw,L and Schauer.R (1988) The biosynthesis of A'-glycolylneuraminic acid occurs by hydroxylation of the CMP-glycoside of /V-acetylneuraminic acid. Biol. Chem. Hoppe-Seyter, 369. 477-486 Shoyab.M and Bachhawat.B.K (1967a) Enzymatic degradation of cytidine 5'-monophosphate-A'-acetylneurarrunic acid. Biochem. J., 102, 13a. Shoyab.M. and Bachhawat.B.K. (1967b) Age dependent changes in the level of cytidjne 5'-monophospho-A'-acetyl neuraminic acid synthesizing and degrading enzymes & bound sialic acid in rat liver. Indian J. Biochem., 4, 142—145 Shoyab.M. and Bachhawat.B.K. (1969) Partial purification & properties of cytidine 5'-monophospho-A'-acetyl neuraminic acid degrading enzyme from sheep liver. Indian J. Biochem., 6, 56—62. Shoyab.M , Pattabiraman.T.N. and Bachhawat.B.K. (1964) Purification and properties of the CMP-A'-acetylneuraminic acid synthesizing enzyme from sheep brain. J. Neurochem. 11, 639-646. Sommers.L.W and Hirschberg.C.B. (1982) Transport of sugar nucleotides into rat liver Golgi. J Biol Chem., 257, 10811-10817. Stone.A. and Kolodny,E.H. (1971) Circular dichroism of gangliosides from normal and Tay-Sachs tissue. Chem. Phys. Lipids, 6, 274—279. Troy.F.A., Vijay.I.K. and Tesche.N. (1975) Role of undecaprenyl phosphate in synthesis of polymers containing sialic acid in Eschenchia coli. J. Biol. Chem . 250. 156-163. Van den Eijnden.D.H (1973) The subcellular localization of cytidine 5'-monophospho-A'-acetylneuraminic acid synthetase in calf brain. J. Neurochem., 21. 949-958. Van den Eijnden.D.H., Meems.L. and Roukema.P.A. (1972) The regional distribution of cytidine 5'-rnonophospho-A'-acetylneurarninic acid synthetase in calf brain J. Neurochem., 19. 1649-1658 Van Dijk.W., Ferwerda.W. and van den Eijnden.D.H. (1973) Subcellular and regional distribution of CMP-A'-acetylneuraminic acid synthetase in the calf kidney. Biochim Biophys. Ada. 315, 162-175. Van Dijk.W., Maier.H. and van den Eijnden.D.H. (1976) Properties and subcellular localizauon of CMP-A'-acetylneuraminic acid hydrolase of calf kidney. Biochim. Biophys. Acta. 444, 816-834

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