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α2,8-Linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal α2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-β-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs ∼50–60 or less, a subpopulation of chains with DPs ∼150 to ∼180 and extending to DP ∼400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-β-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

The pre-existence of α2→8-linked disialic acid (di-Sia) and oligosialic acid (oligo-Sia) structures with up to 7 Sia residues was shown to occur on a large number of brain glycoproteins, including neural cell adhesion molecules (N-CAMs), by two highly sensitive chemical methods (Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Anal. Biochem. 261, 191–197; Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1999) Anal. Biochem. 266, 102–109). This unexpected finding was also confirmed using a newly developed antibody prepared using a copolymer of α2→8-linked N-acetylneuraminylp-vinylbenzylamide and acrylamide as an immunogen and known antibodies whose immunospecificities were determined to be di- and oligo-Sia residues with defined chain lengths. The major significance of the new finding that di- and oligo-Sia chains exist on a large number of brain glycoproteins is 2-fold. First, it reveals a surprising diversity in the number and M r of proteins distinct from N-CAM that are covalently modified by these short sialyl glycotopes. Second, it suggests that synthesis of di- and/or oligo-Sia units may be catalyzed by α2→8-sialyltransferase(s) that are distinct from the known polysialyltransferases, STX and PST, which are partially responsible for polysialylation of N-CAM.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.

The nature of endogenous acceptor molecules implicated in the membrane-directed synthesis of the polysialic acid (polySia) capsule in Escherichia coli K1 serotypes is not known. The capsule contains at least 200 sialic acid (Sia) residues that are elongated by the addition of new Sia residues to the nonreducing termini of growing nascent chains (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342). Presumably, chain growth starts when activated Sia residues are transferred to acceptors that are not already sialylated. In the present study, we used an acapsular mutant defective in synthesis of CMP-NeuAc to label acceptors with [14C]NeuAc and an anti-polySia-specific antibody (H.46) to identify the molecules to which the polySia was attached. [14C]Sia-labeled acceptors were solubilized with 2% Triton X-100, immunoprecipitated with H.46, and partially depolymerized with poly-alpha-2,8-endo-N-acetylneuraminidase. Approximately 5% of the [14C]Sia incorporated remained attached to endogenous acceptors. Double-labeling experiments were used to show that the non-Sia moiety of the acceptor was labeled in vivo with [14C]leucine and elongated in vitro with CMP-[3H]NeuAc. Concomitant with desialylation of the [3H]polySia-[14C]Leu acceptor was the appearance of a new [14C]Leu-labeled protein at 20 kDa. After strong acid hydrolysis, the 20-kDa labeled protein was shown to contain [14C]Leu. The acceptor molecules were not labeled metabolically with D-[3H]GlcN, 35SO4, or 32PO4, indicating that they do not appear to contain lipopolysaccharide, peptidoglycan, phosphatidic acid, or phospholipid. Based on these results, we conclude that the endogenous acceptor molecule is a membrane protein of about 20 kDa. The nature of attachment of polySia to acceptor is unknown. There are only 400-500 acceptor molecules/cell, which is about 100-fold fewer than the 50,000 polySia chains/cell. This suggests that each acceptor molecule may participate in the shuttling of about 100 polySia chains/cell. We hypothesize that the acceptor protein may function to translocate polySia chains from their site of synthesis on the cytoplasmic surface of the inner membrane to the periplasm.

Membrane-associated sialytransferase complexes from Escherichia coli K-235 catalyze the incorporation of N-acetylneuraminic acid (NeuNAc) from cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NeuNAc) into polymeric products and a lipid fraction. Reconstitution of enzyme activity in lipid-depleted membrane complexes had an absolute dependence on a purified phospholipid which was characterized by high resolution mass spectrometry following dephosphorylation as undecaprenol. An identical mass spectrum was obtained on the phosphorylated lipid confirming that the active derivative was undecaprenyl phosphate. This C55-isoprenoid alcohol accounted for 95% of the polyisoprenol and contained 11 isoprene units, each one unsaturated. The remaining 5% was composed of the C60 homologue, dodecaprenol. Ficarprenyl phosphate, an isomer of undecaprenyl phosphate, also restored enzymatic activity although on a molar basis, it was less active than undecaprenyl phosphate. These results provide direct evidence that sialyl polymer synthesis has an obligatory requirement for undecaprenyl phosphate, a membrane-bound lipid coenzyme which functions as an intermediate carrier of glycosyl residues in the biosynthesis of a variety of microbial cell surface polysaccharides. These data extend further the general class of polysaccharides whose synthesis involves undecaprenyl phosphate and are in accord with the hypothesis that this lipid acts as an intermediate carrier of sialyl residues in sialyl polymer synthesis according to the following reaction: CMP-NeuNAc plus P-undecaprenol in equilibrium NeuNAc-P-undecaprenol plus CMP Although the sialylated lipid remains to be characterized, evidence in support of this conclusion was obtained by kinetic analysis of N-acetylneuraminic acid transfer into the lipid soluble fraction and sialyl polymers. These studies showed a rapid incorporation of N-acetylneuraminic acid into the lipid-soluble fraction prior to maximal sialyl polymer formation, an observation consistent with a possible precursortion, an observation consistent with a possible precursor-product relationship. Confirmation that the radioactivity in the lipid-soluble fraction was lipid-linked N-acetylneuraminic acid was provided by the demonstration of the formation of a transitory sialyl-lipid with chromatographic properties expected of sialyl-undecaprenyl phosphate. Three additional lines of investigation implicated a functional role for undecaprenyl phosphate in sialyl polymer synthesis...

The polysialosyl capsule in Escherichia coli K1 is not synthesized when cells are grown at 15 degrees C. Membranous sialyltransferase complexes isolated from cells grown at 15 degrees C spontaneously gain the ability to synthesize sialyl polymers when incubated at 33 degrees C for 2-4 h. Activation of the endogenous synthesis of polysialic acid is localized in a low-density vesicle fraction (Whitfield, C., Adams, D.A., and Troy, F.A. (1984) J. Biol. Chem. 259, 12769-12775). The low density vesicles catalyzed protein synthesis, and spontaneous activation required protein synthesis. Immune blotting confirmed the presence of protein synthesis initiation factor, IF2, and ribosomal protein S6, in the low-density vesicle fraction. Temperature-upshift experiments identified four membrane proteins whose synthesis was temporally correlated with in vitro activation. These proteins have apparent molecular masses of 40, 21, 17.5, and 16 kDA. Although the function of these proteins is not known, they may relate to a sialyltransferase activity required to initiate sialyl polymer assembly, to an endogenous acceptor, or to a porin that may facilitate channeling of the polysialosyl chains through the outer membrane.

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.

The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000. This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity. The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM). Endo-N activity is inhibited by DNA and several other poly-anions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)

A cell envelope particulate fraction from an encapsulated strain of Bacillus licheniformis ATCC 9945A contains a polyglutamyl synthetase which catalyzes the polymerization of l-glutamic acid to form a high molecular weight polymer of γ-d-glutamic acid. The reaction, which is specific for l-glutamic acid, requires ATP and Mg2+ ion and is stimulated by K+ ion and dithiothreitol. A nearly 2-fold increase in activity is observed in the presence of optimal levels of glycerol (0.1 m) and dimethylsulfoxide (0.1 m). The Km for l-glutamic acid is 0.5 mm. d-Glutamic acid is not incorporated nor does it influence the polymerization of the l isomer. Glutamyl dipeptides inhibited polyglutamyl synthetase activity. The cell envelope-associated polyglutamyl synthetase does not require an RNA template since it is refractory to treatment with RNase. It is also not inhibited by chloramphenicol, actinomycin D, puromycin, or rifampicin. Thus, the mechanism of capsular polymer formation is uniquely different from that involved in normal protein synthesis. Pre-treatment of the particulate fraction with Pronase destroyed its ability to synthesize the capsular polymer. Since a molar excess of [12C]α-ketoglutarate had no effect on the incorporation of l-[14C]glutamic acid, the subsequent interconversion of the glutamyl optical antipodes does not involve a series of glutamyl transamination and alanine racemization reactions in which α-ketoglutarate is a key intermediate. These reactions have been demonstrated in soluble extracts from B. licheniformis and have been shown to be one mechanism to account for the formation of d-glutamic acid from the l isomer. The observation that l-glutamine also has no effect on l-glutamic acid incorporation rules out the possible involvement of a transamidation reaction between glutamine and l- or d-glutamic acid. Polymer formation does not involve a series of transpeptidation reactions between glutamyl dipeptides since dipeptides inhibit polymer synthesis. Although the precise mechanism of activation, racemization, and polymerization is not fully understood, the results of these studies establish that the poly(γ-d-glutamyl) capsule is synthesized by a sequence of membrane-associated enzymatic reactions. No evidence for the involvement of small molecular weight oligopeptides as free intermediates has been obtained. The enzymatically synthesized α-d-glutamyl polymers were readily isolated by virtue of their complete resistance to hydrolysis by Pronase. After treatment with this protease, molecular sieve chromatography resulted in the quantitative isolation of the capsular polymers. Glutamic acid was the only labeled amino acid recovered following acid-catalyzed hydrolysis of the purified product. The specific activity of the glutamic acid was 90% that of the initial specific activity which indicates a considerable amount of de novo synthesis. Subsequent chemical, physical, and enzymatic analyses have confirmed that the enzymatically synthesized polymers are structurally identical with the capsular γ-d-glutamyl polymers synthesized in vivo.

Treatment of 14C-uniformly labeled cell walls of Penicillium chrysogenum with chitobiase-containing chitinase resulted in 73% solubilization. The solubilized components were isolated, identified, and determined quantitatively. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose ((GlcNAc)2) accounted for 42% of the cell wall. Since kinetic analysis showed that GlcNAc arose from (GlcNAc)2, at least 42% of the cell wall contains GlcNAc in β-d-(1 → 4)-glycosidic linkage. When the same experiment was performed with a chitobiase-free chitinase preparation nearly identical results were obtained, except, as expected, essentially all of the GlcNAc was present as (GlcNAc)2. Small amounts of glucose and laminaritriose were also released. In addition, a nondialyzable component, Fraction 5A, accounting for 14% of the wall, was isolated. This fraction, devoid of GlcNAc, contains 20 times the amount of galactose and 2 times the amount of mannose with respect to glucose found in cell wall hydrolysates. An additional 13% of the wall was solubilized when the cell wall residue, following treatment with chitinase, was treated with chitinase-free β-d-(1 → 3)-glucanase. Therefore, 86% of the wall was solubilized when treated first with chitinase, then with β-d-(1 → 3)-glucanase. Initial treatment of cell walls with β-d-(1 → 3)-glucanase resulted in 48% solubilization. From an analysis of the solubilized components the conclusion was reached that at least 40% of the wall contains glucose in β-d-(1 → 3)-glycosidic linkage. An additional 44% of the wall was solubilized when the residue, following treatment with β-d-(1 → 3)-glucanase, was treated with chitinase. Thus, 92% of the wall was solubilized when treated first with β-d-(1 → 3)-glucanase, then with chitinase. The observations that β-d-(1 → 3)-glucanase-free chitinase released in addition to GlcNAc and (GlcNAc)2 also glucose, laminaritriose, and a fraction containing mannose, galactose, and glucose (Fraction 5A) and that treatment of this fraction with β-d-(1 → 3)-glucanase released glucose and laminaribiose suggest that glucose may exist in cell walls in one or more forms other than the major glucan. From the available data it is not clear whether Fraction 5A contains one or several polymers. An analysis of the monomeric cell wall constituents released by acid hydrolysis is in excellent agreement with the results obtained by enzymatic digestion. These results showed the cell wall to contain about 48% glucosamine and about 52% neutral hexoses, which consisted of glucose, galactose, and mannose in the molar proportion of 10:1:2. Thus, if 40% of the wall is glucose, then galactose and mannose account for 4% and 8% of the wall, respectively.

Aerobacter aerogenes produces a capsular polysaccharide composed of tetrasaccharide repeating units containing galactose, mannose, and glucuronic acid in a molar ratio of 2:1:1. Particulate cell envelope fractions of the organism catalyze the incorporation of galactose, mannose, and glucuronic acid from their respective nucleotide derivatives into capsular polysaccharide and into a lipid fraction. Kinetic studies of galactose-14C transfer from UDP-galactose-14C into the lipid fraction and into capsular polysaccharide suggested that a lipid-bound oligosaccharide intermediate is a precursor of the capsular polysaccharide. The biosynthesis of the intermediate oligosaccharide was shown to be initiated by the incorporation of galactose into an endogenous phospholipid. This reaction was characterized as a phosphogalactosyltransferase on the basis of its reversibility by UMP, but not UDP, and the alkali-catalyzed release of cyclic galactose 1,2-phosphate from the intermediate, indicating that galactose is bound to the lipid by a pyrophosphate linkage. The carrier phospholipid was isolated and purified by column chromatography on silicic acid and DEAE-cellulose, and finally, by preparative thin layer chromatography. The pure phospholipid, identified as undecaprenyl phosphate by mass spectrometry, restored the polysaccharide-synthesizing capacity of enzyme preparations which had been depleted of endogenous lipid by treatment with organic solvents. The particulate fraction catalyzes the sequential transfer of mannose, glucuronic acid, and a second galactose moiety to galactosyl-P-P-undecaprenol to form the di-, tri-, and tetrasaccharide derivatives, each of which was isolated and characterized, establishing the following sequence: UDP-Gal + P-lipid ⇌ Gal-P-P-lipid + UMP Gal-P-P-lipid + GDP-Man → Man-Gal-P-P-lipid + (GDP) Man-Gal-P-P-lipid + UDP-GlcUA → GlcUA-Man-Gal-P-P-lipid + (UDP) GlcUA-Man-Gal-P-P-lipid + UDP-Gal → Gal-(GlcUA)-Man-Gal-P-P-lipid + (UDP) The lipid-linked tetrasaccharide is polymerized to form a macromolecular product which is identical with native capsular polysaccharide, based on its composition and its susceptibility to degradation by a specific, phage-induced depolymerase.