Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells

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)     

Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units

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.     

On the biosynthesis of alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid catalyzed by the sialyltransferase of Escherichia coli Bos-12.

Escherichia coli Bos-12 synthesizes a heteropolymer of sialic acids with alternating alpha-2,9/alpha-2,8 glycosidic linkages (1). In this study, we have shown that the polysialyltransferase of the E. coli Bos-12 recognizes an alpha-2,8 glycosidic linkage of sialic acid at the nonreducing end of an exogenous acceptor of either the alpha-2,8 homopolymer of sialic acid or the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid and catalyzes the transfer of Neu5Ac from CMP-Neu5Ac to this residue. When the exogenous acceptor is an alpha-2,8-linked oligomer of sialic acid, the main product synthesized is derived from the addition of a single residue of [14C]Neu5Ac to form either an alpha-2,8 glycosidic linkage or an alpha-2,9 glycosidic linkage at the nonreducing end, at an alpha-2, 8/alpha-2,9 ratio of approximately 2:1. When the acceptor is the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid, chain elongation takes place four to five times more efficiently than the alpha-2,8-linked homopolymer of sialic acid as an acceptor. It was found that the alpha-2,9-linked homopolymer of sialic acid and the alpha-2,8/alpha-2,9-linked hetero-oligomer of sialic acid with alpha-2,9 at the nonreducing end not only failed to serve as an acceptor for the E. coli Bos-12 polysialyltransferase for the transfer of [14C]Neu5Ac, but they inhibited the de novo synthesis of polysialic acid catalyzed by this enzyme. The results obtained in this study favor the proposal that the biosynthesis of the alpha-2, 9/alpha-2,8 heteropolymer of sialic acid catalyzed by the E. coli Bos-12 polysialyltransferase involves a successive transfer of a preformed alpha-2,8-linked dimer of sialic acid at the nonreducing terminus of the acceptor to form an alpha-2,9 glycosidic linkage between the incoming dimer and the acceptor. The glycosidic linkage at the nonreducing end of the alternating alpha-2,9/alpha-2,8 heteropolymer of sialic acid produced by E. coli Bos-12 should be an alpha-2,8 glycosidic bond and not an alpha-2,9 glycosidic linkage.     

Giant liposomes as model membranes for immunological studies: spontaneous insertion of purified K1-antigen (poly-alpha-2,8-NeuAc) of Escherichia coli

A flow chamber has been constructed to use giant liposomes (diameter 5-50 microns) as model membranes for immunological studies and other experiments involving the interaction with water-soluble compounds. As an example of immunological importance, the insertion of purified K-antigen from Escherichia coli K1 has been studied. Despite its large hydrophilic part (poly-alpha-2,8-NeuAc), which is capped at its potential reducing end with phosphatidic acid acting as a lipid anchor group, this water-soluble material is readily incorporated into liposomal membranes of dimyristoylphosphatidylcholine (DMPC). The incorporation has been proven by immunofluorescence using a FITC-labeled monoclonal anti-K1-IgG. Without the lipid residue, however, no binding of poly-alpha-2,8-NeuAc to the liposomes has been observed. This could be shown by using colominic acid, an oligomeric form of alpha-2,8-NeuAc with free reducing ends instead of purified K1-antigen. The possibility for further manipulation of this model system has been shown by using a poly-alpha-2,8-NeuAc cleaving enzyme (endoneuraminidase). The function of the endoneuraminidase has been proven by showing no binding of the antibody after enzyme treatment of K1-bearing liposomes as well as by rapid loss of fluorescence of a previously bound FITC-antibody.     

Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms

A rat brain Golgi sialyltransferase activity capable of the differentiation-dependent control of N-CAM sialylation state is described. The specific activity of Golgi sialyltransferase was found to be developmentally regulated with respect to both endogenous and exogenous protein acceptors, with a particular elevation on postnatal days 10-12 when the heavily sialylated or 'embryonic' form of N-CAM is re-expressed. The subsequent developmental decrease in activity was associated with a significant decrease in apparent Km for the CMP-NeuNAc substrate, but not for the asialofetuin exogenous acceptor, which could not be attributed to the temporal expression of an endogenous competitive inhibitor. The apparent Vmax remained constant for CMP-NeuNAc but was significantly reduced for asialofetuin. Sialyltransferase activity, which was optimal at pH 7.0-7.5, was also modulated by various cations. Zinc abolished enzyme function, in contrast to ferric ions which stimulated activity fourfold-sevenfold. The marked activation of the adult form of the enzyme by potassium and magnesium ions, together with the alterations in kinetic constants, suggested this activity to be distinct from that derived from postnatal day-12 tissue. The kinetics of [14C]sialic acid incorporation into immuno-precipitated N-CAM demonstrated the individual polypeptides to be sialylated, possibly by addition of polysialosyl units, in a developmental sequence. The presence of four distinct sialyltransferase activities was demonstrated by non-denaturing gel electrophoresis followed by solid-phase enzyme assay. These isoforms were temporally expressed during development, two being correlated with the postnatal reexpression of the 'embryonic' form of N-CAM.     

Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain.

We have studied alpha 2,8-linked polysialic acid (polySia) and the neural cell adhesion molecule (N-CAM) in the adult rat brain by immunohistochemistry and Western blot analysis. Both molecules were widely distributed but not ubiquitous. Various brain regions showed colocalization of polySia and N-CAM. Strong immunoreactivity for polySia was seen in regions which were negative for N-CAM, such as the main and accessory olfactory bulbs. Immunohistochemical evidence for the heterogeneity of polySia expression in different brain regions was confirmed by immunoblotting. We present evidence that N-CAM is not the only polySia bearing protein in adult rat brain. Specifically, immunoprecipitation using the polySia-specific monoclonal antibody mAb 735 precipitated not only N-CAM isoforms carrying polySia, but also the sodium channel alpha subunit. Immunoblotting using sodium channel alpha subunit antibody (SP20) revealed a smear from 250 kDa upwards. PolySia removal using an endoneuraminidase specific for alpha 2,8-linked polysialic acid of 8 or more residues long, reduced this smear to a single band at 250 kDa. Thus both N-CAM and sodium channels carry homopolymers of alpha 2,8-linked polysialic acid in adult rat brain.     

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.     

Differentiation-Dependent Sialylation of Individual Neural Cell Adhesion Molecule Polypeptides During Postnatal Development

The postnatal sialylation of individual neural cell adhesion molecule (N-CAM) polypeptides by a developmentally regulated sialyltransferase in Golgi-enriched fractions isolated from rat brain is described. The 120-kilodalton polypeptide of N-CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140- and 180-kilodalton N-CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N-CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated "embryonic" form of N-CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The "embryonic" form of N-CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N-CAM was virtually undetectable in the immunoblots of postnatal material.     

Cell-free biosynthesis of the O-acetylated N-acetylneuraminic acid capsular polysaccharide of group C meningococci.

A cell-free system was established to study the biosynthesis of group C meningococcal capsular polysaccharide, an alpha-2 leads to 9-linked N-acetylneuraminic acid (NeuAc) homopolymer containing O-acetyl groups at either C7 or C8. Sialyltransferase activity, isolated from group C meningococcus strain C-11, catalyzed incorporation of [14C]NeuAc from CMP (CMP--[14C]NeuAc) into polymeric form. This sialyltransferase was stimulated by addition of meningococcus group C and Escherichia coli K92 capsular polysaccharides, the latter being an alpha-2 leads to 8- and alpha-2 leads to 9-linked NeuAc heteropolymer. Group C meningococcal sialyltransferase did not require divalent ions but was stimulated by Mn2+. Attempts to demonstrate a lipid-soluble intermediate in the biosynthesis of this NeuAc polymer were unsuccessful. Meningococcal group C sialyltransferase incorporated NeuAc into a membrane-associated product. The polysaccharide can be extracted from the membrane-bound fraction with Triton X-100. The newly synthesized polysaccharide coprecipitates with authentic group C antigen in meningococcal group C antiserum and is degraded by sodium metaperiodate, indicating that the NeuAc polymer synthesized by the cell-free system consists of alpha-2 leads to 9 linkage. Meningococcal group C spheroplast membranes contain an O-acetylase that can catalyze the transfer of acetyl groups from acetyl coenzyme A to the in vitro-synthesized polysaccharide.     

Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule

A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).     

Successive glycosyltransfer of sialic acid by Escherichia coli K92 polysialyltransferase in elongation of oligosialic acceptors

Escherichia coli K92 produces a capsular polysialic acid with alternating alpha2,8 alpha2,9 NeuNAc linkages. This polysaccharide is cross-reactive with the neuroinvasive pathogen Neisseria meningitidis Group C. The K92 polysialyltransferase (PST) catalyzes the synthesis of the polysialic acid with alternating linkages by the transfer of NeuNAc from CMP-NeuNAc to the nonreducing end of the growing polymer. We used a fluorescent-based high-performance liquid chromatography assay to characterize the process of chain extension. The PST elongates the acceptor GT3-FCHASE in a biphasic fashion. The initial phase polymers are characterized by accumulation of product containing 1-8 additional sialic acid residues. This phase is followed by a very rapid formation of high-molecular weight (MW) polymer as the accumulated oligosaccharides containing 8-10 sialic acids are consumed. The high-MW polymer contains 90-100 sialic acids and is sensitive to degradation by periodate and K1-5 endoneuraminidase, suggesting that the polymer contains the alternating structure. The polymerization reaction does not appear to be strictly processive, since oligosaccharides of each intermediate size were detected before accumulation of high-molecular weight polymer. Synthesis can be blocked by CMP-9-azido-NeuNAc. These results suggest that the K92 PST forms both alpha2,8 and alpha2,9 linkages in a successive and nonprocessive fashion.     

A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.     

Biosynthesis of the polysialic acid capsule in Escherichia coli K1. The endogenous acceptor of polysialic acid is a membrane protein of 20 kDa

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.     

The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.     

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.     

Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain

A sialyltransferase involved in the biosynthesis in vitro of LD1c (NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-NAc beta 1-3Gal beta 1-4Glc-Cer) has been characterized from 9 to 11-day-old embryonic chicken brains. The CMP-[14C]NeuAc:LM1(alpha 2-8)sialyltransferase (SAT-2) sedimented (75%) at the junction of 0.75 and 1.2 M on a discontinuous sucrose density gradient when still membrane bound. In addition to the biosynthesis of LD1c, the detergent-solubilized (0.4% Nonidet P-40) preparation also catalyzes the transfer of sialic acid to O-8 of sialic acid in GM3 to form GD3 (NeuAc alpha 2-8NeuAc alpha 2 - 3Gal beta 1 - 4Glc - Cer). Substrate inhibition studies indicated that these two reactions are probably catalyzed by the same enzyme, SAT-2. The kinetic parameters of SAT-2 activity were determined. The Km values were 70 and 63 microM with CMP-[14C]NeuAc and LM1, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The (alpha 2-8)-linkage between the terminal and penultimate sialic acids was determined using nonradioactive CMP-NeuAc and [Ac-14C]LM1 as substrates (Higashi, H., and Basu, S. (1982) Anal. Biochem. 120, 159-164) for the enzyme, followed by identification of the permethylated [14C]sialic acid of the product by radioautography. At 0.5 mM N-ethylmaleimide, the SAT-2 activity was inhibited 50% whereas SAT-1 and SAT-3 activities (Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. (1982) J. Biol. Chem. 257, 12765-12769) remained uninhibited.     

Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

2-Deoxyglucose Incorporation into Rat Brain Glycogen During Measurement of Local Cerebral Glucose Utilization by the 2-Deoxyglucose Method

The incorporation of 14C into glycogen in rat brain has been measured under the same conditions that exist during the measurement of local cerebral glucose utilization by the autoradiographic 2-[14C]deoxyglucose method. The results demonstrate that approximately 2% of the total 14C in brain 45 min after the pulse of 2-[14C]deoxyglucose is contained in the glycogen portion, and, in fact, incorporated into alpha-1-4 and alpha-1-6 deoxyglucosyl linkages. When the brain is removed by dissection, as is routinely done in the course of the procedure of the 2-[14C]deoxyglucose method to preserve the structure of the brain for autoradiography, the portion of total brain 14C contained in glycogen falls to less than 1%, presumably because of postmortem glycogenolysis which restores much of the label to deoxyglucose-phosphates. In any case, the incorporation of the 14C into glycogen is of no consequence to the validity of the autoradiographic deoxyglucose method, not because of its small magnitude, but because 2-[14C]deoxyglucose is incorporated into glycogen via [14C]deoxyglucose-6-phosphate, and the label in glycogen represents, therefore, an additional "trapped" product of deoxyglucose phosphorylation by hexokinase. With the autoradiographic 2-[14C]deoxyglucose method, in which only total 14C concentration in the brain tissue is measured by quantitative autoradiography, it is essential that all the labeled products derived directly or indirectly from [14C]deoxyglucose phosphorylation by hexokinase be retained in the tissue; their chemical identity is of no significance.     

Three-dimensional structural analysis of the group B polysaccharide of Neisseria meningitidis 6275 by two-dimensional NMR: the polysaccharide is suggested to exist in helical conformations in solution

The solution conformations of the group B polysaccharide of Neisseria meningitidis were analyzed by DQF-COSY and pure absorption 2D NOE NMR with three mixing times. The pyranose ring of the sialic acid residue was found to be in the 2C5 conformation. The DQF-COSY analysis indicated that the orientations of H6 and H7 and of H7 and H8 are both gauche. In order to overcome the difficulties in analyzing the NOE data due to the two sets of proton overlaps, molecular modeling of alpha-2,8-linked sialic acid oligomers was carried out to investigate possible conformers, and theoretical NOE calculations were performed by using CORMA (complete relaxation matrix analysis). Our analysis suggests that the polysaccharide adopts helical structures for which the phi (defined by O6-C2-O8-C8) and psi (C2-O8-C8-C7) angles are in the following ranges: phi -60 to 0 degrees, psi 115-175 degrees or phi 90-120 degrees, psi 55-175 degrees. The weak affinity of anti-B antibodies for smaller alpha-2,8-linked oligosaccharides may be due to the fact that such oligomers are more flexible and may not form an ordered structure as the poly(sialic acid) does.