Acid-catalyzed Lactonization of α2,8-Linked Oligo/Polysialic Acids Studied by High Performance Anion-exchange Chromatography

Background:The sialic acid N-glycolylneuraminic acid (Neu5Gc) shows conserved suppression of expression in vertebrate brains, suggesting brain-specific toxicity. Results: ␣2-8-Linked Neu5Gc incorporated into the neural glycan polysialic acid (polySia) resists sialidase breakdown through conformational effects. Conclusion: Neu5Gc in brain would prevent rapid turnover of surface polySia. Significance: This mechanism potentially underlies the evolutionary suppression of Neu5Gc expression in vertebrate brains.

Section: ␣2-8-linked Polymers Of Neu5gc Are More Sensitive To Acid Hymentioning

confidence: 99%

“…It has been previously shown that at low pH, the polymers of Sia will form lactones that stabilize the molecule to acid breakdown (107). Modeling of a poly-Neu5Ac lactone showed that and angles of Ϫ45°and Ϫ50°and 56°and 63°, respectively, would be required within the transition state of lactonization (Fig.…”

Section: Statementioning

confidence: 99%

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

Davies

Pearce

Tessier

et al. 2012

“…lactonization of ␣2,8-linked oligomeric sialic acid (33,34), the carboxyl group of the nonreducing end sialic acid may exist as an open form (Fig. 9).…”

Section: Figmentioning

confidence: 99%

Expression of α2,8/2,9-Polysialyltransferase fromEscherichia coli K92

Shen¹,

Datta²,

Izumi

et al. 1999

The capsular polysaccharide of Escherichia coli K92 contains alternating -8-NeuAc␣2-and -9-NeuAc␣2-linkages. The enzyme catalyzing this polymerizing reaction has been cloned from the genomic DNA of E. coli K92. The 1.2-kilobase polymerase chain reaction fragment was subcloned in pRSET vector and the protein was expressed in the BL21(DE3) strain of E. coli with a hexameric histidine at its N-terminal end. The enzyme was isolated in the supernatant after lysis of the cells and fractionated by ultracentrifugation. Western blotting using anti-histidine antibody showed the presence of a band that migrated at about 47.5 kDa on both reducing and nonreducing SDS-polyacrylamide gel electrophoresis, indicating a monomeric enzyme. Among the carbohydrate acceptors tested, N-acetylneuraminic acid and the gangliosides G D3 and G Q1b were preferred substrates. The cell-free enzyme reaction products obtained were characterized by NMR and mass spectrometry, which indicated the presence of both ␣2,9-and ␣2,8-linked polysialyl structure. The K92 neuS gene was used to transform the K1 strain of E. coli, the capsule of which contains only -8-NeuAc␣2-linkages. Analysis of the polysaccharides isolated from these transformed cells is consistent with the presence of both -8-NeuAc␣2-and -9-NeuAc␣2-linkages. Our results suggest that the neuS gene product of E. coli K92 catalyzes the synthesis of polysialic acid with ␣2,9-and ␣2,8-linkages in vitro and in vivo.

“…Analysis of polySia by HPAEC with pulsed electrochemical detector (PED) was as described previously (28). To improve the yield of higher polymers, lyophilized samples (12 g of Neu5Ac) were first treated with 10 l of 1 M HCl at ambient temperature for 2 h to facilitate lactonization of polySia (32), dried on a centrifugal vacuum evaporator, and then subjected to controlled hydrolysis in 100 l of 0.1 M acetic acid for 15 min at 60°C. To the hydrolysate, 50 l of 0.5 M NaOH was added and a 100-l portion (8 g of Neu5Ac) was injected into a CarboPac PA-100 column.…”

Section: Preparation Of Sialoglycopeptide Fractions From Embryonic Chmentioning

confidence: 99%

Chemical Analysis of the Developmental Pattern of Polysialylation in Chicken Brain

Inoue

Lin

Inoue

2000

Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40-50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Ac␣2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both diand polySia were found to show distinctive changes during embryonic development.Polysialic acid (polySia) 1 is a structurally and functionally unique glycotope expressed on the surface of living cells (1). In higher vertebrates, the ␣2,8-linked homopolymer of Neu5Ac is the only reported structure of polySia, although diverse structures of polySia differing in C-5 substitution of Sia residues and in the inter-residue linkages have been discovered in bacteria, invertebrates, and lower vertebrates (2). In embryonic vertebrate brain, the neural cell adhesion molecule (N-CAM) is a major carrier protein of polySia. A hypothesis that the presence of polySia on N-CAM attenuates the adhesive function of this molecule (3, 4) is supported by temporally regulated expression of polySia on embryonic N-CAM, its spatially limited expression in the olfactory bulb, hippocampus, and cerebellum of adult mammalian brain (where continuous plasticity is required). PolySia is also an oncodevelopmental antigen that is re-expressed on a number of human tumors, including neuroblastomas (5) and Wilms tumor (6). The presence of polySia on N-CAM not only functions as a negative regulator of N-CAMmediated homotypic cell-cell adhesion but also decreases interactions with other cells. Although the molecular details of how polySia affects cell interactions has not been fully elucidated, it has been hypothesized to depend on the physical properties of this negatively charged and heavily hydrated polymer (7,8).Recent studies have shown that two polysialyltransferases (polySTs), designated PST-1 (PST/ST8SiaIV) and STX (ST8SiaII), catalyze the polysialylation of N-CAM. The genes encoding both enzymes have been cloned from several species and sequenced (9 -13). The availability of cDNAs encoding these enzymes has facilitated new approaches to study the function, mechanism, and regulation of polysial...