Chemical Diversity in the Sialic Acids and Related α-Keto Acids: An Evolutionary Perspective

Sialic acids are a subset of nonulosonic acids, which are nine-carbon α-keto aldonic acids. Natural existing sialic acid-containing structures are presented in different sialic acid forms, various sialyl linkages, and on diverse underlying glycans. They play important roles in biological, pathological, and immunological processes. Sialobiology has been a challenging and yet attractive research area. Recent advances in chemical and chemoenzymatic synthesis, as well as large-scale E. coli cell-based production, have provided a large library of sialoside standards and derivatives in amounts sufficient for structure−activity relationship studies. Sialoglycan microarrays provide an efficient platform for quick identification of preferred ligands for sialic acid-binding proteins. Future research on sialic acid will continue to be at the interface of chemistry and biology. Research efforts not only will lead to a better understanding of the biological and pathological importance of sialic acids and their diversi...

Advances in the Biology and Chemistry of Sialic Acids

A multifunctional sialyltransferase has been cloned from Pasteurella multocida strain P-1059 and expressed in E. coli as a truncated C-terminal His6-tagged recombinant protein (tPm0188Ph). Biochemical studies indicate that the obtained protein is (1) an alpha2,3-sialyltransferase (main function), (2) an alpha2,6-sialyltransferase, (3) an alpha2,3-sialidase, and (4) an alpha2,3-trans-sialidase. The recombinant tPm0188Ph is a powerful tool in the synthesis of structurally diverse sialoside libraries due to its relaxed substrate specificity, high solubility, high expression level, and multifunctionality.

A multifunctional Pasteurella multocida sialyltransferase: a powerful tool for the synthesis of sialoside libraries.

Recent advances in o-sialylation.

The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.

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The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.

Sialyltransferase 0160, a bacterial sialyltransferase which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6, is produced by Photobacterium damsela JT0160. The gene coding for sialyltransferase 0160 (bst) was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase 0160 gene contains an open reading frame of 2,028 base pairs encoding a protein of 675 amino acid residues. The deduced amino acid sequence of sialyltransferase 0160 did not contain the sialylmotif and had no significant similarity to mammalian sialyltransferases. Crude extracts of cultured E. coli MV1184 cells carrying an expression plasmid for the sialyltransferase 0160 gene showed sialyltransferase activity, which was identified as beta-galactoside alpha2,6-sialyltransferase activity by enzymatic reaction product analysis. In addition, when mutant genes, lacking 3'-coding regions for COOH-terminal portions of the protein, which are thought to form alpha-helix structures, were expressed in E. coli MV1184, soluble-form enzymes were obtained. This implies that the COOH-terminal portion of sialyltransferase 0160 is required for membrane binding.

Cloning and Expression of a Marine Bacterial β-Galactoside α2, 6-Sialyltransferase Gene from Photobacterium damsela JT0160

A bacterial sialyltransferase, named sialyltransferase 0160, was purified from a marine bacterium that had been isolated from seawater from Sagami Bay, Kanagawa. This strain has been identified as Photobacterium damsela, and named P. damsela JT0160. Sialyltransferase 0160 was purified 688-fold to homogeneity from the crude extract of the cells with a yield of 19% using a combination of anion exchange chromatography, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography. The purified enzyme migrated as a single band (61 kDa) on sodium dodecyl sulfate-polyacrylamide gel. This sialyltransferase was found to be a beta-galactoside alpha 2,6-sialyltransferase [EC 2.4.99.1] which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6 on the basis of an analysis of the enzymatic reaction products with HPLC, 1H-, 13C-NMR spectroscopy, and fast atom bombardment mass spectroscopy.

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Purification and Characterization of a Marine Bacterial .BETA.-Galactoside .ALPHA.2,6-Sialyltransferase from Photobacterium damsela JT0160.

The substrate specificity and enzymatic sialylation ability of the bacterium α-2,6-sialyltransferase were examined. The enzyme assay displayed a remarkable ability to catalyze sialyl transfer to type-II oligosaccharides possessing fucoside or sialoside at the 2 or 3 position of the terminal galactoside. Enzymatic syntheses were performed in order to confirm the structure of unusual assay products found when using Neu5Ac β2,3Galβ1,4Glc and Fuc α1,2Galβ1,4Glc as the sialyl acceptors. Both sialylation reactions (10 μmol scales) were run using 83 munits of enzyme, were complete in 2 h, and afforded the sialoside analogues Neu5Ac α2,6(Fuc α1,2) Galβ1,4Glc (88%) and Neu5Ac α2,6(Neu5Ac β2,3) Galβ1,4Glc (92%).

A Novel α-2,6-Sialyltransferase: Transfer of Sialic Acid to Fucosyl and Sialyl Trisaccharides

This invention provides to a novel gene encoding a protein aving the activity of beta-galactoside-alpha2,6-sialyltransferase.The gene of the invention encodes a protein which having the amino acid sequence of SEQ ID NO:1, or a protein having the amino acid sequence of SEQ ID NO:1 which have been modified by deletion, substitution or addition of at least one amino acid residue in said sequence, while maintaining substantially the same beta-galactoside-alpha2,6-sialyltransferase activity.The present invention uses said gene to further provide a vector for expressing a protein having the beta-galactoside-alpha2,6-sialyltransferase activity, host cells and a recombinant protein. The protein encoded by the gene of the invention does not have any substantial homology with known sialyltransferases, and in addition, the membrane binding region is located in the C-terminal unlike known sialyltransferases.

beta-galactoside-alpha2, 6-sialyltransferase gene

A novel gene encoding a protein having the beta -galactoside alpha -2,6-sialyltransferase activity. This gene has the amino acid sequence represented by SEQ ID NO:1 optionally undergoing the deletion, substitution or addition of one or more amino acids and encodes the protein with the beta -galactoside alpha -2,6-sialyltransferase activity. The invention further provides expression vectors for the beta -galactoside alpha -2,6-sialyltransferase protein with the use of the above gene and host cells and recombinant proteins thereof. The amino acid sequence of the enzyme encoded by the above gene is not homologous with that of the conventionally known sialyltransferases. Moreover, the membrane-binding region thereof is located at the C-end differing from those of the conventionally known enzymes.

Motoko Nakashizuka

Papers

Cloning and Expression of a Marine Bacterial β-Galactoside α2, 6-Sialyltransferase Gene from Photobacterium damsela JT0160

Purification and Characterization of a Marine Bacterial .BETA.-Galactoside .ALPHA.2,6-Sialyltransferase from Photobacterium damsela JT0160.

A Novel α-2,6-Sialyltransferase: Transfer of Sialic Acid to Fucosyl and Sialyl Trisaccharides

beta-galactoside-alpha2, 6-sialyltransferase gene

Gene encoding beta-galactoside alpha-2,6-sialyltransferase