Hitomi Kajiwara

Tamavidin, a versatile affinity tag for protein purification and immobilization

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity and is highly produced in soluble form in Escherichia coli. By contrast, widely used biotin-binding proteins avidin and streptavidin are rarely produced in soluble form in E. coli. In this study, we describe an efficient system for one-step purification and immobilization of recombinant proteins using tamavidin 2 as an affinity tag. A bacterial sialyltransferase and soybean agglutinin were fused to tamavidin 2 and expressed in E. coli and tobacco BY-2 cells, respectively. High-level expressions of the fusion proteins were detected (80 mg l(-1)E. coli culture for bacterial sialyltransferase-tamavidin 2 and 2 mg l(-1) BY-2 cell culture for soybean agglutinin-tamavidin 2). To immobilize and purify the fusion proteins, biotinylated magnetic microbeads were incubated with the soluble extract from each recombinant host producing the fusion protein and then washed thoroughly. As the result, both fusion proteins were immobilized tightly on the microbeads without substantial loss of activity and simultaneously highly purified (90-95% purity) on the microbeads. Biotin with a longer linker contributed to higher affinity between the fusion protein and biotin. These results suggest that tamavidin fusion technology is a powerful tool for production, purification, and immobilization of recombinant proteins.

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β‐Galactoside α2,3‐sialyltransferase produced by Photobacterium phosphoreum JT‐ISH‐467 is a unique enzyme that catalyzes the transfer of N‐acetylneuraminic acid residue from cytidine monophosphate N‐acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono‐ and di‐saccharides as acceptor substrates, and can transfer Neu5Ac to both α‐galactoside and β‐galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3‐sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase‐B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.

An α2,6-sialyltransferase cloned from Photobacterium leiognathi strain JT-SHIZ-119 shows both sialyltransferase and neuraminidase activity

We cloned, expressed, and characterized a novel beta-galactoside alpha2,6-sialyltransferase from Photobacterium leiognathi strain JT-SHIZ-119. The protein showed 56-96% identity to the marine bacterial alpha2,6-sialyltransferases classified into glycosyltransferase family 80. The sialyltransferase activity of the N-terminal truncated form of the recombinant enzyme was 1477 U/L of Escherichia coli culture. The truncated recombinant enzyme was purified as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis through 3 column chromatography steps. The enzyme had distinct activity compared with known marine bacterial alpha2,6-sialyltransferases. Although alpha2,6-sialyltransferases cloned from marine bacteria, such as Photobacterium damselae strain JT0160, P. leiognathi strain JT-SHIZ-145, and Photobacterium sp. strain JT-ISH-224, show only alpha2,6-sialyltransferase activity, the recombinant enzyme cloned from P. leiognathi strain JT-SHIZ-119 showed both alpha2,6-sialyltransferase and alpha2,6-linkage-specific neuraminidase activity. Our results provide important information toward a comprehensive understanding of the bacterial sialyltransferases belonging to the group 80 glycosyltransferase family in the CAZy database.

Sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates

Bacterial sialyltransferases (STs) from marine sources were characterized using glycosphingolipids (GSLs). Bacterial STs were found to be beta-galacotoside STs. There were two types of STs: (1) ST obtained from strains such as ishi-224, 05JTC1 (#1), ishi-467, 05JTD2 (#2), and faj-16, 05JTE1 (#3), which form alpha2-3 sialic acid (Sia) linkages, named alpha2-3ST, (2) ST obtained from strains such as ISH-224, N1C0 (#4), pda-rec, 05JTB2 (#5), and pda-0160, 05JTA2 (#6), which form alpha2-6 Sia linkages, named alpha2-6ST. All STs showed affinity to neolacto- and lacto-series GSLs, particularly in neolactotetraosyl ceramide (nLc(4)Cer). No large differences were observed in the pH and temperature profiles of enzyme activities. Kinetic parameters obtained by Lineweaver-Burk plot analysis showed that #3 and #4 STs had practical synthetic activity and thus it became easily possible to achieve large-scale ganglioside synthesis (100-300 muM) using these recombinant enzymes. Gangliosides synthesized from nLc(4)Cer by alpha2-3 and alpha2-6STs were structurally characterized by several analytical and immunological methods, and they were identified as IV(3)alphaNeuAc-nLc(4)Cer(S2-3PG) and IV(6)alphaNeuAc-nLc(4)Cer (S2-6PG), respectively. Further characterization of these STs using lactotetraosylceramide (Lc(4)Cer), neolactohexaosylceramide (i antigen), and IV(6)kladoLc(8)Cer (I antigen) showed the synthesis of corresponding gangliosides as well. Synthesized gangliosides showed binding activity to the influenza A virus [A/panama/2007/99 (H3N2)] at a similar level to purified S2-3PG and S2-6PG from mammalian sources. The above evidence suggests that these STs have unique features, including substrate specificities restricted to lacto- and neolactoseries GSLs, as well as catalytic potentials for ganglioside synthesis. This demonstrates that efficient in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing Sias modifications, thereby permitting the exploration of unknown functions.

An α2,3-Sialyltransferase from Photobacterium sp. JT-ISH-224 Transfers N-Acetylneuraminic Acid to Both the O-2 and O-3′ Hydroxyl Groups of Lactose

We found that α2,3-sialyltransferase from Photobacterium sp. JT-ISH-224 produced a regio-mistaken sialyl-transferred by-product. Spectroscopic analysis of the purified by-product indicated that it contained two N-acetylneuraminic acids: one attached to the O-3′ hydroxyl group of lactose, and the other attached to the O-2 hydroxyl group of lactose. The relative configuration between the C-1 and C-3 of the α-glucopyranose residue is superimposable with that between C-4 and C-2 of galactopyranoside. Therefore, formation of this by-product, designated 2,3′-disialyllactose, was simply rationalized as a regio-mistaken reaction of bacterial α2,3-sialyltransferase. This finding indicates that this bacterial α2,3-sialyltransferase has a possibility to synthesize several unusual sialosides.

Loss-of-Function Mutation in Bi-Functional Marine Bacterial Sialyltransferase

An α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a bi-functional enzyme showing both α2,3-sialyltransferase and α2,3-linkage specific sialidase activity. To date, the crystal structures of several sialyltransferases have been solved, but the roles of amino acid residues around the catalytic site have not been completely clarified. Hence we performed a mutational study using α2,3-sialyltransferase cloned from P. phosphoreum JT-ISH-467 as a model enzyme to study the role of the amino acid residues around the substrate-binding site. It was found that a mutation of the glutamic acid at position 342 in the sialyltransferase resulted in a loss of sialidase activity, although the mutant showed no decrease in sialyltransferase activity. Based on this result, it is strongly expected that the Glu342 of the enzyme is an important amino acid residue for sialidase activity.

Purification, crystallization and preliminary crystallographic characterization of the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224

Crystallization of the α2,6-sialyltransferase from Photobacterium.

Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases.

On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.

Isolation of fucosyltransferase-producing bacteria from marine environments.

Fucose-containing oligosaccharides on the cell surface of some pathogenic bacteria are thought to be important for host-microbe interactions and to play a major role in the pathogenicity of bacterial pathogens. Here, we screened marine bacteria for glycosyltransferases using two methods: a one-pot glycosyltransferase assay method and a lectin-staining method. Using this approach, we isolated marine bacteria with fucosyltransferase activity. There have been no previous reports of marine bacteria producing fucosyltransferase. This paper thus represents the first report of fucosyltransferase-producing marine bacteria.

A CMP-N-acetylneuraminic Acid Synthetase Purified from a Marine Bacterium, Photobacterium leiognathi JT-SHIZ-145

A cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase was found in a crude extract prepared from Photobacterium leiognathi JT-SHIZ-145, a marine bacterium that also produces a β-galactoside α2,6-sialyltransferase. The CMP-Neu5Ac synthetase was purified from the crude extract of the cells by a combination of anion-exchange and gel filtration column chromatography. The purified enzyme migrated as a single band (60 kDa) on sodium dodecylsulfate–polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at 35 °C at pH 9.0, and the synthetase required Mg2+ for activity. Although these properties are similar to those of other CMP-Neu5Ac synthetases isolated from bacteria, this synthetase produced not only CMP-Neu5Ac from cytidine triphosphate and Neu5Ac, but also CMP-N-glycolylneuraminic acid from cytidine triphosphate and N-glycolylneuraminic acid, unlike CMP-Neu5Ac synthetase purified from Escherichia coli.