Photobacterium sp. JT-ISH-224 produces two sialyltransferases, alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-sialyltransferase and an alpha2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-sialyltransferase, whereas the alpha2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).     

A beta-galactoside alpha2,6-sialyltransferase produced by a marine bacterium, Photobacterium leiognathi JT-SHIZ-145, is active at pH 8

A gene encoding a sialyltransferase produced by Photobacterium leiognathi JT-SHIZ-145 was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase gene contained an open reading frame of 1494 base pairs (bp) encoding a predicted protein of 497 amino acid residues. The deduced amino acid sequence of the sialyltransferase had no significant similarity to mammalian sialyltransferases and did not contain sialyl motifs, but did show high homology to another marine bacterial sialyltransferase, a beta-galactoside alpha2,6-sialyltransferase produced by P. damselae JT0160. The acceptor substrate specificity of the new enzyme was similar to that of the alpha2,6-sialyltransferase from P. damselae JT0160, but its activity was maximal at pH 8. This property is quite different from the properties of all mammalian and bacterial sialyltransferases reported previously, which have maximal activity at acidic pH. In general, both sialosides and cytidine-5'-monophospho-N-acetylneuraminic acid, the common donor substrate of sialyltransferases, are more stable under basic conditions. Therefore, a sialyltransferase with an optimum pH in the basic range should be useful for the preparation of sialosides and the modification of glycoconjugates, such as asialo-glycoproteins and asialo-glycolipids. Thus, the sialyltransferase obtained from P. leiognathi JT-SHIZ-145 is a promising tool for the efficient production of sialosides.     

Molecular cloning, expression and properties of an alpha/beta-Galactoside alpha2,3-sialyltransferase from Vibrio sp. JT-FAJ-16

We cloned, expressed and characterized a novel alpha/beta-galactoside alpha2,3-sialyltransferase from Vibrio sp. bacterium JT-FAJ-16. Using a alpha2,3-sialyltransferase gene from a marine bacterium as a probe, a DNA sequence encoding a 402-amino-acid protein was identified from the JT-FAJ-16 genomic library. The protein showed 27.3-64.7% identity to the bacterial sialyltransferases classified into glycosyltransferase family 80. The protein showed sialyltransferase activity when expressed in Escherichia coli. The N-terminal truncated form of the enzyme was amplified in E. coli and its recovered activity was 215.7 unit/l culture medium. It was purified as a single band on SDS-PAGE through the three chromatographic steps. The specific activity of the purified recombinant enzyme reached 57.5 unit/mg protein. The alpha2,3sialylation was confirmed by (1)H- and (13)C-NMR analyses of the reaction products. The enzyme was optimally active at pH 5.5 and at 20 degrees C. Interestingly, the enzyme used both the alpha- and beta-anomers of galactosides as acceptors, suggesting that it can be described as an alpha/beta-galactoside alpha2,3-sialyltransferase. The enzyme had a wide range of acceptor substrate specificities. It transferred N-acetylneuraminic acid (NeuAc) to various monosaccharides and various oligosaccharides, and both N-linked and O-linked asialo-glycoprotein. These results suggest that the enzyme can be used as a powerful tool for the study for glycotechnology.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.     

Purification of lumazine proteins from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties

Bright strains of the marine bioluminescent bacterium Photobacterium leiognathi produce a "lumazine protein" in amounts comparable to that previously found in Photobacterium phosphoreum. New protocols are developed for the purification to homogeneity of the proteins from both species in yields up to 60%. In dimmer strains the amounts of lumazine protein in extracts are less, and also there is an accompanying shift of the bioluminescence spectral maximum to longer wavelength, 492 nm. Both types of lumazine proteins have identical fluorescence spectra, with maxima at 475 nm, so it is suggested that, whereas lumazine protein is the major emitter in bright strains, there is a second emitter also present with a fluorescence maximum at longer wavelength. The two species of lumazine protein have the same 276 nm/visible absorbance ratio, 2.2, but differ in visible maxima: P. phosphoreum, 417 nm; P. leiognathi, 420 nm. For the latter the bound lumazine has epsilon 420 = 10 100 M-1 cm-1, practically the same as in free solution. The two lumazine proteins also differ quantitatively in their effect on the in vitro bioluminescence reaction, i.e., at blue shifting the bioluminescence spectrum or altering the kinetics. The P. phosphoreum lumazine protein is more effective with its homologous luciferase or with P. leiognathi luciferase than is the lumazine protein from P. leiognathi. These differences may have an electrostatic origin.     

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae

We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.     

Identification and functional expression of a second human beta-galactoside alpha2,6-sialyltransferase, ST6Gal II.

BLAST analysis of the human and mouse genome sequence databases using the sequence of the human CMP-sialic acid:beta-galactoside alpha-2,6-sialyltransferase cDNA (hST6Gal I, EC2.4.99.1) as a probe allowed us to identify a putative sialyltransferase gene on chromosome 2. The sequence of the corresponding cDNA was also found as an expressed sequence tag of human brain. This gene contained a 1590 bp open reading frame divided in five exons and the deduced amino-acid sequence didn't correspond to any sialyltransferase already known in other species. Multiple sequence alignment and subsequent phylogenic analysis showed that this new enzyme belonged to the ST6Gal subfamily and shared 48% identity with hST6Gal-I. Consequently, we named this new sialyltransferase ST6Gal II. A construction in pFlag vector transfected in COS-7 cells gave raise to a soluble active form of ST6Gal II. Enzymatic assays indicate that the best acceptor substrate of ST6Gal II was the free disaccharide Galbeta1-4GlcNAc structure whereas ST6Gal I preferred Galbeta1-4GlcNAc-R disaccharide sequence linked to a protein. The alpha2,6-linkage was confirmed by the increase of Sambucus nigra agglutinin-lectin binding to the cell surface of CHO transfected with the cDNA encoding ST6Gal II and by specific sialidases treatment. In addition, the ST6Gal II gene showed a very tissue specific pattern of expression because it was found essentially in brain whereas ST6Gal I gene is ubiquitously expressed.     

Crystallization and preliminary X-ray diffraction studies of a flavoprotein, FP390, from a luminescent bacterium, Photobacterium phosphoreum.

A flavoprotein, FP390, obtained from a luminescent bacterium, Photobacterium phosphoreum, in the purification of luciferase has been crystallized by the vapor-diffusion procedure. Crystals obtained from polyethylene glycol 4000 solutions, whose X-ray photographs show powder diffraction patterns, were unsuitable for further crystallographic work. However, tetragonal crystals grown from potassium phosphate solution well diffracted X-rays beyond 3 A resolution. The space group of this crystal is P4(1)22 or P4(3)22 with unit-cell dimensions of a = b = 76.8 and c = 241 A. Assuming two or three molecules in an asymmetric unit, the value for the crystal volume per unit molecular mass, Vm, is calculated as 3.3 or 2.2 A3/Da, respectively. A total of 13,555 independent reflections for the native crystal was collected up to 3 A resolution using a Weissenberg camera attached to the synchrotron radiation source, the merging R factor being 0.077 for 79,335 measurements.     

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.     

Purification and properties of an alginate lyase from a marine bacterium.

An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.     

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 Mg(2+) 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.     

Molecular cloning and expression of Galbeta1,3GalNAc alpha2, 3-sialyltransferase from human fetal liver.

Based on the sequences of the highly conserved segments in the previously cloned sialyltransferases, a cDNA encoding Galbeta1, 3GalNAc alpha2,3-sialyltransferase (SIATFL) has been isolated from human fetal liver. Expression analysis of the gene has been performed with various carcinoma cell lines, fetal tissues, fetal and adult liver and both hepatoma and the surrounding tissue from the same liver. The SIATFL gene was expressed poorly in fetal liver and in adult liver, slightly in hepatoma and highly in the surrounding tissue of hepatoma. The cDNA encoding the putative active domain was expressed in COS-1, Escherichia coli, and Pichia pastoris. The recombinant protein expressed in COS-1 could catalyse the transfer of NeuAc from CMP-NeuAc to asialo-fetuin. No enzyme activity was detected with a 32-kDa protein in E. coli and both 32-kDa and 41-kDa proteins in P. pastoris. These results suggested that correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity.     

Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.     

Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides

Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.     

Purification and partial characterization of an alpha-2,8-sialyltransferase from human erythroleukemia K562 cells.

An alpha-2,8-sialyltransferase (ST8), the enzyme involved in the biosynthesis of polysialic acid chains, has been purified and partly characterized from undifferentiated human erythroleukemia K562 cells. Purification, based on a key step of affinity chromatography utilizing immobilized colominic acid, was greater than 1000-fold. The enzyme molecular weight determined by SDS-PAGE was estimated to be about 40 kDa, in good agreement with literature data. For the determination of the main kinetic parameters (Vmax and K(M)), fetuin turned out to be the unique substrate acceptor. In fact, other compounds such as asialofetuin, transferrin, alpha1-acid glycoprotein, and G(M3), routinely used to explore the different ST8 isoforms' activities, did not serve as substrate acceptors. In all cases, contrary to the routinely adopted protocol where a radioactive substrate donor is employed, for our purpose a non-radioactive, fluorescent substrate donor such as cytidine-5'-monophospho-9-(3-fluoresceinylthioureido)-9-deoxy-N-acetyl-neuraminic acid (CMP-9-fluoresceinyl-NeuAc) was used. Thus, under our experimental conditions, by using fetuin, data reported in a typical Lineweaver-Burk plot gave a Vmax value of about 4 nkatal/mg of protein and a K(M) value around 0.61 mM. Just as with the estimated molecular weight, these kinetic data were also in good agreement with those already reported for the ST8 purified from human neuroblastoma CHP-134 cells. In particular, in both cases, Vmax values were almost similar (4 nkatal/mg of protein for our ST8 purified from K562 cells and 4.35 nkatal/mg of protein for ST8 purified from CHP-134 cells); conversely, the K(M) value we found was about 3.25-fold lower than that found by Stoykova and Glick (0.61 mM vs. 2 mM). Then, although our purification was lower than that obtained by Stoykova and Glick (1080-fold vs. 2910-fold), the enzyme we purified showed a greater apparent affinity.     

Purification and characterization of alanine dehydrogenase from a marine bacterium, Vibrio proteolyticus

Alanine dehydrogenase of Vibrio proteolyticus DSM 30189 shows high activity toward β-hydroxypyruvate, and the enzyme is applicable to the production of -serine. We have cloned the enzyme gene from the bacterium into Escherichia coli TG1 with a vector plasmid, pUC118. The enzyme was overproduced by the transformed cells and purified to homogeneity with a yield of 46%. The molecular mass of the enzyme was about 230 kDa and consisted of six identical subunits. The enzyme showed broad specificity toward α-keto acids in the reductive amination. The relative activities of the enzyme for pyruvate, β-fluoropyruvate, and β-hydroxypyruvate were 100%, 74%, and 54%, respectively. The enzyme retained more than 90% of the activity after incubation at 65 °C for 60 min in the presence of 2.0 M NaCl, but 98% of its original activity was lost in the absence of NaCl. RbCl, as well as NaCl, significantly stabilized the enzyme. On the other hand, LiCl and KCl were not as effective as stabilizers such as RbCl and NaCl.     

Isolation and identification of Photobacterium phosphoreum from an unexpected niche: migrating salmon

Six luminous bacteria were isolated from migrating salmon in the Yukon River, Alaska. All isolates were identified as Photobacterium phosphoreum. Previous studies suggest that P. phosphoreum is an exclusively marine bacterium, while our Alaskan isolates are from salmon which migrated up to 1,228 km from the marine environment.