Decreasing the sialidase activity of multifunctional Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) by site-directed mutagenesis

Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) is a multifunctional enzyme which has α2-6-sialyltransferase, α2-3-sialidase, and α2-3-trans-sialidase activities in addition to its major α2-3-sialyltransferase activity. The presence of the α2-3-sialidase activity of PmST1 complicates its application in enzymatic synthesis of α2-3-linked sialosides as the product formed can be hydrolyzed by the enzyme. Herein we show that the α2-3-sialidase activity of PmST1 can be significantly decreased by protein crystal structure-based site-directed mutagenesis. A PmST1 double mutant E271F/R313Y showed a significantly (6333-fold) decreased sialidase activity without affecting its α2-3-sialyltransferase activity. The double mutant E271F/R313Y, therefore, is a superior enzyme for enzymatic synthesis of α2-3-linked sialosides.     

Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmST_WT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmST_E271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmST_E271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.     

PmST3 from <Emphasis Type="Italic">Pasteurella multocida</Emphasis> encoded by <Emphasis Type="Italic">Pm1174</Emphasis> gene is a monofunctional α2–3-sialyltransferase

Pasteurella multocida (Pm) strain Pm70 has three putative sialyltransferase genes including Pm0188, Pm0508, and Pm1174. A Pm0188 gene homolog in Pm strain P-1059 encodes a multifunctional α2–3-sialyltransferase, PmST1, that prefers oligosaccharide acceptors. A Pm0508 gene homolog in the same strain encodes a monofunctional sialyltransferase PmST2 that prefers glycolipid acceptors. Here, we report that the third sialyltransferase from Pm (PmST3) encoded by gene Pm1174 in strain Pm70 is a monofunctional α2–3-sialyltransferase that can use both oligosaccharides and glycolipids as efficient acceptors. Despite the existence of both Pm0188 and Pm0508 gene homologs encoding PmST1 and PmST2, respectively, in Pm strain P-1059, a Pm1174 gene homolog appears to be absent from Pm strains P-1059 and P-934. PmST3 was successfully obtained by cloning and expression using a synthetic gene of Pm1174 with codons optimized for Escherichia coli expression system as the DNA template for polymer chain reactions. Truncation of 35 amino acid residues from the carboxyl terminus was shown to improve the expression of a soluble and active enzyme in E. coli as a C-His₆-tagged fusion protein. This sialidase-free monofunctional α2–3-sialyltransferase is a useful tool for synthesizing sialylated oligosaccharides and glycolipids.     

3-(3-Phenoxybenzyl)amino-β-carboline: a novel antitumor drug targeting α-tubulin

3-(3-Phenoxybenzyl)amino-β-carboline 2h showed extremely-high activity; the IC₅₀ value was 0.074 μM. To verify 2h-induced cell death types, we observed the chromatin condensation, the DNA fragmentation and activated caspase-3 using Hoechst 33342, agarose electrophoresis and western blot, and suggesting 2h-induced cell death type was apoptosis. Flow cytometry showed that 2h-treated cell was induced SubG1 cell population after G2/M cell cycle arrest. In addition, using affinity chromatography and peptide mass fingerprinting, we found that interacting protein with this compound was α-tubulin protein.     

Enzymatic characterization of CMP-NeuAc:Galβ1-4GlcNAc-R α(2-3)-sialyltransferase from human placenta

In this report we present the enzymatic characterization of CMP-NeuAc:Gal1-4GlcNAc-R (2-3)-sialyltransferase from human placenta using placenta membranes as an enzyme preparation. This sialyltransferase is highly sensitive to detergents and prefers type 2 chain (Gal1-4GlcNAc) over type 1 chain (Gal1-3GlcNAc) acceptors. Oligosaccharides and glycopeptides were better acceptor substrates than glycoproteins. Of the branched oligosaccharides, those with a bisectedN-acetylglucosamine (GlcNAc) structure appeared to be poorer substrates, while triantennary structures containing a Gal1-4GlcNAc1-4Man1-3Man branch were preferred. Product characterization, using 400 MHz¹H-NMR spectroscopy, confirmed that sialic acid was introduced into the Gal1-4GlcNAc-R units of the acceptor substrates in an (2-3) linkage, and revealed that this sialytransferase does not prefer either of the two branches of a complex type diantennary glycopeptide acceptor for sialic acid attachment. These properties distinguish this enzyme from all other sialyltransferases characterized to date.Abbreviations NeuAc N-acetylneuraminic acid - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - GP-F2 and GP-F4 diantennary complex type glycopeptides from desialylated fibrinogen - GP-Trf diantennary complex type glycopeptide from desialylated transferrin - LNT Gal1-3GlcNAc1-3Gal1-4Glc (lacto-N-tetraose) - 6-sialytransferase CMP-NeuAc:Gal1-4GlcNAc-R (2-6)-sialytransferase - 3-sialytransferaseO CMP-NeuAc:Gal1-3GalNAc-R (2-3)-sialyltransferase - 3-sialytransferase I CMP-NeuAc:Gal1-3(4)GlcNAc-R (2-3)-sialyltransferase - 3-sialytransferase II CMP-NeuAc:Gal1-4GlcNAc-R (2-3)-sialytransferase     

Cloning and characterization of a viral α2-3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami? B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-α-D-glucose biosynthesis enzyme

Pasteurella multocida strains are classified into 16 different lipopolysaccharide (LPS) serovars using the Heddleston serotyping scheme. Ongoing studies in our laboratories on the LPS aim to determine the core oligosaccharide (OS) structures expressed by each of the Heddleston type strains and identify the genes and transferases required for the biosynthesis of the serovar-specific OSs. In this study, we have determined the core OS of the LPS expressed by the Heddleston serovar 9 type strain, P2095. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy and mass spectrometry revealing the following structure: . The serovar 9 OS contains an inner core that is conserved among P. multocida strains with an elaborate outer core extension containing rhamnose (Rha), a D-glycero-D-manno isomer of heptose, and the unusual deoxyamino sugar, 3-acetamido-3,6-dideoxy-α-D-glucose (Qui3NAc). Genetic analyses of the LPS outer core biosynthesis locus revealed that in addition to the glycosyltransferases predicted to transfer the sugars to the nascent LPS molecule, the locus also contained the complete set of genes required for the biosynthesis of the nucleotide sugar donors dTDP-Rha and dTDP-Qui3NAc. One of the genes identified as part of the dTDP-Qui3NAc biosynthesis pathway, qdtD, encodes a proposed bi-functional enzyme with N-terminal amino acid identity to dTDP-4-oxo-6-deoxy-D-glucose-3,4-oxoisomerase and C-terminal amino acid identity to dTDP-3-oxo-6-deoxy-α-D-glucose transacetylase.     

Sialyltransferase activity in FR3T3 cells transformed withras oncogene: decreased CMP-Neu5Ac:Galβ1-3GalNAc α-2,3-sialyltransferase

We have investigated the activity of CMP-Neu5Ac:Gal\1-3GalNAc -2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the -galactosidase -2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activatedras gene decreases the activity of this specific -2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of activeO-glycan -2,3-sialyltransferase polypeptides inras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface ofras-transformed FR3T3 suggesting that no change in the sialylation ofO-glycan core 1 appeared in these cells, although the activity of the -2,3-sialyltransferase was decreased.Abbreviations -2,3-ST(O) CMP-Neu5Ac:Gal1-3GalNAc-R -2,3-sialyltransferase - -2,3-ST(N/O) CMP-Neu5Ac:Gal1-3/4GlcNAc-R -2,3-sialyltransferase - -2,6-ST(N) CMP-Neu5Ac:Gal1-4GlcNAc-R -2,6-sialyltransferase - -2,6-ST(O)I CMP-Neu5Ac:R-GalNAc(1-O)Ser -2,6-sialyltransferase - -2,6-ST(O)II CMP-Neu5Ac:Neu5Ac2-3Gal1-3GalNAc-R -2,6-sialyltransferase - ASFet asialofetuin - FR3T3 Fisher rat fibroblast - FRras Ha-ras-transfected FR3T3 fibroblasts - NaCl/P_i sodium phosphate 10mm, NaCl 0.15m, pH 7.4, buffer - pNp p-nitrophenol     

Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2

An aerobic bacterial strain P11-2 with high amylolytic activity was isolated from soil sample collected from wheat field of Jiyuan, China. The strain was identified as Bacillus methylotrophicus by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. The α-amylase was purified to homogeneity by a combination of 80% (NH₄)₂SO₄ precipitation, DEAE FF anion exchange, and superdex 75 10/300 GL gel filtration chromatography. The purified α-amylase exhibited specific activity of 330.7 U/mg protein that corresponds to 13.1 fold purification. The relative molecular mass of the α-amylase was 44.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for enzyme activity were 7.0 and 70 °C, respectively. The α-amylase activity was stimulated by Mg²⁺, Ba²⁺, Al³⁺ and dl-dithiothreitol (DTT), however, Ca²⁺ almost had no activation or inhibition on the α-amylase. After 4 h of reaction toward soluble starch, the end products were glucose, maltose and maltotriose. The 10 residues of the N-terminal sequence of the purified α-amylase were SVKNGQILHA, which showed no homology to other reported α-amylases from Bacillus strain.     

Specific stimulation of α2-6 sialyltransferase activity by a novel cytosolic factor from rat colon

A factor present in the 100 000 g supernatant from the homogenate of rat colon stimulated the activity of purified GaIβ1-4GlcNAc α2,6 sialyltransferase [α2-6ST(N)] from rat liver and α2-6ST(N) from either liver microsomes or Golgi membrane. The stimulation of α2-6ST(N) activity by the colon factor using protein acceptors was about four-fold and highly reproducible when the reaction product of the α2-6ST(N) was assayed by either precipitation or affinity chromatography. In contrast, the colon factor did not stimulate the GaIβ1-4GlcNAc α2,3 sialyltransferase [α2-3ST (N)], from rat jejunum microsomes or purified Galβ1-3GalNAc α2,3 sialyltransferase [α2-3ST (O)] from porcine liver, or purified β1,4 galactosyltransferase (GT) from bovine milk. In addition to rat colon, the 100 000 g supernatant from the homogenates of rat brain and kidney also stimulated the α2-6ST(N) activity. The stimulation of α2-6ST(N) by the colon factor resulted in a decrease in the K_m (by about two-fold) and an increase in V_max (about 2- to 3-fold) for desialylated α₁ acid glycoprotein and CMP-[¹⁴C]N-acetylneuraminic acid. The stimulation of α2-6ST(N) activity by the colon factor was temperature dependent, protease sensitive and was inhibited by CTP, but did not need the presence of either metal ions or detergent. The cytosolic factor was partially purified by ion-exchange chromatography with the retention of the activator activity in the peaks containing low molecular weight proteins, but the activity was lost on attempts to further purification. A specific marked stimulation of the α2-6ST(N) activity by cytosolic factors in certain tissues might suggest a physiological role for these factors in the regulation of α2-6ST(N) activity.     

Synthesis of hexyl α-glucoside and α-polyglucosides by a novel Microbacterium isolate

Alkyl-glucosides and alkyl-polyglucosides are the new-generation biodegradable surfactants with good emulsifying and wetting properties. The α-forms of these glucosides occur in antibiotics and also stimulate nasal absorption of many drugs. In this paper, we report the synthesis of hexyl α-glucoside and α-polyglucosides using cell-bound α-glucosidase activity of a novel strain of Microbacterium paraoxydans. A number of cell-bound glycosyl hydrolase activities were detected in the isolate with the maximum hydrolytic activity of 180 IU g^?1 dry wt cells on p-nitrophenyl-α-d-glucopyranoside. In a micro-aqueous system, at a water activity of 0.69, 1.8 g l^?1 of hexyl α-glucoside (corresponding to about 25 % yield) was synthesized by whole cells with maltose and hexanol as substrates. The concentration was enhanced to 11 g l^?1 (~60 % yield) in a biphasic system at a water content of 60 %. ¹H and ¹³C NMR spectra of the purified compound confirmed the synthesized product to be hexyl-α-d-glucopyranoside, while the presence of hexyl di- and tri-glucosides was confirmed by electrospray ionization mass spectrometry. The cell-driven synthesis makes this an extremely attractive alternative for synthesis of such compounds.     

Characterization of a novel GH2 family α-l-arabinofuranosidase from hyperthermophilic bacterium Thermotoga thermarum

The 2,367-bp ORF of TtAFase from Thermotoga thermarum DSM 5069 encodes a calculated 90-kDa α-l-arabinofuranosidase (TtAFase), which does not belonging to any reported glycosyl hydrolase families α-l-arabinofuranosidases in the database and represents a novel one of glycosyl hydrolase family 2. The purified recombinant TtAFase produced in Escherichia coli BL21 (DE3) had optimum activity at pH 5.5 and at 80 °C. It was stable up to 80 °C and from pH 4.5–8.5. Kinetic experiments at 80 °C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a K _m of 0.77 mM, V _max of 2.3 μmol mg^?1 min^?1 and k _cat of 4.5 s^?1. The enzyme had no apparent requirement of metal ions for activity, and its activity was significantly inhibited by Cu²⁺ or Zn²⁺.     

Prolyl hydroxylase 2: a novel regulator of β2 -adrenoceptor internalization

Adrenergic receptor (AR)-mediated signalling is modulated by oxygen levels. Prolyl hydroxylases (PHDs) are crucial for intracellular oxygen sensing and organism survival. However, it remains to be clarified whether or how PHDs are involved in the regulation of β(2) -adrenoceptor (β(2) -AR) signalling. Here we show that PHD2 can modulate the rate of β(2) -AR internalization through interactions with β-arrestin 2. PHD2 hydroxylates β-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of β-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with β(2) -AR into the cytosol. β(2) -AR internalization is critical to control the temporal and spatial aspects of β(2) -AR signalling. Identifying novel regulators of β(2) -AR internalization will enable us to develop new strategies to manipulate receptor signalling and provide potential targets for drug development in the prevention and treatment of diseases associated with β(2) -AR signalling dysregulation.     

Integrin αIIbβ3: A novel effector of Gα13

Under physiological conditions, circulating platelets are discoid in shape.¹ On these platelets, the fibrinogen receptor (integrin α_IIbβ₃) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin α_IIbβ₃ through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling.² Outside-in signaling control processes, such as platelet spreading and clot retraction, by regulating small G-proteins such as RhoA, Rac and cdc42.Key words: platelets, integrin α_IIbβ₃, Galpha13, RhoA, clot retraction, thrombin, fibrinogenThe majority of the physiological platelet agonists (except collagen) induce inside-out signaling by binding to specific G-protein-coupled receptors (GPCRs). A G-protein plays a crucial role in translating the signal from GPCR to downstream effector molecules, ultimately leading to affinity modulation of integrin α_IIbβ₃. Platelets express nine Gα subunits; namely G_q, G_i1, G_i2, G_i3, G_z, G₁₂, G₁₃, G_s and G₁₆. Previous studies have shown that a small G-protein, RhoA, is activated by the G_12/13 family and plays a crucial role in calcium-independent platelet shape change.³ However, RhoA is also activated by α_IIbβ₃ and inhibits platelet spreading to trigger clot retraction.⁴ Recently, in a series of elegant experiments, Gong et al. have described the dynamic regulation of RhoA through a signaling crosstalk between Gα₁₃ and α_IIbβ₃.⁵By generating mice in which the platelets were depleted of Gα₁₃ using siRNA technology, Gong et al. investigated the role of Gα₁₃-mediated signaling on platelet spreading on immobilized Fg.⁵ The confocal images very clearly showed that, in the absence of Gα₁₃, platelets spread poorly on Fg, which was rescued by pretreatment with the Rho-kinase inhibitor Y27632, confirming previous findings that RhoA activated downstream of integrin α_IIbβ₃ inhibits platelet spreading. Interestingly, Gα₁₃-depleted platelets failed to activate c-Src but accelerated RhoA activation. From these observations, the authors infer that Gα₁₃ is important for integrin-mediated c-Src activation and RhoA inhibition, leading to increased cell spreading.⁵Since Gα₁₃ regulates integrin-mediated cell spreading and c-Src activation, Gong et al. examined the interaction of Gα₁₃ with α_IIbβ₃ using co-immunoprecipitation and GST pull-down assays.⁵ They found that the GTP-bound form of Gα₁₃ shows enhanced interaction with the integrin β₃ subunit. This interaction is required for the activation of c-Src and the inhibition of RhoA. However, they found that the inhibition of RhoA is transient. RhoA activation is suppressed for the first 15 min of platelet spreading, after which RhoA is activated. This initial suppression is rescued by blocking Gα₁₃ and β₃ cytoplasmic domain (β3-CD) interaction. Furthermore, they observed that RhoA activation parallels clot retraction.⁵ These findings indicate that Gα₁₃ is a key regulator of platelet spreading and clot retraction phenomena.According to Gong et al., thrombin-induced inside-out signaling through GPCR leads to GTP loading of Gα₁₃ (Fig. 1A). This GTP-bound Gα₁₃ interacts with integrin β3-CD of ligand-bound integrin, thus facilitating c-Src activation, which leads to platelet spreading. Blockade of the interaction between Gα₁₃ and β3-CD or cleavage of β3-CD by calpain results in clot retraction (Fig. 1B).Open in a separate windowFigure 1Schematic representation of the dynamic regulation of RhoA by Gα₁₃ during platelet activation. (A) Activation of platelets by thrombin receptors coupled to Gα₁₃ leads to the activation of RhoA, leading to platelet shape change. (B) The change in the conformation of integrin to a high-affinity form results in fibrinogen binding to α_IIbβ₃. Active Gα13 binds to the cytoplasmic domain of β₃ leading to the activation of c-Src, resulting in platelet spreading. The rise in intracellular calcium activates calpain, which cleaves the β₃ cytoplasmic domain, releasing c-Src, which, resulting in the activation of RhoA, leads to cell retraction. *Denotes GTP-bound active form of G-proteins.Perhaps the most significant and novel finding of the study is the identification of integrin α_IIbβ₃ as an effector of Gα₁₃. The study also convincingly shows that Gα₁₃ bound to integrin regulates RhoA via c-Src. Furthermore, achieving 80% knockdown of Gα₁₃ in an in vivo setting using siRNA represents a technological advancement. Since Gα₁₃ binds to integrin β3-CD in a 1:1 stoichiometry, it appears that only a small population of integrin is regulated by Gα₁₃, as there are far less Gα₁₃ molecules in a single platelet than the number of α_IIbβ₃ molecules. This will require further investigation. Gong et al. also finds that an appreciable amount of Gα₁₃ is associated with β₃ in resting platelets, which requires some explanation.⁵ It is also not clear if Gα₁₃ remains bound to β3-CD or dissociates from the integrin during clot retraction.Overall, this is a paradigm-shifting study that establishes the importance of the dynamic regulation of RhoA by Gα₁₃ in order to achieve efficient platelet spreading and clot retraction.     

A new thermostable α-l-arabinofuranosidase from a novel thermophilic bacterium

An alpha-L-arabinofuranosidase gene was identified in a sequenced genome of a novel thermophilic bacterium, which belongs to the recently described phylum of Thermomicrobia. Amino acid sequence comparison of the enzyme (designated AraF) revealed similarity to glycoside hydrolases of family 51. The gene was cloned into Escherichia coli and its recombinant product expressed and purified. The enzyme appeared to be a hexamer. AraF was optimally active at 70 degrees C (over 10 min) and pH 6 having 92% residual activity after 1 h at 70 degrees C. AraF had a Km) value of 0.6 mM and V(max) value of 122 U mg(-1) on p-nitrophenyl-alpha-L-arabinofuranoside. AraF was almost equally active on branched arabinan and debranched arabinan, properties not previously found in alpha-L-arabinofuranosidases in GH family 51.     

Purification and properties of a novel recombinant human hybrid interferon, σ-4 α2/α1

The human interferon (huIFN) σ-4 α2_5–62/α1_64–166 is a genetically engineered hybrid that consists of residues 5–62 of huIFN α2 and residues 64–166 of huIFN α1. This variant contains four cysteine residues at positions 29, 86, 99 and 139, but does not contain the cysteine at position 1 that is characteristic of naturally occurring huIFN α subtypes. This novel recombinant hybrid was purified fromEscherichia coli to greater than 95% homogeneity. The purification was based on ethanol extraction of a trichloroacetic acid precipitate and Matrex Gel Blue A chromatography followed by either a selective precipitation or DEAE-Sepharose chromatography. The purified protein that was treated with 2-mercaptoethanol exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 17 800 and 17 100, both of which exhibited antiviral activity. Electrophoresis performed without prior reduction with 2-mercaptoethanol indicated only a minor extent of intermolecular disulfide bonding. The purified protein exhibited a high specific antiviral activity of 7·10⁷ units/mg when assayed on human fibroblast cells and, in distinction to the parental huIFN α2, it also demonstrated antiviral activity on murine L929 cells. The level of antiproliferative activity of huIFN δ-4 α2_5–62/α1_64–166 on various cell lines of different histological origin appeared to be more comparable to that of huIFN α1 than huIFN α2. The data suggest that huIFN δ-4 α2_5–62/α1_64–166 hybrid may be a useful tool for understanding huIFN structure-function relations.     

Identification and characterization of a lipopolysaccharide α,2,3-sialyltransferase from the human pathogen Helicobacter bizzozeronii

Terminal sialic acid in the lipopolysaccharides (LPSs) of mucosal pathogens is an important virulence factor. Here we report the characterization of a Helicobacter sialyltransferase involved in the biosynthesis of sialylated LPS in Helicobacter bizzozeronii, the only non-pylori gastric Helicobacter species isolated from humans thus far. Starting from the genome sequences of canine and human strains, we identified potential sialyltransferases downstream of three genes involved in the biosynthesis of N-acetylneuraminic acid. One of these candidates showed monofunctional α,2,3-sialyltransferase activity with a preference for N-acetyllactosamine as a substrate. The LPSs from different strains were shown by SDS-PAGE and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to contain sialic acid after neuraminidase treatment. The expression of this sialyltransferase and sialyl-LPS appeared to be a phase-variable characteristic common to both human and canine H. bizzozeronii strains. The sialylation site of the LPSs of two H. bizzozeronii strains was determined to be NeuAc-Hex-HexNAc, suggesting terminal 3'-sialyl-LacNAc. Moreover, serological typing revealed the possible presence of sialyl-Lewis X in two additional strains, indicating that H. bizzozeronii could also mimic the surface glycans of mammalian cells. The expression of sialyl-glycans may influence the adaptation process of H. bizzozeronii during the host jump from dogs to humans.     

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 2: α-Branched quaternary amines

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 μg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC. As such, pyrazinoyl quaternary amines represent a promising new class of ENaC blockers for the treatment of cystic fibrosis that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride.