RESUMO
Raw horsemeat has the potential to induce food poisoning which often presents with diarrheal symptoms. A sample of horsemeat was found to be infected with Sarcocystis fayeri, and a 15-kDa protein isolated from the cysts of S. fayeri was found to clearly show its diarrhea-inducing activity. A nested polymerase chain reaction was used to clone the cDNA of the 15-kDa protein. The deduced amino acid sequence showed homology to actin-depolymerizing factor (ADF). A recombinant 15-kDa protein depolymerized prepolymerized actins in a test tube. The 15-kDa protein possessed conserved amino acid sequences of ADF of Toxoplasma gondii and Eimeria tenella. These characteristics indicate that the 15-kDa protein of S. fayeri belongs to the ADF/cofilin protein family. The recombinant 15-kDa protein evoked fluid accumulation in the looped ileum, resulting in diarrhea, but it did not kill the cultured fibroblast cells, macrophages or intestinal mucosal cells. In addition, the culture supernatant of the macrophages treated with the recombinant 15-kDa protein killed the fibroblast L929 cells. This fact indicates that ADF of S. fayeri induced cytotoxic substances, such as tumor necrosis factor-α, according to the published reports. Although further experiments are needed now to elucidate the enterotoxic mechanism of S. fayeri's ADF, our findings may offer new insight into research on parasites and parasite-instigated food poisoning.
Assuntos
Fatores de Despolimerização de Actina/toxicidade , Diarreia/parasitologia , Proteínas de Protozoários/toxicidade , Sarcocystis/patogenicidade , Toxinas Biológicas/toxicidade , Fatores de Despolimerização de Actina/química , Animais , Linhagem Celular , Sequência Conservada , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Domínios Proteicos , Proteínas de Protozoários/química , Coelhos , Toxinas Biológicas/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC4S conjugates glutathione to LTA4, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA4 that distinguishes LTC4S from other MGSTs. The LTC4S monomer has four transmembrane alpha-helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA4 would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA4 to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4). These findings provide a structural basis for the development of LTC4S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.
Assuntos
Cisteína/química , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Leucotrienos/biossíntese , Leucotrienos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glutationa/química , Glutationa/metabolismo , Humanos , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).
Assuntos
Arginina/química , Glutationa Transferase/química , Glutationa/química , Leucotrieno C4/química , Arginina/genética , Arginina/metabolismo , Asma/enzimologia , Asma/genética , Sítios de Ligação , Cristalografia por Raios X , Glutationa/genética , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Leucotrieno C4/biossíntese , Leucotrieno C4/genética , Mutação , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
A gene, mf1, encoding a novel cholinephosphotransferase in glycoglycerophospholipid (GGPL) biosynthesis of Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, and expressed in Escherichia coli. The mf1 gene comprises an open reading frame of 777 bp encoding 258 amino acids. The mf1 gene product, Mf1, has 23% amino acid homology with LicD of Haemophilus influenzae but no homology with genes of other Mycoplasma species in the GenBank database. The reaction product of Mf1 using alpha-glucopyranosyl-1,2-dipalmitoilglycerol and cytidine 5'-diphosphocholine (CDP-choline) as substrates showed the specific protonated molecule at m/z 896, which corresponded to GGPL-I as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem mass spectrometry (MS) analysis of protonated molecules at m/z 896. These results identified mf1 as a novel cholinephosphotransferase and showed that the phosphocholine transfer step is involved in the GGPL biosynthesis pathway of M. fermentans. This is the first report of a GGPL biosynthesis enzyme.
Assuntos
Proteínas de Bactérias/metabolismo , Clonagem Molecular , Diacilglicerol Colinofosfotransferase/metabolismo , Expressão Gênica , Glicerofosfatos/biossíntese , Mycoplasma fermentans/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Diacilglicerol Colinofosfotransferase/química , Diacilglicerol Colinofosfotransferase/genética , Glicerofosfatos/química , Dados de Sequência Molecular , Mycoplasma fermentans/química , Mycoplasma fermentans/classificação , Mycoplasma fermentans/genética , Filogenia , Especificidade por SubstratoRESUMO
An immunosensor based on surface plasmon resonance was applied to detect mast cells expressing c-Kit. Sufficient detection of the mast cells was achieved by covalent immobilization of gelatin firstly on the sensor surface and followed by covalent binding of the anti-c-Kit antibody to lysine residues in the gelatin molecules through bis(sulfosuccinimidyl)suberate (BS3) treatment. By using BS3, which is a homo-bifunctional reagent, the lysine residues of the anti-c-Kit antibody easily bound to the lysine residues of the gelatin in the physiological condition. The lower limit of detection was 104 cells/mL.
Assuntos
Anticorpos Imobilizados/química , Gelatina/química , Regulação da Expressão Gênica , Imunoensaio/métodos , Mastócitos/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Imobilizados/imunologia , Ácidos Decanoicos/química , Humanos , Limite de Detecção , Mastócitos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/imunologia , Succinimidas/químicaRESUMO
An immunosensor based on surface plasmon resonance was developed for detection of c-Kit expressed on a cell surface. The combination of the antibody solution modified with gelatin before immobilization to the sensor chip and its blocking with gelatin drastically decreased the nonspecific reaction. The condition may be useful for the detection of various cells by using antibody against cell surface marker including the c-Kit.
Assuntos
Regulação da Expressão Gênica , Imunoensaio/métodos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Proto-Oncogênicas c-kit/imunologiaRESUMO
The cell adhesion molecule (CADM) family of the immunoglobulin superfamily (IgSF) comprises four members, CADM1-CADM4, and participates in the formation of epithelial and synaptic adhesion through cell-cell homophilic and heterophilic interactions. To identify the partners that interact with each member of the CADM family proteins, we set up a platform for multiple detection of the extracellular protein-protein interactions using surface plasmon resonance imaging (SPRi) and analyzed the interactions between the CADM family proteins and 10 IgSF of their structurally related cell adhesion molecules. SPRi analysis identified a new interaction between CADM1 and CADM4, where this heterophilic interaction was shown to be involved in morphological spreading of adult T-cell leukemia (ATL) cells expressing CADM1 when incubated on CADM4-coated glass. Moreover, class-I MHC-restricted T-cell-associated molecule (CRTAM) was identified to show the highest affinity to CADM1 among its binding partners by comparing the dissociation constants calculated from the SPR sensorgrams. These results suggest that the SPRi platform would provide a novel screening tool to characterize extracellular protein-protein interactions among cell-surface and secreted proteins, including IgSF molecules.
RESUMO
Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.
Assuntos
Bufo marinus/metabolismo , Plexo Corióideo/enzimologia , Oxirredutases Intramoleculares/química , Sequência de Aminoácidos , Animais , Pigmentos Biliares/metabolismo , DNA Complementar , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Lipocalinas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinoides/metabolismo , Hormônios Tireóideos/metabolismo , XenopusRESUMO
Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia-actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and ß/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and ß/γ-actin, but its sensitivity towards ß/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates ß/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and ß/γ-actin.
Assuntos
Clostridium perfringens/metabolismo , Enterotoxinas/química , Enterotoxinas/genética , Actinas/química , Actinas/metabolismo , Difosfato de Adenosina , Sequência de Aminoácidos , Sítios de Ligação , Clostridium perfringens/genética , Cristalografia por Raios X , Enterotoxinas/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/química , NAD/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
Leukotriene (LT) C(4) synthase, an 18 kDa integral membrane enzyme, conjugates LTA(4) with reduced glutathione to form LTC(4), the parent compound of all cysteinyl leukotrienes that play a crucial role in the pathobiology of bronchial asthma. We have calculated a projection map of recombinant human LTC(4) synthase at a resolution of 4.5 A by electron crystallography, which shows that the enzyme is a trimer. A map truncated at 7.5 A visualizes four transmembrane alpha helices per protein monomer. The densities in projection indicate that most of the alpha helices run nearly perpendicular to the plane of the membrane. At this resolution, LTC(4) synthase is strikingly similar to microsomal glutathione S-transferase 1, which belongs to the same gene family but bears little sequence identity and no resemblance in substrate specificity to the LTC(4) synthase. These results provide new insight into the structure and function of membrane proteins involved in eicosanoid and glutathione metabolism.
Assuntos
Glutationa Transferase/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação Proteica , RatosRESUMO
There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.
Assuntos
Infecções por Clostridium/microbiologia , Clostridium perfringens/genética , Enterotoxinas/genética , Doenças Transmitidas por Alimentos/microbiologia , Fatores de Ribosilação do ADP/biossíntese , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , Infecções por Clostridium/epidemiologia , Clostridium perfringens/isolamento & purificação , Sequência Conservada , Surtos de Doenças , Enterotoxinas/biossíntese , Enterotoxinas/imunologia , Doenças Transmitidas por Alimentos/epidemiologia , Expressão Gênica , Humanos , Íleo/microbiologia , Masculino , Dados de Sequência Molecular , NAD+ Nucleosidase/biossíntese , NAD+ Nucleosidase/genética , NAD+ Nucleosidase/imunologia , Coelhos , Análise de Sequência de DNA , Tóquio , Células VeroRESUMO
Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.
Assuntos
Sistema Hematopoético/enzimologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/química , Sais de Tetrazólio/química , Sais de Tetrazólio/farmacologia , Benzotiazóis , Cristalografia por Raios X , Humanos , Lipocalinas , Modelos MolecularesRESUMO
Lipocalin-type prostaglandin D synthase is the key enzyme for the production of prostaglandin D(2), a potent endogenous somnogen, in the brain. We cloned, produced, and crystallized the native enzyme and selenomethionyl Cys(65)Ala mutants of the recombinant mouse protein by the hanging drop vapor-diffusion method with both malonate and citrate as precipitants. The native crystals obtained with malonate belong to orthorhombic space group P2(1)2(1)2(1) with lattice constants a = 46.2, b = 66.8, and c = 105.3 A. The selenomethionyl crystals obtained with citrate belong to orthorhombic space group C222(1) with lattice constants a = 45.5, b = 66.8, and c = 104.5 A. The native crystals diffracted beyond 2.1 A resolution.
Assuntos
Oxirredutases Intramoleculares/química , Animais , Encéfalo/enzimologia , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Expressão Gênica , Oxirredutases Intramoleculares/biossíntese , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/ultraestrutura , Lipocalinas , Camundongos , Prostaglandina D2/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestruturaRESUMO
Food poisoning has been reported after the consumption of raw horsemeat in Japan. Diarrhea with a short incubation period is a common symptom in such cases of food poisoning. Cysts found in horsemeat ingested by patients have been identified as Sarcocystis fayeri based on morphological and genetic evaluation and findings from experimental feeding of cysts to dogs, which resulted in the excretion of sporocysts. The extracts of the horsemeat containing the cysts produced a positive enterotoxic response in the rabbit ileal loop test. Intravenous injection of a 15-kDa protein isolated from the cysts induced diarrhea and lethal toxicity in rabbits, and the protein produced enterotoxicity in the ileal loop test as did the extracts of the horsemeat containing the cysts. The partial amino acid sequence of the 15-kDa protein was homologous to the actin-depolymerizing factor of Toxoplasma gondii and Eimeria tenella. These findings indicate that the 15-kDa protein of S. fayeri is a toxin that causes food poisoning after consumption of parasitized horsemeat.
Assuntos
Doenças Transmitidas por Alimentos/parasitologia , Doenças dos Cavalos/parasitologia , Carne/parasitologia , Sarcocystis/metabolismo , Sarcocistose/parasitologia , Sarcocistose/veterinária , Toxinas Biológicas/isolamento & purificação , Adulto , Animais , Cães , Contaminação de Alimentos/análise , Cavalos , Humanos , Japão , Masculino , Carne/análise , Dados de Sequência Molecular , Peso Molecular , Oocistos/química , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Coelhos , Sarcocystis/química , Sarcocystis/crescimento & desenvolvimento , Sarcocystis/isolamento & purificação , Toxinas Biológicas/química , Toxinas Biológicas/toxicidadeRESUMO
More than 27 outbreaks per year of food poisoning caused by consuming horse meat were reported in Kumamoto Prefecture (including Kumamoto City) from January 2009 to September 2011. It was found that the causative agent of the outbreaks was a protein with a molecular weight of 15 kDa that had originated from bradyzoites of Sarcocystis fayeri parasitizing the horse meat. Rabit ileal loop tests showed that pepsin treatment of homogenates of frozen horse meat containing the cysts of S. fayeri induced loss of toxicity, presumably by digestion of the proteinous causative agent(s). Slices of horse meat containing the cysts were frozen at below -20°C for various periods. The cysts were collected after thawing the slices, then treated in an artificial stomach juice containing pepsin. The bradyzoites of the cysts kept at -20°C for 48 hr or more completely disappeared. Simultaneously, the 15 kDa protein also disappeared in the frozen cysts. After notifying the public and recommending freezing treatment of horse meat, no subsequent cases of food poisoning were reported. This indicates that freezing of horse meat is effective to prevent the occurrence of food poisoning caused by consuming raw horse meat containing S. fayeri.
Assuntos
Manipulação de Alimentos/métodos , Doenças Transmitidas por Alimentos/prevenção & controle , Doenças Transmitidas por Alimentos/parasitologia , Congelamento , Carne/intoxicação , Carne/parasitologia , Sarcocystis/patogenicidade , Sarcocistose/prevenção & controle , Sarcocistose/parasitologia , Animais , Doenças Transmitidas por Alimentos/epidemiologia , Cavalos , Humanos , Japão/epidemiologia , Coelhos , Sarcocystis/isolamento & purificaçãoRESUMO
A gene, mf3, encoding glycosyltransferase in glycoglycerophospholipid (GGPL; GGPL-I and GGPL-III) biosynthesis in Mycoplasma fermentans PG18 was identified by genomic analysis, cloned, modified codon usage, and expressed in Escherichia coli. The mf3 gene consists of an open reading frame of 1221 bp encoding 406 amino acids. The mf3 gene product, Mf3, has 27% amino acid homology with glycosyltransferase of Borrelia burgdorferi but no homology to genes of other Mycoplasma species in the GenBank database. The reaction product of Mf3 using 1,2-dipalmitoilglycerol and UDP-glucose as substrates showed a specific sodium adducted ion at m/z 753, which corresponded to glucopyranosyl dipalmitoilglycerol as determined by MALDI-TOF mass spectrometry. Furthermore, in the reaction product by Mf3 and Mf1 which was a cholinephosphotransferase and previously cloned from M. fermentans PG18, an ion at m/z 896 corresponding to GGPL-I was detected by mass spectrometry. The product ions of choline, phosphocholine, and hexose-bound phosphocholine were detected by tandem MS analysis of protonated molecules at m/z 896. From these results, mf3 was identified as a glycosyltransferase. It was suggested that glucose transfer and phosphocholine transfer to 1,2-dipalmitoylglycerol are involved in the GGPL biosynthesis pathway of M. fermentans PG18.
Assuntos
Diglicerídeos/metabolismo , Glicolipídeos/biossíntese , Mycoplasma fermentans/genética , Mycoplasma fermentans/metabolismo , Sequência de Bases , Primers do DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Glicolipídeos/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Mycoplasma fermentans/enzimologia , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.
Assuntos
Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/antagonistas & inibidores , Lipocalinas/metabolismo , Pneumonia/enzimologia , Cicatrização/efeitos dos fármacos , Ferimentos Perfurantes/enzimologia , Administração Oral , Animais , Calcimicina/farmacologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprosta/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Eosinófilos/enzimologia , Humanos , Oxirredutases Intramoleculares/genética , Ionóforos/farmacologia , Lipocalinas/genética , Masculino , Megacariócitos/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Monócitos/enzimologia , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Prostaglandina D2/biossíntese , Ferimentos Perfurantes/tratamento farmacológico , Ferimentos Perfurantes/genéticaRESUMO
Lipocalin type prostaglandin D synthase (L-PGDS) is a multifunctional protein acting as a somnogen (PGD2)-producing enzyme, an extracellular transporter of various lipophilic ligands, and an amyloid-beta chaperone in human cerebrospinal fluid. In this study, we determined the crystal structures of two different conformers of mouse L-PGDS, one with an open cavity of the beta-barrel and the other with a closed cavity due to the movement of the flexible E-F loop. The upper compartment of the central large cavity contains the catalytically essential Cys65 residue and its network of hydrogen bonds with the polar residues Ser45, Thr67, and Ser81, whereas the lower compartment is composed of hydrophobic amino acid residues that are highly conserved among other lipocalins. SH titration analysis combined with site-directed mutagenesis revealed that the Cys65 residue is activated by its interaction with Ser45 and Thr67 and that the S45A/T67A/S81A mutant showed less than 10% of the L-PGDS activity. The conformational change between the open and closed states of the cavity indicates that the mobile calyx contributes to the multiligand binding ability of L-PGDS.
Assuntos
Oxirredutases Intramoleculares/fisiologia , Lipocalinas/química , Peptídeos beta-Amiloides/metabolismo , Animais , Catálise , Cristalografia por Raios X/métodos , Cisteína/química , Escherichia coli/metabolismo , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Lipocalinas/fisiologia , Camundongos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.
Assuntos
Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Animais , Dicroísmo Circular , Guanidina , Cinética , Lipocalinas , Espectroscopia de Ressonância Magnética , Camundongos , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , UreiaRESUMO
Here we report the crystal structures of human hematopoietic prostaglandin (PG) D synthase bound to glutathione (GSH) and Ca2+ or Mg2+. Using GSH as a cofactor, prostaglandin D synthase catalyzes the isomerization of PGH2 to PGD2, a mediator for allergy response. The enzyme is a homodimer, and Ca2+ or Mg2+ increases its activity to approximately 150% of the basal level, with half maximum effective concentrations of 400 microM for Ca2+ and 50 microM for Mg2+. In the Mg2+-bound form, the ion is octahedrally coordinated by six water molecules at the dimer interface. The water molecules are surrounded by pairs of Asp93, Asp96 and Asp97 from each subunit. Ca(2+) is coordinated by five water molecules and an Asp96 from one subunit. The Asp96 residue in the Ca2+-bound form makes hydrogen bonds with two guanidium nitrogen atoms of Arg14 in the GSH-binding pocket. Mg2+ alters the coordinating water structure and reduces one hydrogen bond between Asp96 and Arg14, thereby changing the interaction between Arg14 and GSH. This effect explains a four-fold reduction in the K(m) of the enzyme for GSH. The structure provides insights into how Ca2+ or Mg2+ binding activates human hematopoietic PGD synthase.