Bifunctional enzyme which catalyzes both the conversion of PGH2 to PGD2, a prostaglandin involved in smooth muscle contraction/relaxation and a potent inhibitor of platelet aggregation, and the conjugation of glutathione with a wide range of aryl halides and organic isothiocyanates. Also exhibits low glutathione-peroxidase activity towards cumene hydroperoxide.
Prostaglandin (PG) D synthase activity was found in the four human megakaryocytic cell lines CMK86, CMK, CMK11.5, and Dami cells. The PGD synthase in these cells was identified to be the hematopoietic-type and not the lipocalin-type, as judged by a GSH requirement for the enzyme activity, its immunoreactivity, and Northern blot analysis. The PGD synthase activity and the mRNA level of these cells increased 2.5 approximately 4.5- and 1.7 approximately 4.5-fold respectively when treated with 12-O-tetradecanoyl-phorbol-13-acetate at 0.1 microM for 72 h, indicating that the enzyme was inducible. The expression level of the enzyme was the highest in CMK86 cells and was less in CMK, CMK11-5, and Dami cells in that order. On the other hand, the expression of platelet glycoprotein Ib (CD42b), a marker of megakaryocytic maturation, was observed in those cell lines in the reverse order. These results suggest that hematopoietic PGD synthase is a useful marker for identifying the differentiation stage of human megakaryocytes.
Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.
We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg2+, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45-A resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp104 and by its piperidine group with GSH and Arg14 through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg2+. HQL-79 inhibited human H-PGDS competitively against the substrate PGH2 and non-competitively against GSH with Ki of 5 and 3 microm, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg2+ (Kd, 0.8 microm) than in their absence. Mutational studies revealed that Arg14 was important for the Mg2+-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp104, Lys112, and Lys198 were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD2 production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC50 value of about 100 microm but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD2, without affecting the production of PGE2 and PGF2alpha, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD2 production specifically.
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.
J. Biol. Chem. 272, 28263-28266 (1997)[PubMed:9353279]
The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.
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.
Eur. J. Biochem. 267, 3315-3322 (2000)[PubMed:10824118]
Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.
GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.
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.
Catalysis of the reaction: R-X + glutathione = H-X + R-S-glutathione. R may be an aliphatic, aromatic or heterocyclic group; X may be a sulfate, nitrile or halide group.
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.
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.
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.
The specific movement from place to place of an organism in response to external or internal stimuli. Locomotion of a whole organism in a manner dependent upon some combination of that organism's internal state and external conditions.
To examine the function of prostaglandin (PG) D synthase (PGDS) gene, as well as endogenously produced PGD(2) in sleep regulation in vivo, we generated transgenic (TG) mice that overexpress human PGDS gene to study their sleep behavior. Although no difference was observed in the sleep/wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye movement (NREM), but not in rapid eye movement (REM), sleep was observed in two independent lines of TG mice after stimulation by tail clipping. Concomitantly, the spontaneous locomotor activity of TG animals was drastically decreased in response to the tail clip. Induction of NREM sleep in TG mice was positively correlated with the PGD(2) production in the brain. Sleep, locomotion, and PGD(2) content were essentially unchanged in wild-type mice after tail clipping. The results with TG mice demonstrate the involvement of the PGDS gene in the regulation of NREM sleep.
The chemical reactions and pathways resulting in the formation of prostaglandins, any of a group of biologically active metabolites which contain a cyclopentane ring.
The chemical reactions and pathways involving prostaglandins, any of a group of biologically active metabolites which contain a cyclopentane ring due to the formation of a bond between two carbons of a fatty acid. They have a wide range of biological activities.
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.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
J. Biol. Chem. 272, 28263-28266 (1997)[PubMed:9353279]
The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.
We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg2+, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45-A resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp104 and by its piperidine group with GSH and Arg14 through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg2+. HQL-79 inhibited human H-PGDS competitively against the substrate PGH2 and non-competitively against GSH with Ki of 5 and 3 microm, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg2+ (Kd, 0.8 microm) than in their absence. Mutational studies revealed that Arg14 was important for the Mg2+-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp104, Lys112, and Lys198 were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD2 production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC50 value of about 100 microm but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD2, without affecting the production of PGE2 and PGF2alpha, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD2 production specifically.
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.
Prostaglandin (PG) D synthase activity was found in the four human megakaryocytic cell lines CMK86, CMK, CMK11.5, and Dami cells. The PGD synthase in these cells was identified to be the hematopoietic-type and not the lipocalin-type, as judged by a GSH requirement for the enzyme activity, its immunoreactivity, and Northern blot analysis. The PGD synthase activity and the mRNA level of these cells increased 2.5 approximately 4.5- and 1.7 approximately 4.5-fold respectively when treated with 12-O-tetradecanoyl-phorbol-13-acetate at 0.1 microM for 72 h, indicating that the enzyme was inducible. The expression level of the enzyme was the highest in CMK86 cells and was less in CMK, CMK11-5, and Dami cells in that order. On the other hand, the expression of platelet glycoprotein Ib (CD42b), a marker of megakaryocytic maturation, was observed in those cell lines in the reverse order. These results suggest that hematopoietic PGD synthase is a useful marker for identifying the differentiation stage of human megakaryocytes.
GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.
J. Biol. Chem. 272, 28263-28266 (1997)[PubMed:9353279]
The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.
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.
Eur. J. Biochem. 267, 3315-3322 (2000)[PubMed:10824118]
Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.
Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.
Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.
J. Biol. Chem. 272, 28263-28266 (1997)[PubMed:9353279]
The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.
Eur. J. Biochem. 267, 3315-3322 (2000)[PubMed:10824118]
Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.
We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg2+, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45-A resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp104 and by its piperidine group with GSH and Arg14 through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg2+. HQL-79 inhibited human H-PGDS competitively against the substrate PGH2 and non-competitively against GSH with Ki of 5 and 3 microm, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg2+ (Kd, 0.8 microm) than in their absence. Mutational studies revealed that Arg14 was important for the Mg2+-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp104, Lys112, and Lys198 were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD2 production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC50 value of about 100 microm but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD2, without affecting the production of PGE2 and PGF2alpha, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD2 production specifically.
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.
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.
Prostaglandin (PG) D synthase activity was found in the four human megakaryocytic cell lines CMK86, CMK, CMK11.5, and Dami cells. The PGD synthase in these cells was identified to be the hematopoietic-type and not the lipocalin-type, as judged by a GSH requirement for the enzyme activity, its immunoreactivity, and Northern blot analysis. The PGD synthase activity and the mRNA level of these cells increased 2.5 approximately 4.5- and 1.7 approximately 4.5-fold respectively when treated with 12-O-tetradecanoyl-phorbol-13-acetate at 0.1 microM for 72 h, indicating that the enzyme was inducible. The expression level of the enzyme was the highest in CMK86 cells and was less in CMK, CMK11-5, and Dami cells in that order. On the other hand, the expression of platelet glycoprotein Ib (CD42b), a marker of megakaryocytic maturation, was observed in those cell lines in the reverse order. These results suggest that hematopoietic PGD synthase is a useful marker for identifying the differentiation stage of human megakaryocytes.
GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.
Prostaglandin (PG) D synthase activity was found in the four human megakaryocytic cell lines CMK86, CMK, CMK11.5, and Dami cells. The PGD synthase in these cells was identified to be the hematopoietic-type and not the lipocalin-type, as judged by a GSH requirement for the enzyme activity, its immunoreactivity, and Northern blot analysis. The PGD synthase activity and the mRNA level of these cells increased 2.5 approximately 4.5- and 1.7 approximately 4.5-fold respectively when treated with 12-O-tetradecanoyl-phorbol-13-acetate at 0.1 microM for 72 h, indicating that the enzyme was inducible. The expression level of the enzyme was the highest in CMK86 cells and was less in CMK, CMK11-5, and Dami cells in that order. On the other hand, the expression of platelet glycoprotein Ib (CD42b), a marker of megakaryocytic maturation, was observed in those cell lines in the reverse order. These results suggest that hematopoietic PGD synthase is a useful marker for identifying the differentiation stage of human megakaryocytes.
J. Biol. Chem. 272, 28263-28266 (1997)[PubMed:9353279]
The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.
GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.
Eur. J. Biochem. 267, 3315-3322 (2000)[PubMed:10824118]
Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.
Prostaglandin PGD2 synthesis is stimulated by calcium and magnesium ions. One calcium or magnesium ion is bound between the subunits of the homodimer. The interactions with the protein are for the most part mediated via water molecules. Magnesium increases the affinity for glutathione, while calcium has no effect on the affinity for glutathione.
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.
Protein involved in the synthesis of fatty acids, long chain organic acids of the general formula CH3(CnHx)COOH. They are constituents of lipids and can be saturated or unsaturated. The esterified forms are important both as energy storage molecules and structural molecules.
Protein involved in the biochemical reactions with fatty acids. Fatty acids are long chain organic acids of the general formula CH3(CnHx)COOH. They are constituents of lipids and can be saturated or unsaturated. The esterified forms are important both as energy storage molecules and structural molecules.
Protein involved in the synthesis of lipids, a diverse class of compounds which are insoluble in water but soluble in organic solvents. They include fats, oils, triacylglycerols, fatty acids, glycolipids, phospholipids and steroids.
Protein involved in the biochemical reactions of lipids. Lipids are a diverse class of compounds which are insoluble in water but soluble in organic solvents. They include fats, oils, triacylglycerols, fatty acids, glycolipids, phospholipids and steroids.
Protein involved in the biosynthesis of prostaglandins. Prostaglandins are fatty acids composed of 20 carbons with a substituted cyclopentane ring. There are four major classes of prostaglandin, which differ in the position of the double bonds and/or the oxygen substituents on the ring: PGA, PGB, PGE, and PGF. They are found in many mammalian tissues.
Protein involved in a biochemical reaction with prostaglandins. Prostaglandins are fatty acids composed of 20 carbons with a substituted cyclopentane ring. There are four major classes of prostaglandin, which differ in the position of the double bonds and/or the oxygen substituents on the ring: PGA, PGB, PGE, and PGF. They are found in many mammalian tissues.
Enzyme that catalyzes the 1,1-, 1,2- or 1,3-hydrogen shift. The 1,1- hydrogen shift is an inversion at an asymmetric carbon center (racemases, epimerases). The 1,2-hydrogen shift involved a hydrogen transfer between two adjacent carbon atoms, one undergoing oxidation, the other reduction (aldose-ketose isomerases). The 1,3-hydrogen shifts are allylic or azaallylic (when nitrogen is one of the three atoms) isomerizations.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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