Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precurson of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central nervous system and the peripheral tissues where it is involved in the maintenance of the body temperature, regulation of nerve cell function, regulation of the sleep wake cycle, tactile pain sensitivity, allergic asthma, inhibition of platelet aggregation and chemotactic recruitment of inflammatory cells. L-PGDS belongs to the lipocalin family and it is able to bind and transport small hydrophobic molecules; it is also the first member of this important family to be recognized as an enzyme. Recently L-PGDS was identified to be already known as the β-trace protein, which is the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, human testis and prostate, endothelial cells, placenta cells, heart tissue and even in macrophages infiltrated to atherosclerotic plaques. In those tissues L-PGDS is involved in many physiological activities as well as in the response to diseases such as diabetes, cardiovascular lesions, multiple sclerosis, Alzheimer’s disease and tumors. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. The aim of this work was to express, purify and crystallize recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction experiments. Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion chromatography and hydrophobic interaction chromatography. The purification method was improved to obtain highly homogeneous protein suitable for preliminary crystallization trials. Crystallization conditions were optimized to obtain large and highly ordered crystals that were tested by X-ray diffraction using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement technique was used to solve the phase problem and heavy atom derivatives were obtained by soaking. An unidentified electron density was observed that seemed to interact with lysine 59 inside the L-PGDS-C65A cavity. It was not possible, at that moment, to characterize this residual molecule although different protocols were tested, including the use of physiological ligands. The L-PGDS-C65A/K59A structure showed a completely free cavity. It was noticed that L-PGDS-C65A/K59A crystals grew without PEG as precipitant, which instead was necessary for the L-PGDS-C65A crystals, suggesting that PEG could be the foreign molecule. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. A high resolution data set of a L-PGDS-C65A gave us the possibility to well define the boundaries of the unknown electron density showing that the foreign molecule was probably PEG. An important crystal structure was obtained by mixing L-PGDS-C65A/K59A with the amyloid β peptide (1-40). Although structure refinement is work in progress, it was the first structural evidence of the interaction of L-PGDS with the amyloid β peptide (1-40). Wild type L-PGDS and L-PGDS C89/186A were purified but they were not homogeneous and no crystals grew in any of the crystallization trials.

Structural Characterization of Human Lipocalin-type Prostaglandin D Synthase (L-PGDS)

PERDUCA, Massimiliano
2009-01-01

Abstract

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precurson of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central nervous system and the peripheral tissues where it is involved in the maintenance of the body temperature, regulation of nerve cell function, regulation of the sleep wake cycle, tactile pain sensitivity, allergic asthma, inhibition of platelet aggregation and chemotactic recruitment of inflammatory cells. L-PGDS belongs to the lipocalin family and it is able to bind and transport small hydrophobic molecules; it is also the first member of this important family to be recognized as an enzyme. Recently L-PGDS was identified to be already known as the β-trace protein, which is the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, human testis and prostate, endothelial cells, placenta cells, heart tissue and even in macrophages infiltrated to atherosclerotic plaques. In those tissues L-PGDS is involved in many physiological activities as well as in the response to diseases such as diabetes, cardiovascular lesions, multiple sclerosis, Alzheimer’s disease and tumors. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. The aim of this work was to express, purify and crystallize recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction experiments. Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion chromatography and hydrophobic interaction chromatography. The purification method was improved to obtain highly homogeneous protein suitable for preliminary crystallization trials. Crystallization conditions were optimized to obtain large and highly ordered crystals that were tested by X-ray diffraction using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement technique was used to solve the phase problem and heavy atom derivatives were obtained by soaking. An unidentified electron density was observed that seemed to interact with lysine 59 inside the L-PGDS-C65A cavity. It was not possible, at that moment, to characterize this residual molecule although different protocols were tested, including the use of physiological ligands. The L-PGDS-C65A/K59A structure showed a completely free cavity. It was noticed that L-PGDS-C65A/K59A crystals grew without PEG as precipitant, which instead was necessary for the L-PGDS-C65A crystals, suggesting that PEG could be the foreign molecule. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. A high resolution data set of a L-PGDS-C65A gave us the possibility to well define the boundaries of the unknown electron density showing that the foreign molecule was probably PEG. An important crystal structure was obtained by mixing L-PGDS-C65A/K59A with the amyloid β peptide (1-40). Although structure refinement is work in progress, it was the first structural evidence of the interaction of L-PGDS with the amyloid β peptide (1-40). Wild type L-PGDS and L-PGDS C89/186A were purified but they were not homogeneous and no crystals grew in any of the crystallization trials.
LIPOCALIN-TYPE PROSTAGLANDIN D SYNTHASE; PGDS; LIPOCALIN; PROTEIN STRUCTURE
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/927100
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