-
Teal S Hallstrand,
Ying Lai,
William A Altemeier,
Cara L Appel,
Brian Johnson,
Charles W Frevert,
Kelly L Hudkins,
James G Bollinger,
Prescott G Woodruff,
Dallas M Hyde,
William R Henderson, Michael H Gelb
[show abstract]
[hide abstract]
ABSTRACT: Rationale: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A2 group X (sPLA2-X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models. Objectives: We sought to examine sPLA2-X in the airway epithelium and airway wall of patients with asthma, the relationship to AHR in humans, and the regulation and function of sPLA2-X within the epithelium. Methods: We precisely phenotyped 34 asthmatics (19 with and 15 without EIB), and 10 normal controls to examine in vivo differences in epithelial gene expression, quantitative morphometry of endobronchial biopsies, and levels of secreted protein. The regulation of sPLA2-X gene (PLA2G10) expression was examined in primary airway epithelial cell cultures. The function of epithelial sPLA2-X in eicosanoid formation was examined using PLA2 inhibitors and murine tracheal epithelial cells with Pla2g10 deletion. Measurements and Main Results: We found that sPLA2-X protein is increased in the airways of patients with asthma, and that epithelial-derived sPLA2-X may be increased in association with indirect AHR. The expression of sPLA2-X increases during in vitro epithelial differentiation, is regulated by inflammatory signals including TNF, IL-13, and IL-17, and is both secreted from the epithelium and directly participates in the release of arachidonic acid by epithelial cells. Conclusions: These data reveal a relationship between epithelial-derived sPLA2-X and indirect AHR in asthma, and that sPLA2-X serves as an epithelial regulator of inflammatory eicosanoid formation. Therapies targeting epithelial sPLA2-X may be useful in asthma.
American Journal of Respiratory and Critical Care Medicine 04/2013; · 11.08 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: OBJECTIVE: To assess the performance of a tandem mass spectrometry (MS/MS) technology in a newborn screening laboratory to simultaneously measure α-galactosidase, acid-α-glucosidase, and α-L-iduronidase for the detection of infants at risk to develop Fabry, Pompe, or mucopolysaccharidosis (MPS)-I diseases. STUDY DESIGN: Enzyme activity was assayed from a 3.2-mm punch from 100 000+ anonymous newborn blood spots. Punches with low enzyme activity were further evaluated by nucleotide sequence analysis of the responsible gene. Confirmation of affected infants was dependent on identification of mutations compatible with diminished enzyme activity. RESULTS: The technology for simultaneously measuring multiple enzyme activities by MS/MS was successful. The confirmation of diagnosis for Fabry, Pompe, or MPS-I, by DNA sequencing estimated the prevalence of Fabry disease at 1/7800 males (95% CI 1/17 800-1/3600); Pompe disease at 1/27 800 newborns (95% CI 1/90 000-1/10 200); and MPS-I at 1/35 500 newborns (95% CI 1/143 000-1/11 100). These estimates of prevalence are 2 to 4 times greater than the prevalence estimated by clinical diagnosis. The combined prevalence for the 3 disorders was 1/7500 newborns (95% CI 1/13 500-1/4500). CONCLUSIONS: MS/MS for the simultaneous assay of multiple lysosomal enzymes can be successfully introduced into a routine newborn screening laboratory. The technology has a positive predictive value equal to, or better, than methods currently used for the detection of nonlysosomal disorders. Using newborn blood spots, the combined prevalence of Fabry, Pompe, and MPS-I is estimated at 1/7500 newborns based on low-enzyme activity and confirmation by mutation analysis.
The Journal of pediatrics 03/2013; · 4.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND: There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS).METHODS: The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode.RESULTS: Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories.CONCLUSIONS: HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.
Clinical Chemistry 01/2013; · 7.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Previous work has shown that disruption of the gene for group X secreted phospholipase A (sPLA-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLAs in the mammalian genome, the involvement of other sPLAs in the asthma model is possible - in particular, the group V sPLA (sPLA-V) that like sPLA-X is highly active at hydrolyzing membranes of mammalian cells.
The allergen-driven asthma phenotype was significantly reduced in sPLA-V-deficient mice but to a lesser extent than observed previously in sPLA-X-deficient mice. The most striking difference observed between the sPLA-V and sPLA-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA) in the sPLA-V mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V mice diminishes Th2 cytokine responses in the airways.
This paper illustrates the diverse roles of sPLAs in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA-V as a potential new therapy to treat asthma and other allergic disorders.
PLoS ONE 01/2013; 8(2):e56172. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Alterations in the airway epithelium have been associated with the development of asthma in elite athletes and in subjects that are susceptible to exercise-induced bronchoconstriction (EIB). The syndrome of EIB refers to acute airflow obstruction that is triggered by a period of physical exertion. Asthmatics who are susceptible to EIB have increased levels of cysteinyl leukotrienes (CysLTs, i.e., LTs C₄, D₄, and E₄) in induced sputum and exhaled breath condensate, and greater shedding of epithelial cells into the airway lumen. Exercise challenge in individuals susceptible to this disorder initiates a sustained increase in CysLTs in the airways, and secreted mucin release and smooth muscle constriction, which may be mediated in part through activation of sensory nerves. We have identified a secreted phospholipase A₂ (sPLA₂) with increased levels in the airways of patients with EIB called sPLA₂ group X(sPLA₂-X).We have found that sPLA₂-X is strongly expressed in the airway epithelium in asthma. Further,we discovered that transglutaminase 2 (TGM2) is expressed at increased levels in asthma and serves asa regulator of sPLA₂-X. Finally, we demonstrated that sPLA₂-X acts on target cells such as eosinophils to initiate cellular eicosanoid synthesis. Collectively, these studies identify a novel mechanism linking the airway epithelium to the production of inflammatory eicosanoids by leukocytes.
Pulmonary Pharmacology & Therapeutics 12/2012; 25(6):432-7. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We explored the inhibition mode of group IIA secreted phospholipase A(2) (GIIA sPLA(2)) selective inhibitors and tested their ability to inhibit GIIA sPLA(2) activity as chemical conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole sPLA(2) inhibitor were developed in which the carboxylate group on the inhibitor scaffold, which has been shown to coordinate to a Ca(2+) ligand in the enzyme active site, was replaced with other functionality. Replacing the carboxylate group with amine, amide, or hydroxyl groups had no effect on human GIIA (hGIIA) sPLA(2) inhibition potency but dramatically lowered inhibition potency against hGV and hGX sPLA(2)s. An alkylation protection assay was used to probe active site binding of carboxylate and noncarboxylate inhibitors in the presence and absence of Ca(2+) and/or lipid vesicles. We observed that carboxylate-containing inhibitors bind the hGIIA sPLA(2) active site with low nanomolar affinity, but only when Ca(2+) is present. Noncarboxylate, GIIA sPLA(2) selective inhibitors also bind the hGIIA sPLA(2) active site in the nanomolar range. However, binding for GIIA sPLA(2) selective inhibitors was dependent on the presence of a lipid membrane and not Ca(2+). These results indicate that GIIA sPLA(2) selective inhibitors exert their inhibitory effects by binding to the hGIIA sPLA(2) active site. An HA-linked GIIA inhibitor conjugate was developed using peptide coupling conditions and found to be less potent and selective against hGIIA sPLA(2) than the unconjugated inhibitor. Compounds reported in this study are some of the most potent and selective GIIA sPLA(2) active site binding inhibitors reported to date.
Biochemistry 09/2012; · 3.42 Impact Factor
-
Frederick S Buckner,
Maria Terezinha Bahia,
Praveen Kumar Suryadevara,
Karen L White,
David M Shackleford,
Naveen Kumar Chennamaneni,
Matthew A Hulverson,
Joy U Laydbak,
Eric Chatelain,
Ivan Scandale,
Christophe L M J Verlinde,
Susan A Charman,
Galina I Lepesheva, Michael H Gelb
[show abstract]
[hide abstract]
ABSTRACT: Chagas disease, caused by the protozoan pathogen Trypanosoma cruzi, remains a challenging infection due to the unavailability of safe and efficacious drugs. Inhibitors of the trypanosome sterol 14α-demethylase enzyme (CYP51), including azole antifungal drugs, are promising candidates for development as anti-Chagas disease drugs. Posaconazole is under clinical investigation for Chagas disease, although the high cost of this drug may limit its widespread use. We have previously reported that the human protein farnesyltransferase (PFT) inhibitor tipifarnib has potent anti-T. cruzi activity by inhibiting the CYP51 enzyme. Furthermore, we have developed analogs that minimize the PFT-inhibitory activity and enhance the CYP51 inhibition. In this paper, we describe the efficacy of the lead tipifarnib analog compared to that of posaconazole in a murine model of T. cruzi infection. The plasma exposure profiles for each compound following a single oral dose in mice and estimated exposure parameters after repeated twice-daily dosing for 20 days are also presented. The lead tipifarnib analog had potent suppressive activity on parasitemia in mice but was unsuccessful at curing mice, whereas posaconazole as well as benznidazole cured 3 of 5 and 4 of 6 mice, respectively. The efficacy results are consistent with posaconazole having substantially higher predicted exposure than that of the tipifarnib analog after repeat twice-daily administration. Further changes to the tipifarnib analogs to reduce plasma clearance are therefore likely to be important. A crystal structure of a trypanosomal CYP51 bound to a tipifarnib analog is reported here and provides new insights to guide structure-based drug design for further optimized compounds.
Antimicrobial Agents and Chemotherapy 07/2012; 56(9):4914-21. · 4.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The clinical phenotype of Sanfilippo Syndrome is caused by one of four enzyme deficiencies that are associated with a defect in mucopolysaccharide metabolism. The four subtypes (A, B, C, and D) are each caused by an enzyme deficiency involved in the degradation of heparan sulfate. We have developed a highly efficient synthesis of the substrates and internal standards required for the enzymatic assay of each of the four enzymes. The synthesis of the substrates involves chemical modification of a common intermediate. The substrates and internal standards allow the measurement of the enzymes relevant to heparan N-sulfatase (type A); N-acetyl-α-glucosaminidase (type B); acetyl-CoA:α-glucosamide N-acetyltransferase (type C); and N-acetylglucosamine 6-sulfatase (type D). The internal standards are similar to the substrates and allow for the accurate quantification of the enzyme assays using tandem mass spectrometry. The synthetic substrates incorporate a coumarin moiety and can also be used in fluorometric enzyme assays. We confirm that all four substrates can detect the appropriate Sanfilippo Syndrome in fibroblast lysates, and the measured enzyme activities are distinctly lower by a factor of 10 when compared to fibroblast lysates from unaffected persons.
Bioconjugate Chemistry 02/2012; 23(3):557-64. · 4.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at cleaving the various bonds in phospholipids, namely phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction, lipid mediators production, and digestion in humans. Although all phospholipases target phospholipids as substrates, they vary in the site of action on the phospholipids molecules, physiological function, mode of action, and their regulation. Significant studies on phospholipases characterization, physiological role, and industrial potential have been conducted worldwide. Some of them have been directed for biotechnological advances, such as gene discovery and functional enhancement by protein engineering. Others reported phospholipases as virulence factors and major causes of pathophysiological effects. In this introductory chapter, we provide brief details of different phospholipases.
Methods in molecular biology (Clifton, N.J.) 01/2012; 861:63-85.
-
[show abstract]
[hide abstract]
ABSTRACT: Attempts to characterize, quantify, and/or modulate the activity of the secreted phospholipase A(2) family of enzymes result from the diversity of physiological roles for which these enzymes have been implicated. The 1-palmitoyl-2-(10-pyrenedecanoyl)-phosphatidylglycerol (pyrenePG)-based fluorometric assay is a sensitive and readily adaptable method for further elucidating phospholipase function under various experimental conditions, as well as a tool for screening chemical libraries for potent inhibitors of this enzymatic activity. This assay is based on the observed difference in fluorescent emission of pyrene aggregated in vesicles compared to sequestered in monomeric form by binding to bovine serum albumin after lipolytic activity, thus allowing direct quantification of hydrolyzed fatty acids by the measurement of the corresponding monomeric emission intensity. The assay can be carried out in multiwell plates for high-throughput screening of compound libraries.
Methods in molecular biology (Clifton, N.J.) 01/2012; 861:149-58.
-
Norbert Degousee,
David J. Kelvin,
Gerd Geisslinger,
David M. Hwang,
Eva Stefanski,
Xing-Hua Wang,
Ali Danesh,
Carlo Angioni,
Helmut Schmidt,
Thomas F. Lindsay, Michael H. Gelb,
James Bollinger,
Christine Payré,
Gérard Lambeau,
Jonathan P. Arm,
Armand Keating,
Barry B. Rubin
[show abstract]
[hide abstract]
ABSTRACT: Group V-secreted phospholipase A2 (GV sPLA2) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA2 in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA2 after pulmonary E. coli infection. GV−/− mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10,
and TNF-α levels than GV+/+ mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV+/+ and GV−/− mice after lung E. coli infection. In contrast, GV+/+ mice have higher levels of prostaglandin D2 (PGD2), PGF2α, and 15-keto-PGE2 in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV−/− mice after lung infection with E. coli. Selective deletion of GV sPLA2 in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA2 in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA2 in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate
in the regulation of the innate immune response to pulmonary infection with E. coli.
Journal of Biological Chemistry 10/2011; 286(41):35650-35662. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Structures of tert-butylcarbamate ions in the gas-phase and methanol solution were studied for simple secondary and tertiary carbamates as well as for carbamate-containing products and internal standards for lysosomal enzyme assays used in newborn screening of a α-galactosidase A deficiency (Fabry disease), mucopolysaccharidosis I (Hurler disease), and mucopolysaccharidosis II (Hunter disease). The protonation of simple t-butylcarbamates can occur at the carbonyl group, which is the preferred site in the gas phase. Protonation in methanol solution is more favorable if occurring at the carbamate nitrogen atom. The protonation of more complex t-butylcarbamates occurs at amide and carbamate carbonyl groups, and the ions are stabilized by intramolecular hydrogen bonding, which is affected by solvation. Tertiary carbamates containing aminophenol amide groups were calculated to have substantially greater gas-phase basicities than secondary carbamates containing coumarin amide groups. The main diagnostically important ion dissociation by elimination of 2-methylpropene (isobutylene, i-C(4)H(8)) and carbon dioxide is shown by experiment and theory to proceed in two steps. Energy-resolved collision-induced dissociation of the Hurler's disease enzymatic product ion, which is a coumarin-diamine linker-t-butylcarbamate conjugate (3a(+)), indicated separate energy thresholds for the loss of i-C(4)H(8) and CO(2). Computational investigation of the potential energy surface along two presumed reaction pathways indicated kinetic preference for the migration of a t-butyl hydrogen atom to the carbamate carbonyl resulting in the isobutylene loss. The consequent loss of CO(2) required further proton migrations that had to overcome energy barriers.
Biological Mass Spectrometry 10/2011; 46(10):1089-98. · 3.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Among mammalian secreted phospholipases A(2) (sPLA(2)s), group X sPLA(2) has the most potent hydrolyzing activity toward phosphatidylcholine and is involved in arachidonic acid (AA) release. Group X sPLA(2) is produced as a proenzyme and contains a short propeptide of 11 amino acids ending with a dibasic motif, suggesting cleavage by proprotein convertases. Although the removal of this propeptide is clearly required for enzymatic activity, the cellular location and the protease(s) involved in proenzyme conversion are unknown. Here we have analyzed the maturation of group X sPLA(2) in HEK293 cells, which have been extensively used to analyze sPLA(2)-induced AA release. Using recombinant mouse (PromGX) and human (ProhGX) proenzymes; HEK293 cells transfected with cDNAs coding for full-length ProhGX, PromGX, and propeptide mutants; and various permeable and non-permeable sPLA(2) inhibitors and protease inhibitors, we demonstrate that group X sPLA(2) is mainly converted intracellularly and releases AA before externalization from the cell. Most strikingly, the exogenous proenzyme does not elicit AA release, whereas the transfected proenzyme does elicit AA release in a way insensitive to non-permeable sPLA(2) inhibitors. In transfected cells, a permeable proprotein convertase inhibitor, but not a non-permeable one, prevents group X sPLA(2) maturation and partially blocks AA release. Mutations at the dibasic motif of the propeptide indicate that the last basic residue is required and sufficient for efficient maturation and AA release. All together, these results argue for the intracellular maturation of group X proenzyme in HEK293 cells by a furin-like proprotein convertase, leading to intracellular release of AA during secretion.
Journal of Biological Chemistry 08/2011; 286(42):36509-21. · 4.77 Impact Factor
-
Norbert Degousee,
David J Kelvin,
Gerd Geisslinger,
David M Hwang,
Eva Stefanski,
Xing-Hua Wang,
Ali Danesh,
Carlo Angioni,
Helmut Schmidt,
Thomas F Lindsay, Michael H Gelb,
James Bollinger,
Christine Payré,
Gérard Lambeau,
Jonathan P Arm,
Armand Keating,
Barry B Rubin
[show abstract]
[hide abstract]
ABSTRACT: Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.
Journal of Biological Chemistry 08/2011; 286(41):35650-62. · 4.77 Impact Factor
-
Jia-Shu Yang,
Carmen Valente,
Roman S Polishchuk,
Gabriele Turacchio,
Emilie Layre,
D Branch Moody,
Christina C Leslie, Michael H Gelb,
William J Brown,
Daniela Corda,
Alberto Luini,
Victor W Hsu
[show abstract]
[hide abstract]
ABSTRACT: Intracellular transport occurs through two general types of carrier, either vesicles or tubules. Coat proteins act as the core machinery that initiates vesicle formation, but the counterpart that initiates tubule formation has been unclear. Here, we find that the coat protein I (COPI) complex initially drives the formation of Golgi buds. Subsequently, a set of opposing lipid enzymatic activities determines whether these buds become vesicles or tubules. Lysophosphatidic acid acyltransferase-γ (LPAATγ) promotes COPI vesicle fission for retrograde vesicular transport. In contrast, cytosolic phospholipase A2-α (cPLA2α) inhibits this fission event to induce COPI tubules, which act in anterograde intra-Golgi transport and Golgi ribbon formation. These findings not only advance a molecular understanding of how COPI vesicle fission is achieved, but also provide insight into how COPI acts in intra-Golgi transport and reveal an unexpected mechanistic relationship between vesicular and tubular transport.
Nature Cell Biology 07/2011; 13(8):996-1003. · 19.49 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.
The Journal of Immunology 07/2011; 187(1):482-9. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We report a comparative study of triplex tandem mass spectrometry (MS/MS) based assays of lysosomal enzymes in dried blood spots for the early detection of Pompe, Fabry, and Hurler diseases in newborns. Four methods have been evaluated that differed in sample handling and the equipment used. A newly developed method uses assay quenching with acetonitrile to precipitate blood proteins followed by analysis on an LC-electrospray/MS/MS system capable of multiple consecutive sample injections on two parallel chromatographic columns. This method requires 1.5 min per a triplex analysis of enzyme products and internal standards, which matches the throughput of the previously reported flow injection method. LC separation reduces matrix effects and allows for more facile sample workup. The new LC-based method showed figures of merit that were superior to those of the currently used method based on liquid-liquid extraction into ethyl acetate and flow injection into the mass spectrometer. The other methods we investigated for comprehensive comparison involved liquid-liquid extraction into ethyl acetate followed by LC-ESI-MS/MS and acetonitrile quenching followed by direct flow injection. Both methods using acetonitrile quenching were found to be robust and provide good quality data while requiring fewer liquid transfer steps and less disposable material and labor than did the extraction methods. The individual merits of the new methods are discussed to present an evaluated alternative approach to high-throughput analysis in newborn screening laboratories.
Analytical Chemistry 06/2011; 83(12):4822-8. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Group X (GX) phospholipase A(2), a member of a large group of secreted phospholipases A(2) (sPLA(2)s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA(2) significantly impairs development of the asthma phenotype. In this study, we generated mGX-sPLA(2)(-/-) mice with knock-in of human GX (hGX)-sPLA(2) (i.e. hGX-sPLA(2)(+/+) knock-in mice) to understand more fully the role of GX-sPLA(2) in these allergic pulmonary responses and to assess the effect of pharmacological blockade of the GX-sPLA(2)-mediated responses. Knock-in of hGX-sPLA(2) in mGX-sPLA(2)(-/-) mice restored the allergen-induced airway infiltration by inflammatory cells, including eosinophils, goblet cell metaplasia, and hyperresponsiveness to methacholine in the mGX-sPLA(2)-deficient mice. This knock-in mouse model enabled the use of a highly potent indole-based inhibitor of hGX-sPLA(2), RO061606 (which is ineffective against mGX-sPLA(2)), to assess the potential utility of GX-sPLA(2) blockade as a therapeutic intervention in asthma. Delivery of RO061606 via mini-osmotic pumps enabled the maintenance in vivo in the mouse asthma model of plasma inhibitor concentrations near 10 μm, markedly higher than the IC(50) for inhibition of hGX-sPLA(2) in vitro. RO061606 significantly decreased allergen-induced airway inflammation, mucus hypersecretion, and hyperresponsiveness in the hGX-sPLA(2)(+/+) knock-in mouse. Thus, development of specific hGX-sPLA(2) inhibitors may provide a new pharmacological opportunity for the treatment of patients with asthma.
Journal of Biological Chemistry 06/2011; 286(32):28049-55. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have developed a tandem mass spectrometry based assay of iduronate-2-sulfatase (IdS) activity for the neonatal detection of mucopolysaccharidosis II (MPS-II, Hunter Syndrome). The assay uses a newly designed synthetic substrate (IdS-S) consisting of α-L-iduronate-2-sulfate, which is glycosidically conjugated to a coumarin and a linker containing a tert-butyloxycarbamido group. A short synthesis of the substrate has been developed that has the potential of being scaled to multigram quantities. Sulfate hydrolysis of IdS-S by IdS found within a 3 mm dried blood spot specifically produces a nonsulfated product (IdS-P) which is detected by electrospray tandem mass spectrometry and quantified using a deuterium-labeled internal standard, both carried out in positive ion mode. Analysis of DBS from 75 random human newborns showed IdS activities in the range of 4.8-16.2 (mean 9.1) μmol/(h L of blood), which were clearly distinguished from the activities measured for 14 MPS-II patients at 0.17-0.52 (mean 0.29) μmol/(h L of blood). The assay shows low blank activity, 0.15 ± 0.03 μmol/(h L of blood). The within-assay coefficient of variation (CV) was 3.1% while the interassay CV was 15%.
Analytical Chemistry 02/2011; 83(3):1152-6. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Group IVA cytosolic phospholipase A(2) (cPLA(2)α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)α activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)α. Our results define how the mutations negatively influence cPLA(2)α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.
Biochemistry 02/2011; 50(10):1731-8. · 3.42 Impact Factor
