Suvorexant Acutely Decreases Tau Phosphorylation and Aß in the Human CNS.

OBJECTIVE: In Alzheimer's disease, hyperphosphorylated tau is associated with formation of insoluble paired helical filaments that aggregate as neurofibrillary tau tangles and are associated with neuronal loss and cognitive symptoms. Dual orexin receptor antagonists decrease soluble amyloid-ß levels and amyloid plaques in mouse models overexpressing amyloid-ß, but have not been reported to affect tau phosphorylation. In this randomized controlled trial, we tested the acute effect of suvorexant, a dual orexin receptor antagonist, on amyloid-ß, tau, and phospho-tau. METHODS: Thirty-eight cognitively unimpaired participants aged 45 to 65 years were randomized to placebo (N = 13), suvorexant 10 mg (N = 13), and suvorexant 20 mg (N = 12). Six milliliters of cerebrospinal fluid were collected via an indwelling lumbar catheter every 2 hours for 36 hours starting at 20:00. Participants received placebo or suvorexant at 21:00. All samples were processed and measured for multiple forms of amyloid-ß, tau, and phospho-tau via immunoprecipitation and liquid chromatography-mass spectrometry. RESULTS: The ratio of phosphorylated-tau-threonine-181 to unphosphorylated-tau-threonine-181, a measure of phosphorylation at this tau phosphosite, decreased ~10% to 15% in participants treated with suvorexant 20 mg compared to placebo. However, phosphorylation at tau-serine-202 and tau-threonine-217 were not decreased by suvorexant. Suvorexant decreased amyloid-ß ~10% to 20% compared to placebo starting 5 hours after drug administration. INTERPRETATION: In this study, suvorexant acutely decreased tau phosphorylation and amyloid-ß concentrations in the central nervous system. Suvorexant is approved by the US Food and Drug Administration to treatment insomnia and may have potential as a repurposed drug for the prevention of Alzheimer's disease, however, future studies with chronic treatment are needed. ANN NEUROL 2023;94:27-40.

Apolipoprotein E O-glycosylation is associated with amyloid plaques and APOE genotype.

Although the APOE ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (AD), the relationship between apolipoprotein (apoE) and AD pathophysiology is not yet fully understood. Relatively little is known about the apoE protein species, including post-translational modifications, that exist in the human periphery and CNS. To better understand these apoE species, we developed a LC-MS/MS assay that simultaneously quantifies both unmodified and O-glycosylated apoE peptides. The study cohort included 47 older individuals (age 75.6 ± 5.7 years [mean ± standard deviation]), including 23 individuals (49%) with cognitive impairment. Paired plasma and cerebrospinal fluid samples underwent analysis. We quantified O-glycosylation of two apoE protein residues - one in the hinge region and one in the C-terminal region - and found that glycosylation occupancy of the hinge region in the plasma was significantly correlated with plasma total apoE levels, APOE genotype and amyloid status as determined by CSF Aß42/Aß40. A model with plasma glycosylation occupancy, plasma total apoE concentration, and APOE genotype distinguished amyloid status with an AUROC of 0.89. These results suggest that plasma apoE glycosylation levels could be a marker of brain amyloidosis, and that apoE glycosylation may play a role in the pathophysiology of AD.

Acute sleep loss decreases CSF-to-blood clearance of Alzheimer's disease biomarkers.

INTRODUCTION: Sleep deprivation increases cerebrospinal fluid (CSF) amyloid beta (Aß) and tau levels; however, sleep's effect on Aß and tau in plasma is unknown. METHODS: In a cross-over design, CSF Aß and tau concentrations were measured in five cognitively normal individuals who had blood and CSF collected every 2 hours for 36 hours during sleep-deprived and normal sleep control conditions. RESULTS: Aß40, Aß42, unphosphorylated tau threonine181 (T181), unphosphorylated tau threonine-217 (T217), and phosphorylated T181 (pT181) concentrations increased ∼35% to 55% in CSF and decreased ∼5% to 15% in plasma during sleep deprivation. CSF/plasma ratios of all Alzheimer's disease (AD) biomarkers increased during sleep deprivation while the CSF/plasma albumin ratio, a measure of blood-CSF barrier permeability, decreased. CSF and plasma Aß42/40, pT181/T181, and pT181/Aß42 ratios were stable longitudinally in both groups. DISCUSSION: These findings show that sleep loss alters some plasma AD biomarkers by lowering brain clearance mechanisms and needs to be taken into account when interpreting individual plasma AD biomarkers but not ratios.

Comparative analytical performance of multiple plasma Aß42 and Aß40 assays and their ability to predict positron emission tomography amyloid positivity.

INTRODUCTION: This report details the approach taken to providing a dataset allowing for analyses on the performance of recently developed assays of amyloid beta (Aß) peptides in plasma and the extent to which they improve the prediction of amyloid positivity. METHODS: Alzheimer's Disease Neuroimaging Initiative plasma samples with corresponding amyloid positron emission tomography (PET) data were run on six plasma Aß assays. Statistical tests were performed to determine whether the plasma Aß measures significantly improved the area under the receiver operating characteristic curve for predicting amyloid PET status compared to age and apolipoprotein E (APOE) genotype. RESULTS: The age and APOE genotype model predicted amyloid status with an area under the curve (AUC) of 0.75. Three assays improved AUCs to 0.81, 0.81, and 0.84 (P < .05, uncorrected for multiple comparisons). DISCUSSION: Measurement of Aß in plasma contributes to addressing the amyloid component of the ATN (amyloid/tau/neurodegeneration) framework and could be a first step before or in place of a PET or cerebrospinal fluid screening study. HIGHLIGHTS: The Foundation of the National Institutes of Health Biomarkers Consortium evaluated six plasma amyloid beta (Aß) assays using Alzheimer's Disease Neuroimaging Initiative samples. Three assays improved prediction of amyloid status over age and apolipoprotein E (APOE) genotype. Plasma Aß42/40 predicted amyloid positron emission tomography status better than Aß42 or Aß40 alone.

The Skyline ecosystem: Informatics for quantitative mass spectrometry proteomics.

Skyline is a freely available, open-source Windows client application for accelerating targeted proteomics experimentation, with an emphasis on the proteomics and mass spectrometry community as users and as contributors. This review covers the informatics encompassed by the Skyline ecosystem, from computationally assisted targeted mass spectrometry method development, to raw acquisition file data processing, and quantitative analysis and results sharing.

PECAN: library-free peptide detection for data-independent acquisition tandem mass spectrometry data.

Data-independent acquisition (DIA) is an emerging mass spectrometry (MS)-based technique for unbiased and reproducible measurement of protein mixtures. DIA tandem mass spectrometry spectra are often highly multiplexed, containing product ions from multiple cofragmenting precursors. Detecting peptides directly from DIA data is therefore challenging; most DIA data analyses require spectral libraries. Here we present PECAN (http://pecan.maccosslab.org), a library-free, peptide-centric tool that robustly and accurately detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, which enables detection of low-abundance analytes with poor precursor ion signal. We demonstrate the chromatographic peak picking accuracy and peptide detection capability of PECAN, and we further validate its detection with data-dependent acquisition and targeted analyses. Lastly, we used PECAN to build a plasma proteome library from DIA data and to query known sequence variants.

Human Central Nervous System (CNS) ApoE Isoforms Are Increased by Age, Differentially Altered by Amyloidosis, and Relative Amounts Reversed in the CNS Compared with Plasma.

The risk of Alzheimer's disease (AD) is highly dependent on apolipoprotein-E (apoE) genotype. The reasons for apoE isoform-selective risk are uncertain; however, both the amounts and structure of human apoE isoforms have been hypothesized to lead to amyloidosis increasing the risk for AD. To address the hypothesis that amounts of apoE isoforms are different in the human CNS, we developed a novel isoform-specific method to accurately quantify apoE isoforms in clinically relevant samples. The method utilizes an antibody-free enrichment step and isotope-labeled physiologically relevant lipoprotein particle standards produced by immortalized astrocytes. We applied this method to a cohort of well characterized clinical samples and observed the following findings. The apoE isoform amounts are not different in cerebrospinal fluid (CSF) from young normal controls, suggesting that the amount of apoE isoforms is not the reason for risk of amyloidosis prior to the onset of advanced age. We did, however, observe an age-related increase in both apoE isoforms. In contrast to normal aging, the presence of amyloid increased apoE3, whereas apoE4 was unchanged or decreased. Importantly, for heterozygotes, the apoE4/apoE3 isoform ratio was increased in the CNS, although the reverse was true in the periphery. Finally, CSF apoE levels, but not plasma apoE levels, correlated with CSF ß-amyloid levels. Collectively, these findings support the hypothesis that CNS and peripheral apoE are separate pools and differentially regulated. Furthermore, these results suggest that apoE mechanisms for the risk of amyloidosis and AD are related to an interaction between apoE, aging, and the amount of amyloid burden.

Automated Trapping Column Exchanger for High-Throughput Nanoflow Liquid Chromatography.

As compared to conventional high-performance liquid chromatography (HPLC) techniques, nanoflow HPLC exhibits improved sensitivity and limits of detection. However, nanoflow HPLC suffers from low throughput due to instrument failure (e.g., fitting fatigue and trapping column failure), limiting the utility of the technique for clinical and industrial applications. To increase the robustness of nanoflow HPLC, we have developed and tested a trapping column exchanging robot for autonomous interchange of trapping columns. This robot makes reproducible, automated connections between the active trapping column and the rest of the HPLC system. The intertrapping column retention time is shown to be sufficiently reproducible for scheduled selected reaction monitoring assays to be performed on different trapping columns without rescheduling the selection windows.

Using Data Independent Acquisition (DIA) to Model High-responding Peptides for Targeted Proteomics Experiments.

Targeted mass spectrometry is an essential tool for detecting quantitative changes in low abundant proteins throughout the proteome. Although selected reaction monitoring (SRM) is the preferred method for quantifying peptides in complex samples, the process of designing SRM assays is laborious. Peptides have widely varying signal responses dictated by sequence-specific physiochemical properties; one major challenge is in selecting representative peptides to target as a proxy for protein abundance. Here we present PREGO, a software tool that predicts high-responding peptides for SRM experiments. PREGO predicts peptide responses with an artificial neural network trained using 11 minimally redundant, maximally relevant properties. Crucial to its success, PREGO is trained using fragment ion intensities of equimolar synthetic peptides extracted from data independent acquisition experiments. Because of similarities in instrumentation and the nature of data collection, relative peptide responses from data independent acquisition experiments are a suitable substitute for SRM experiments because they both make quantitative measurements from integrated fragment ion chromatograms. Using an SRM experiment containing 12,973 peptides from 724 synthetic proteins, PREGO exhibits a 40-85% improvement over previously published approaches at selecting high-responding peptides. These results also represent a dramatic improvement over the rules-based peptide selection approaches commonly used in the literature.

A targeted proteomic strategy for the measurement of oral cancer candidate biomarkers in human saliva.

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non-invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically-derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.

Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma.

Secreted phospholipase A2s (sPLA2s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA2s function as enzymes, some of the sPLA2s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA2s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1-/-) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1-/- mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1-/- mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation.

Regulation and function of epithelial secreted phospholipase A2 group X in asthma.

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 patients with asthma (19 with and 15 without EIB) and 10 normal control subjects 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 tumor necrosis factor, 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.

LC/ESI-MS/MS detection of FAs by charge reversal derivatization with more than four orders of magnitude improvement in sensitivity.

Quantitative analysis of fatty acids (FAs) is an important area of analytical biochemistry. Ultra high sensitivity FA analysis usually is done with gas chromatography of pentafluorobenzyl esters coupled to an electron-capture detector. With the popularity of electrospray ionization (ESI) mass spectrometers coupled to liquid chromatography, it would be convenient to develop a method for ultra high sensitivity FA detection using this equipment. Although FAs can be analyzed by ESI in negative ion mode, this method is not very sensitive. In this study, we demonstrate a new method of FA analysis based on conversion of the carboxylic acid to an amide bearing a permanent positive charge, N-(4-aminomethylphenyl)pyridinium (AMPP) combined with analysis on a reverse-phase liquid chromatography column coupled to an ESI mass spectrometer operating in positive ion mode. This leads to an ∼60,000-fold increase in sensitivity compared with the same method carried out with underivatized FAs. The new method is about 10-fold more sensitive than the existing method of gas chromatography/electron-capture mass spectrometry of FA pentafluorobenzyl esters. Furthermore, significant fragmentation of the precursor ions in the nontag portion improves analytical specificity. We show that a large number of FA molecular species can be analyzed with this method in complex biological samples such as mouse serum.

Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model.

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA(2) (sPLA(2)-X), the sPLA(2) with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA(2)-X(-/-) mice, compared with those of sPLA(2)-X(+/+) littermates, had significant reduction in ovalbumin-induced infiltration by CD4(+) and CD8(+) T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA(2)-X as a novel therapeutic target for asthma.

Predicting continuous amyloid PET values with CSF and plasma Aß42/Aß40.

Introduction: Continuous measures of amyloid burden as measured by positron emission tomography (PET) are being used increasingly to stage Alzheimer's disease (AD). This study examined whether cerebrospinal fluid (CSF) and plasma amyloid beta (Aß)42/Aß40 could predict continuous values for amyloid PET. Methods: CSF Aß42 and Aß40 were measured with automated immunoassays. Plasma Aß42 and Aß40 were measured with an immunoprecipitation-mass spectrometry assay. Amyloid PET was performed with Pittsburgh compound B (PiB). The continuous relationships of CSF and plasma Aß42/Aß40 with amyloid PET burden were modeled. Results: Most participants were cognitively normal (427 of 491 [87%]) and the mean age was 69.0 ± 8.8 years. CSF Aß42/Aß40 predicted amyloid PET burden until a relatively high level of amyloid accumulation (69.8 Centiloids), whereas plasma Aß42/Aß40 predicted amyloid PET burden until a lower level (33.4 Centiloids). Discussion: CSF Aß42/Aß40 predicts the continuous level of amyloid plaque burden over a wider range than plasma Aß42/Aß40 and may be useful in AD staging. Highlights: Cerebrospinal fluid (CSF) amyloid beta (Aß)42/Aß40 predicts continuous amyloid positron emission tomography (PET) values up to a relatively high burden.Plasma Aß42/Aß40 is a comparatively dichotomous measure of brain amyloidosis.Models can predict regional amyloid PET burden based on CSF Aß42/Aß40.CSF Aß42/Aß40 may be useful in staging AD.

Blockade of human group X secreted phospholipase A2 (GX-sPLA2)-induced airway inflammation and hyperresponsiveness in a mouse asthma model by a selective GX-sPLA2 inhibitor.

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.

The performance of plasma amyloid beta measurements in identifying amyloid plaques in Alzheimer's disease: a literature review.

The extracellular buildup of amyloid beta (Aß) plaques in the brain is a hallmark of Alzheimer's disease (AD). Detection of Aß pathology is essential for AD diagnosis and for identifying and recruiting research participants for clinical trials evaluating disease-modifying therapies. Currently, AD diagnoses are usually made by clinical assessments, although detection of AD pathology with positron emission tomography (PET) scans or cerebrospinal fluid (CSF) analysis can be used by specialty clinics. These measures of Aß aggregation, e.g. plaques, protofibrils, and oligomers, are medically invasive and often only available at specialized medical centers or not covered by medical insurance, and PET scans are costly. Therefore, a major goal in recent years has been to identify blood-based biomarkers that can accurately detect AD pathology with cost-effective, minimally invasive procedures.To assess the performance of plasma Aß assays in predicting amyloid burden in the central nervous system (CNS), this review compares twenty-one different manuscripts that used measurements of 42 and 40 amino acid-long Aß (Aß42 and Aß40) in plasma to predict CNS amyloid status. Methodologies that quantitate Aß42 and 40 peptides in blood via immunoassay or immunoprecipitation-mass spectrometry (IP-MS) were considered, and their ability to distinguish participants with amyloidosis compared to amyloid PET and CSF Aß measures as reference standards was evaluated. Recent studies indicate that some IP-MS assays perform well in accurately and precisely measuring Aß and detecting brain amyloid aggregates.

Validation of Plasma Amyloid-ß 42/40 for Detecting Alzheimer Disease Amyloid Plaques.

BACKGROUND AND OBJECTIVES: To determine the diagnostic accuracy of a plasma Aß42/Aß40 assay in classifying amyloid PET status across global research studies using samples collected by multiple centers that utilize different blood collection and processing protocols. METHODS: Plasma samples (n = 465) were obtained from 3 large Alzheimer disease (AD) research cohorts in the United States (n = 182), Australia (n = 183), and Sweden (n = 100). Plasma Aß42/Aß40 was measured by a high precision immunoprecipitation mass spectrometry (IPMS) assay and compared to the reference standards of amyloid PET and CSF Aß42/Aß40. RESULTS: In the combined cohort of 465 participants, plasma Aß42/Aß40 had good concordance with amyloid PET status (receiver operating characteristic area under the curve [AUC] 0.84, 95% confidence interval [CI] 0.80-0.87); concordance improved with the inclusion of APOE ε4 carrier status (AUC 0.88, 95% CI 0.85-0.91). The AUC of plasma Aß42/Aß40 with CSF amyloid status was 0.85 (95% CI 0.78-0.91) and improved to 0.93 (95% CI 0.89-0.97) with APOE ε4 status. These findings were consistent across the 3 cohorts, despite differences in protocols. The assay performed similarly in both cognitively unimpaired and impaired individuals. DISCUSSION: Plasma Aß42/Aß40 is a robust measure for detecting amyloid plaques and can be utilized to aid in the diagnosis of AD, identify those at risk for future dementia due to AD, and improve the diversity of populations enrolled in AD research and clinical trials. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that plasma Aß42/Aß40, as measured by a high precision IPMS assay, accurately diagnoses brain amyloidosis in both cognitively unimpaired and impaired research participants.

Interfacial kinetic and binding properties of mammalian group IVB phospholipase A2 (cPLA2beta) and comparison with the other cPLA2 isoforms.

The cytosolic (group IV) phospholipase A(2) (cPLA(2)s) family contains six members. We have prepared recombinant proteins for human α, mouse ß, human γ, human δ, human ε, and mouse ζ cPLA(2)s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA(2)ß action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca(2+) only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca(2+) on the interfacial binding of mouse cPLA(2)ß and its C2 domain to vesicles. Ca(2+)-dependent binding of mouse cPLA(2)ß to cardiolipin-containing vesicles requires a patch of basic residues near the Ca(2+)-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA(2)δ also displays Ca(2+)- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A(1), and phospholipase A(2) activities of the full set of mammalian cPLA(2)s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA(2)δ stands out as having relatively high phospholipase A(1) activity. We also tested the susceptibility of all cPLA(2) family members to a panel of previously reported inhibitors of human cPLA(2)α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA(2)ß. These in vitro studies help determine the regulation and function of the cPLA(2) family members.

Eosinophil cysteinyl leukotriene synthesis mediated by exogenous secreted phospholipase A2 group X.

Secreted phospholipase A(2) group X (sPLA(2)-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C(4), D(4), and E(4)) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA(2)-X. We found that recombinant sPLA(2)-X caused marked AA release and a rapid onset of CysLT synthesis in human eosinophils that was blocked by a selective sPLA(2)-X inhibitor. Exogenous sPLA(2)-X released lysophospholipid species that arise from phospholipids enriched in AA in eosinophils, including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine as well as plasmenyl phosphatidylcholine and phosphatidylethanolamine. CysLT synthesis mediated by sPLA(2)-X but not AA release could be suppressed by inhibition of cPLA(2)α. Exogenous sPLA(2)-X initiated Ser(505) phosphorylation of cPLA(2)α, an intracellular Ca(2+) flux, and translocation of cPLA(2)α and 5-lipoxygenase in eosinophils. Synthesis of CysLTs in response to sPLA(2)-X or lysophosphatidylcholine was inhibited by p38 or JNK inhibitors but not by a MEK 1/2 inhibitor. A further increase in CysLT synthesis was induced by the addition of sPLA(2)-X to eosinophils under conditions of N-formyl-methionyl-leucyl-phenylalanine-mediated cPLA(2)α activation. These results indicate that sPLA(2)-X participates in AA and lysophospholipid release, resulting in CysLT synthesis in eosinophils through a mechanism involving p38 and JNK MAPK, cPLA(2)α, and 5-lipoxygenase activation and resulting in the amplification of CysLT synthesis during cPLA(2)α activation. Transactivation of eosinophils by sPLA(2)-X may be an important mechanism leading to CysLT formation in the airways of patients with asthma.