Mass Spectrometry Defines Lysophospholipids as Ligands for Chicken MHCY Class I Molecules.

Chicken (Gallus gallus) MHCY class I molecules are highly polymorphic yet substantially different from polymorphic MHC class I molecules that bind peptide Ags. The binding grooves in MHCY class I molecules are hydrophobic and too narrow to accommodate peptides. An earlier structural study suggested that ligands for MHCY class I might be lipids, but the contents of the groove were not clearly identified. In this study, lysophospholipids have been identified by mass spectrometry as bound in two MHCY class I isoforms that differ substantially in sequence. The two isoforms, YF1*7.1 and YF1*RJF34, differ by 35 aa in the α1 and α2 domains that form the MHC class I ligand binding groove. Lyso-phosphatidylethanolamine (lyso-PE) 18:1 was the dominant lipid identified in YF1*7.1 and YF1*RJF34 expressed as recombinant molecules and renatured with ß2-microglobulin in the presence of a total lipid extract from Escherichia coli. Less frequently detected were lyso-PE 17:1, lyso-PE 16:1, and lysophosphatidylglycerols 17:1 and 16:0. These data provide evidence that lysophospholipids are candidate ligands for MHCY class I molecules. Finding that MHCY class I isoforms differing substantially in sequence bind the same array of lysophospholipids indicates that the amino acid polymorphism that distinguishes MHCY class I molecules is not key in defining ligand specificity. The polymorphic positions lie mostly away from the binding groove and might define specificity in interactions of MHCY class I molecules with receptors that are presently unidentified. MHCY class I molecules are distinctive in bound ligand and in display of polymorphic residues.

The Plethora of Angiotensin-Converting Enzyme-Processed Peptides in Mouse Plasma.

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

Two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

Fibrinogen and the prediction of residual obstruction manifested after pulmonary embolism treatment.

Residual pulmonary vascular obstruction (RPVO) and chronic thromboembolic pulmonary hypertension (CTEPH) are both long-term complications of acute pulmonary embolism, but it is unknown whether RPVO can be predicted by variants of fibrinogen associated with CTEPH.We used the Akaike information criterion to select the best predictive models for RPVO in two prospectively followed cohorts of acute pulmonary embolism patients, using as candidate variables the extent of the initial obstruction, clinical characteristics and fibrinogen-related data. We measured the selected models' goodness of fit by analysis of deviance and compared models using the Chi-squared test.RPVO occurred in 29 (28.4%) out of 102 subjects in the first cohort and 46 (25.3%) out of 182 subjects in the second. The best-fit predictive model derived in the first cohort (p=0.0002) and validated in the second cohort (p=0.0005) implicated fibrinogen Bß-chain monosialylation in the development of RPVO. When the derivation procedure excluded clinical characteristics, fibrinogen Bß-chain monosialylation remained a predictor of RPVO in the best-fit predictive model (p=0.00003). Excluding fibrinogen characteristics worsened the predictive model (p=0.03).Fibrinogen Bß-chain monosialylation, a common structural attribute of fibrin, helped predict RPVO after acute pulmonary embolism. Fibrin structure may contribute to the risk of developing RPVO.

Lipid Identification by Untargeted Tandem Mass Spectrometry Coupled with Ultra-High-Pressure Liquid Chromatography.

Lipidomics refers to the large-scale study of lipids in biological systems (Wenk, Nat Rev Drug Discov 4(7):594-610, 2005; Rolim et al., Gene 554(2):131-139, 2015). From a mass spectrometric point of view, by lipidomics we understand targeted or untargeted mass spectrometric analysis of lipids using either liquid chromatography (LC) (Castro-Perez et al., J Proteome Res 9(5):2377-2389, 2010) or shotgun (Han and Gross, Mass Spectrom Rev 24(3):367-412, 2005) approaches coupled with tandem mass spectrometry. This chapter describes the former methodology, which is becoming rapidly the preferred method for lipid identification owing to similarities with established omics workflows, such as proteomics (Washburn et al., Nat Biotechnol 19(3):242-247, 2001) or genomics (Yadav, J Biomol Tech: JBT 18(5):277, 2007). The workflow described consists in lipid extraction using a modified Bligh and Dyer method (Bligh and Dyer, Can J Biochem Physiol 37(8):911-917, 1959), ultra high pressure liquid chromatography fractionation of lipid samples on a reverse phase C18 column, followed by tandem mass spectrometric analysis and in silico database search for lipid identification based on MSMS spectrum matching (Kind et al., Nat Methods 10(8):755-758, 2013; Yamada et al., J Chromatogr A 1292:211-218, 2013; Taguchi and Ishikawa, J Chromatogr A 1217(25):4229-4239, 2010; Peake et al., Thermoscientifices 1-3, 2015) and accurate mass of parent ion (Sud et al., Nucleic Acids Res 35(database issue):D527-D532, 2007; Wishart et al., Nucleic Acids Res 35(database):D521-D526, 2007).

Ticagrelor Removal From Human Blood.

The authors devised an efficient method for ticagrelor removal from blood using sorbent hemadsorption. Ticagrelor removal was measured in 2 sets of in vitro experiments. The first set was a first-pass experiment using bovine serum albumin (BSA) solution pre-incubated with ticagrelor, whereas the second set, performed in a recirculating manner, used human blood mixed with ticagrelor. Removal of ticagrelor from BSA solution reached values >99%. The peak removal rate was 99% and 94% from whole blood and 99.99% and 90% from plasma during 10 h and 3 to 4 h of recirculating experiments, respectively. In conclusion, hemadsorption robustly removes ticagrelor from BSA solution and human blood samples.

Receptor-Mediated Mechanism Controlling Tissue Levels of Bioactive Lipid Oxidation Products.

RATIONALE: Oxidative stress is an important contributing factor in several human pathologies ranging from atherosclerosis to cancer progression; however, the mechanisms underlying tissue protection from oxidation products are poorly understood. Oxidation of membrane phospholipids, containing the polyunsaturated fatty acid docosahexaenoic acid, results in the accumulation of an end product, 2-(ω-carboxyethyl)pyrrole (CEP), which was shown to have proangiogenic and proinflammatory functions. Although CEP is continuously accumulated during chronic processes, such as tumor progression and atherosclerosis, its level during wound healing return to normal when the wound is healed, suggesting the existence of a specific clearance mechanism. OBJECTIVE: To identify the cellular and molecular mechanism for CEP clearance. METHODS AND RESULTS: Here, we show that macrophages are able to bind, scavenge, and metabolize carboxyethylpyrrole derivatives of proteins but not structurally similar ethylpyrrole derivatives, demonstrating the high specificity of the process. F4/80(hi) and M2-skewed macrophages are much more efficient at CEP binding and scavenging compared with F4/80(lo) and M1-skewed macrophages. Depletion of macrophages leads to increased CEP accumulation in vivo. CEP binding and clearance are dependent on 2 receptors expressed by macrophages, CD36 and toll-like receptor 2. Although knockout of each individual receptor results in diminished CEP clearance, the lack of both receptors almost completely abrogates macrophages' ability to scavenge CEP derivatives of proteins. CONCLUSIONS: Our study demonstrates the mechanisms of recognition, scavenging, and clearance of pathophysiologically active products of lipid oxidation in vivo, thereby contributing to tissue protection against products of oxidative stress.