N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization.

Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.

Structural Divergence in O-GlcNAc Glycans Displayed on Epidermal Growth Factor-like Repeats of Mammalian Notch1.

Extracellular O-GlcNAc is a novel class of modification catalyzed by epidermal growth factor-like (EGF)-domain specific O-GlcNAc transferase (EOGT). In mammals, EOGT is required for ligand-mediated Notch signaling for vascular development. Previous studies have revealed that O-GlcNAc in mammalian cultured cells is subject to subsequent glycosylation, which may impose additional layers of regulation. This study aimed to analyze the O-GlcNAc glycans of Drosophila EGF20 as model substrates and mouse Notch1 EGF repeats by mass-spectrometry. The analysis of Drosophila EGF20 expressed in HEK293T cells revealed that the majority of the proteins are modified with an elongated form of O-GlcNAc glycan comprising terminal galactose or sialic acid residues. In contrast, recombinant Notch1 EGF repeats isolated from HEK293T cells revealed structural divergence of O-GlcNAc glycans among the different EGF domains. Although the majority of Notch1 EGF2 and EGF20 domains contained the extended forms of the glycan, the O-GlcNAc in many other domains mostly existed as a monosaccharide irrespective of the exogenous EOGT expression. Our results raised a hypothesis that an array of O-GlcNAc monosaccharides may impact the structure and function of Notch receptors.

Comprehensive identification of sphingolipid species by in silico retention time and tandem mass spectral library.

BACKGROUND: Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is used for comprehensive metabolome and lipidome analyses. Compound identification relies on similarity matching of the retention time (RT), precursor m/z, isotopic ratio, and MS/MS spectrum with reference compounds. For sphingolipids, however, little information on the RT and MS/MS references is available. RESULTS: Negative-ion ESI-MS/MS is a useful method for the structural characterization of sphingolipids. We created theoretical MS/MS spectra for 21 sphingolipid classes in human and mouse (109,448 molecules), with substructure-level annotation of unique fragment ions by MS-FINDER software. The existence of ceramides with ß-hydroxy fatty acids was confirmed in mouse tissues based on cheminformatic- and quantum chemical evidences. The RT of sphingo- and glycerolipid species was also predicted for our LC condition. With this information, MS-DIAL software for untargeted metabolome profiling could identify 415 unique structures including 282 glycerolipids and 133 sphingolipids from human cells (HEK and HeLa) and mouse tissues (ear and liver). CONCLUSIONS: MS-DIAL and MS-FINDER software programs can identify 42 lipid classes (21 sphingo- and 21 glycerolipids) with the in silico RT and MS/MS library. The library is freely available as Microsoft Excel files at the software section of our RIKEN PRIMe website (http://prime.psc.riken.jp/).

Effects of sex, age, and fasting conditions on plasma lipidomic profiles of fasted Sprague-Dawley rats.

Circulating lipid molecules reflect biological processes in the body and, thus, are useful tools for preclinical estimation of the efficacy and safety of newly developed drugs. However, background information on profiles of circulating lipid molecules in preclinical animal models is limited. Therefore, we examined the effects of multiple factors such as sex (fasted male vs. female), age (fasted 10 vs. 30 weeks old), and feeding conditions (feeding vs. fasting, 16 vs. 22 hr fasting, 10 AM vs. 4 PM blood collection), on the global profiles of lipid molecules in plasma from Sprague-Dawley rats by using a lipidomic approach. Our assay platform determined 262 lipid molecules (68 phospholipids, 20 sphingolipids, 138 neutral lipids, and 36 polyunsaturated fatty acids and their metabolites) in rat plasma. Multivariate discriminant analysis (orthogonal partial least squares discriminant analysis) and heat maps of statistically significant lipid molecules revealed that the plasma lipid profiles in rats are predominantly influenced by feeding conditions, followed by sex and age. In addition, the fasting duration (16 vs. 22 hr fasting) or the time of blood collection (10 AM vs. 4 PM blood collection) has limited or no contribution on the profiles of lipid molecules in rat plasma. Our results provide useful, fundamental information for exploring and validating biomarkers in future preclinical studies and may help to establish regulatory standards for such studies.

Glucosylceramide and lysophosphatidylcholines as potential blood biomarkers for drug-induced hepatic phospholipidosis.

Drug-induced phospholipidosis is one of the major concerns in drug development and clinical treatment. The present study involved the use of a nontargeting lipidomic analysis with liquid chromatography-mass spectrometry to explore noninvasive blood biomarkers for hepatic phospholipidosis from rat plasma. We used three tricyclic antidepressants (clomipramine [CPM], imipramine [IMI], and amitriptyline [AMT]) for the model of phospholipidosis in hepatocytes and ketoconazole (KC) for the model of phospholipidosis in cholangiocytes and administered treatment for 3 and 28 days each. Total plasma lipids were extracted and measured. Lipid molecules contributing to the separation of control and drug-treated rat plasma in a multivariate orthogonal partial least squares discriminant analysis were identified. Four lysophosphatidylcholines (LPCs) (16:1, 18:1, 18:2, and 20:4) and 42:1 hexosylceramide (HexCer) were identified as molecules separating control and drug-treated rats in all models of phospholipidosis in hepatocytes. In addition, 16:1, 18:2, and 20:4 LPCs and 42:1 HexCer were identified in a model of hepatic phospholipidosis in cholangiocytes, although LPCs were identified only in the case of 3-day treatment with KC. The levels of LPCs were decreased by drug-induced phospholipidosis, whereas those of 42:1 HexCer were increased. The increase in 42:1 HexCer was much higher in the case of IMI and AMT than in the case of CPM; moreover, the increase induced by IMI was dose-dependent. Structural characterization determining long-chain base and hexose delineated that 42:1 HexCer was d18:1/24:0 glucosylceramide (GluCer). In summary, our study demonstrated that d18:1/24:0 GluCer and LPCs are potential novel biomarkers for drug-induced hepatic phospholipidosis.

Plasma and serum lipidomics of healthy white adults shows characteristic profiles by subjects' gender and age.

Blood is a commonly used biofluid for biomarker discovery. Although blood lipid metabolites are considered to be potential biomarker candidates, their fundamental properties are not well characterized. We aimed to (1) investigate the matrix type (serum vs. plasma) that may be preferable for lipid biomarker exploration, (2) elucidate age- and gender-associated differences in lipid metabolite levels, and (3) examine the stability of lipid metabolites in matrix samples subjected to repeated freeze-thaw cycles. Using liquid chromatography-mass spectrometry, we performed lipidomic analyses for fasting plasma and serum samples for four groups (15 subjects/group) of young and elderly (25-34 and 55-64 years old, respectively) males and females and for an additional aliquot of samples from young males, which were subjected to repeated freeze-thaw cycles. Lysophosphatidylcholine and diacylglycerol levels were higher in serum than in plasma samples, suggesting that the clotting process influences serum lipid metabolite levels. Gender-associated differences highlighted that the levels of many sphingomyelin species were significantly higher in females than in males, irrespective of age and matrix (plasma and serum). Age-associated differences were more prominent in females than in males, and in both matrices, levels of many triacylglycerols were significantly higher in elderly females than in young females. Plasma and serum levels of most lipid metabolites were reduced by freeze-thawing. Our results indicate that plasma is an optimal matrix for exploring lipid biomarkers because it represents the original properties of an individual's blood sample. In addition, the levels of some blood lipid species of healthy adults showed gender- and age-associated differences; thus, this should be considered during biomarker exploration and its application in diagnostics. Our fundamental findings on sample selection and handling procedures for measuring blood lipid metabolites is important for ensuring the quality of biomarkers identified and its qualification process.

Lipidomic analysis of brain tissues and plasma in a mouse model expressing mutated human amyloid precursor protein/tau for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD), the most common cause of dementia among neurodegenerative diseases, afflicts millions of elderly people worldwide. In addition to amyloid-beta (Aß) peptide and phosphorylated tau, lipid dysregulation is suggested to participate in AD pathogenesis. However, alterations in individual lipid species and their role in AD disease progression remain unclear. METHODS: We performed a lipidomic analysis using brain tissues and plasma obtained from mice expressing mutated human amyloid precursor protein (APP) and tau protein (Tg2576×JNPL3) (APP/tau mice) at 4 (pre-symptomatic phase), 10 (early symptomatic) and 15 months (late symptomatic). RESULTS: Levels of docosahexaenoyl (22:6) cholesterol ester (ChE) were markedly increased in APP/tau mice compared to controls at all stages examined. Several species of ethanolamine plasmalogens (pPEs) and sphingomyelins (SMs) showed different levels between brains from APP/tau and control mice at various stages of AD. Increased levels of 12-hydroxyeicosatetraenoic acid (12-HETE) during the early symptomatic phase were consistent with previous reports using human AD brain tissue. In addition, 19,20-dihydroxy-docosapentaenoic acid (19,20-diHDoPE) and 17,18-dihydroxy-eicosatetraenoic acid (17,18-diHETE), which are produced from docosahexaenoic acid and eicosapentaenoic acid via 19,20-epoxy-docosapentaenoic acid (19,20-EpDPE) and 17,18-epoxy-eicosatetraenoic acid (17,18-EpETE), respectively, were significantly increased in APP/tau brains during the pre-symptomatic phase, and concomitant increases occurred in plasma. Several arachidonic acid metabolites such as prostaglandin D2 (PGD2) and 15-hydroxyeicosatetraenoic acid (15-HETE), which have potential deteriorating and protective actions, respectively, were decreased in the early symptomatic phase of APP/tau mice. Significant decreases in phosphatidylcholines and PEs with polyunsaturated fatty acids were also detected in the late symptomatic phase, indicating a perturbation of membrane properties. CONCLUSION: Our results provide fundamental information on lipid dysregulation during various stages of human AD.

Plasma and serum from nonfasting men and women differ in their lipidomic profiles.

Biomarkers will play important roles in disease diagnosis, drug development, and the proper use of drugs. Blood is considered the best biofluid for biomarker research because it is easy to access and a wealth of data are available. However, previous studies revealed that several ionic metabolites showed different levels (including presence or absence) in plasma and serum. Thus, attention should be paid to selecting the best biofluid for biomarker exploration. Many lipid molecules have biological significance and thus would be candidate biomarkers. However, no comprehensive study revealing differences in lipid metabolite levels between plasma and serum has been undertaken. Furthermore, gender differences have not been reported. To clarify the difference in the levels of lipid metabolites between human plasma and serum from both genders, we performed lipid metabolomic analysis using liquid chromatography-mass spectrometry-based systems for phospholipids (PLs), lysoPLs, sphingomyelins, ceramides and oxidative fatty acids. Our results revealed that most of the lipid metabolites were present at similar levels in plasma and serum and in males and females. However, several oxidative fatty acid metabolites showed differences. Of the metabolites related to clotting processes, three showed higher levels in serum than in plasma, and three were detected only in serum. Furthermore, four metabolites were present at different levels between males and females, and two were detected only in males. Thus, attention should be paid to the selection of plasma or serum when utilizing these lipid metabolites as biomarkers.

NMR-based metabolomics of urine in a mouse model of Alzheimer's disease: identification of oxidative stress biomarkers.

Alzheimer's disease (AD) is the most common cause of neurodegenerative dementia among elderly patients. A biomarker for the disease could make diagnosis easier and more accurate, and accelerate drug discovery. In this study, NMR-based metabolomics analysis in conjunction with multivariate statistics was applied to examine changes in urinary metabolites in transgenic AD mice expressing mutant tau and ß-amyloid precursor protein. These mice showed significant changes in urinary metabolites throughout the progress of the disease. Levels of 3-hydroxykynurenine, homogentisate and allantoin were significantly higher compared to control mice in 4 months (prior to onset of AD symptoms) and reverted to control values by 10 months of age (early/middle stage of AD), which highlights the relevance of oxidative stress to this neurodegenerative disorder even prior the onset of dementia. The level of these changed metabolites at very early period may provide an indication of disease risk at asymptomatic stage.