Generation of Fabry cardiomyopathy model for drug screening using induced pluripotent stem cell-derived cardiomyocytes from a female Fabry patient.

BACKGROUND: Fabry disease is an X-linked disease caused by mutations in α-galactosidase A (GLA); these mutations result in the accumulation of its substrates, mainly globotriaosylceramide (Gb3). The accumulation of glycosphingolipids induces pathogenic changes in various organs, including the heart, and Fabry cardiomyopathy is the most frequent cause of death in patients with Fabry disease. Existing therapies to treat Fabry disease have limited efficacy, and new approaches to improve the prognosis of patients with Fabry cardiomyopathy are required. METHODS AND RESULTS: We generated induced pluripotent stem cell (iPSC) lines from a female patient and her son. Each iPSC clone from the female patient showed either deficient or normal GLA activity, which could be used as a Fabry disease model or its isogenic control, respectively. Erosion of the inactivated X chromosome developed heterogeneously among clones, and mono-allelic expression of the GLA gene was maintained for a substantial period in a subset of iPSC clones. Gb3 accumulation was observed in iPSC-derived cardiomyocytes (iPS-CMs) from GLA activity-deficient iPSCs by mass-spectrometry and immunofluorescent staining. The expression of ANP was increased, but the cell surface area was decreased in iPS-CMs from the Fabry model, suggesting that cardiomyopathic change is ongoing at the molecular level in Fabry iPS-CMs. We also established an algorithm for selecting proper Gb3 staining that could be used for high-content analysis-based drug screening. CONCLUSIONS: We generated a Fabry cardiomyopathy model and a drug screening system by using iPS-CMs from a female Fabry patient. Drug screening using our system may help discover new drugs that would improve the prognosis of patients with Fabry cardiomyopathy.

A selected reaction monitoring mass spectrometric assessment of biomarker candidates diagnosing large-cell neuroendocrine lung carcinoma by the scaling method using endogenous references.

Selected reaction monitoring mass spectrometry (SRM-MS) -based semi-quantitation was performed to assess the validity of 46 selected candidate proteins for specifically diagnosing large-cell neuroendocrine lung carcinoma (LCNEC) and differentiating it from other lung cancer subtypes. The scaling method was applied in this study using specific SRM peak areas (AUCs) derived from the endogenous reference protein that normalizes all SRM AUCs obtained for the candidate proteins. In a screening verification study, we found that seven out of the 46 candidate proteins were statistically significant for the LCNEC phenotype, including 4F2hc cell surface antigen heavy chain (4F2hc/CD98) (p-ANOVA ≤ 0.0012), retinal dehydrogenase 1 (p-ANOVA ≤ 0.0029), apolipoprotein A-I (p-ANOVA ≤ 0.0004), ß-enolase (p-ANOVA ≤ 0.0043), creatine kinase B-type (p-ANOVA ≤ 0.0070), and galectin-3-binding protein (p-ANOVA = 0.0080), and phosphatidylethanolamine-binding protein 1 (p-ANOVA ≤ 0.0012). In addition, we also identified candidate proteins specific to the small-cell lung carcinoma (SCLC) subtype. These candidates include brain acid soluble protein 1 (p-ANOVA < 0.0001) and γ-enolase (p-ANOVA ≤ 0.0013). This new relative quantitation-based approach utilizing the scaling method can be applied to assess hundreds of protein candidates obtained from discovery proteomic studies as a first step of the verification phase in biomarker development processes.

Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae.

Ceramide is one of the most important intercellular components responsible for the barrier and moisture retention functions of the skin. Because of the risks involved with using products of animal origin and the low productivity of plants, the availability of ceramides is currently limited. In this study, we successfully developed a system that produces sphingosine-containing human ceramide-NS in the yeast Saccharomyces cerevisiae by eliminating the genes for yeast sphingolipid hydroxylases (encoded by SUR2 and SCS7) and introducing the gene for a human sphingolipid desaturase (encoded by DES1). The inactivation of the ceramidase gene YDC1, overexpression of the inositol phosphosphingolipid phospholipase C gene ISC1, and endoplasmic reticulum localization of the DES1 gene product resulted in enhanced production of ceramide-NS. The engineered yeast strains can serve as hosts not only for providing a sustainable source of ceramide-NS but also for developing further systems to produce sphingosine-containing sphingolipids.

A proteomic profiling of laser-microdissected lung adenocarcinoma cells of early lepidic-types.

BACKGROUND: In the new pathologic classification of lung adenocarcinoma proposed by IASLC/ATS/ERS in 2011, lepidic type adenocarcinomas are constituted by three subtypes; adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and lepidic predominant invasive adenocarcinoma (LPIA). Although these subtypes are speculated to show sequential progression from preinvasive lesion to invasive lung cancer, changes of protein expressions during these processes have not been fully studied yet. This study aims to glimpse a proteomic view of the early lepidic type lung adenocarcinomas. METHODS: A total of nine formalin-fixed and paraffin-embedded (FFPE) lepidic type lung adenocarcinoma tissues were selected from our archives, three tissues each in AIS, MIA and LPIA. The tumor and peripheral non-tumor cells in these FFPE tissues were collected with laser microdissection (LMD). Using liquid chromatography-tandem mass spectrometry (MS/MS), protein compositions were compared with respect to the peptide separation profiles among tumors collected from three types of tissues, AIS, MIA and LPIA. Proteins identified were semi-quantified by spectral counting-based or identification-based protein-based approach, and statistical evaluation was performed by pairwise G-tests. RESULTS: A total of 840 proteins were identified. Spectral counting-based semi-quantitative comparisons of all identified proteins through AIS to LPIA have revealed that the protein expression profile of LPIA was significantly differentiated from other subtypes. 70 proteins including HPX, CTTN, CDH1, EGFR, MUC1 were found as LPIA-type marker candidates, 15 protein candidates for MIA-type marker included CRABP2, LMO7, and NPEP, and 26 protein candidates for AIS-type marker included LTA4H and SOD2. The STRING gene set enrichment resulted from the protein-protein interaction (PPI) network analysis suggested that AIS was rather associated with pathways of focal adhesion, adherens junction, tight junction, that MIA had a strong association predominantly with pathways of proteoglycans in cancer and with PI3K-Akt. In contrast, LPIA was associated broadly with numerous tumor-progression pathways including ErbB, Ras, Rap1 and HIF-1 signalings. CONCLUSIONS: The proteomic profiles obtained in this study demonstrated the technical feasibility to elucidate protein candidates differentially expressed in FFPE tissues of LPIA. Our results may provide candidates of disease-oriented proteins which may be related to mechanisms of the early-stage progression of lung adenocarcinoma.

Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

Clinical initiatives linking Japanese and Swedish healthcare resources on cancer studies utilizing Biobank Repositories.

The Tokyo Medical University Hospital in Japan and the Lund University hospital in Sweden have recently initiated a research program with the objective to impact on patient treatment by clinical disease stage characterization (phenotyping), utilizing proteomics sequencing platforms. By sharing clinical experiences, patient treatment principles, and biobank strategies, our respective clinical teams in Japan and Sweden will aid in the development of predictive and drug related protein biomarkers. Data from joint lung cancer studies are presented where protein expression from Neuro- Endocrine lung cancer (LCNEC) phenotype patients can be separated from Small cell- (SCLC) and Large Cell lung cancer (LCC) patients by deep sequencing and spectral counting analysis. LCNEC, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. An establishment of protein targets characteristic of LCNEC is quite helpful for decision of optimal therapeutic strategy by diagnosing individual patients. Proteoform annotation and clinical biobanking is part of the HUPO initiative (http://www.hupo.org) within chromosome 10 and chromosome 19 consortia.

Full-length transcriptome-based H-InvDB throws a new light on chromosome-centric proteomics.

H-Invitational Database (H-InvDB; http://hinv.jp/ ) is an integrated database of all human genes and transcripts that started in an international collaborative research project for establishing a functional annotation database of human full-length cDNAs. Because H-InvDB contains an abundance of information for human transcripts, including not only well-characterized protein-coding transcripts but also those without experimental evidence at the protein level, this will be a useful information resource for identifying novel and uncharacterized human proteins (so-called missing proteins). By extending predicted protein data in H-InvDB, we developed the H-Inv Extended Protein Database (H-EPD; http://hinv.jp/hinv/h-epd/ ). From now on, we plan to carry out a database-driven proteome research that makes full use of H-EPD to promote discoveries in the current and future C-HPP. Furthermore, we will push forward with the integration of genome, transcriptome, and proteome databases using a unique tool for connecting distributed databases and would like to develop a knowledge discovery system by incorporating data mining tools.

Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization.

Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.

A glycan of Ψ-factor from Dictyostelium discoideum contains a bisecting-GlcNAc, an intersecting-GlcNAc, and a core α-1,6-fucose.

A secretory glycoprotein named Ψ-factor that we have purified and cloned from Dictyostelium discoideum is prespore cell-inducing factor. To address its functional significance, it is necessary to examine the attached sites and structures of its glycans as well as its protein structure. Here we identified and isolated a tryptic glycosylated peptide with the 71st to 89th amino acids of Ψ-factor that contained the consensus amino acid sequence for an N-linked glycan (N-T-T). MALDI-TOF mass spectrometry indicated that the major protonated molecular ions, [M+H](+), of the glycopeptide were present at m/z 3,806, the minor m/z 3,603 and 3,400 ions corresponding to the loss of one and two N-acetylhexosamines respectively. Digestion of it with N-glycosidase F gave a molecular mass of 1,766.9 for the whole glycan moiety, which accounts for its composition of five hexoses, four N-acetylhexosamines, and a deoxyhexose. Further digestion experiments on the basis of the substrate specificity of α-mannosidase and ß-N-acetylhexosaminidase allowed us to elucidate the unique structure of the glycan, which contains a bisecting and an intersecting GlcNAc and a core α1,6-fucosyl moiety.

Preferential expression of potential markers for cancer stem cells in large cell neuroendocrine carcinoma of the lung. An FFPE proteomic study.

BACKGROUND: Large cell neuroendocrine carcinoma (LCNEC) of the lung, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. We started a project establishing protein targets characteristic of LCNEC with a proteomic method using formalin fixed paraffin-embedded (FFPE) tissues, which will help make diagnosis convincing. METHODS: Cancer cells were collected by laser microdissection from cancer foci in FFPE tissues of LCNEC (n = 4), SCLC (n = 5), and LCC (n = 5) with definite histological diagnosis. Proteins were extracted from the harvested sections, trypsin-digested, and subjected to HPLC/mass spectrometry. Proteins identified by database search were semi-quantified by spectral counting and statistically sorted by pair-wise G-statistics. The results were immunohistochemically verified using a total of 10 cases for each group to confirm proteomic results. RESULTS: A total of 1981 proteins identified from the three cancer groups were subjected to pair-wise G-test under p < 0.05 and specificity of a protein's expression to LCNEC was checked using a 3D plot with the coordinates comprising G-statistic values for every two group comparisons. We identified four protein candidates preferentially expressed in LCNEC compared with SCLC with convincingly low p-values: aldehyde dehydrogenase 1 family member A1 (AL1A1) (p = 6.1 × 10-4), aldo-keto reductase family 1 members C1 (AK1C1) (p = 9.6x10-10) and C3 (AK1C3) (p = 3.9x10-10) and CD44 antigen (p = 0.021). These p-values were confirmed by non-parametric exact inference tests. Interestingly, all these candidates would belong to cancer stem cell markers. Immunohistochmistry supported proteomic results. CONCLUSIONS: These results suggest that candidate biomarkers of LCNEC were related to cancer stem cells and this proteomic approach via FFPE samples was effective to detect them.

Developments for a growing Japanese patient population: facilitating new technologies for future health care.

Lung cancer, COPD and cardiovascular diseases are highlighted as some of the most common disease that cause mortality, and for that reason are the most active areas for drug development. This perspective paper overviews the urgent need to develop a health care system for a rapidly growing patient population in Japan, including forthcoming demands on clinical care, expecting outcomes, and economics. There is an increasing requirement to build on the strengths of the current health care system, thereby delivering urgent solutions for the future. There is also a declaration from the Ministry of Health, Labour and Welfare (MHLW), to develop new biomarker diagnostics, which is intended for patient stratification, aiding in diagnostic phenotype selection for responders to drug treatment of Japanese patients. This perspective was written by the panel in order to introduce novel technologies and diagnostic capabilities with successful implementation. The next generation of personalized drugs for targeted and stratified patient treatment will soon be available in major disease areas such as, lifestyle-related cancers, especially lung cancers with the highest mortality including a predisposing disorder chronic obstructive pulmonary disease, cardiovascular disease, and other diseases. Mass spectrometric technologies can provide the "phenotypic fingerprint" required for the concept of Personalized Medicine. Mass spectrometry-driven target biomarker diagnoses in combination with high resolution computed tomography can provide a critical pathway initiative facilitated by a fully integrated e-Health infrastructure system. We strongly recommend integrating validated biomarkers based on clinical proteomics, medical imaging with clinical care supported by e-Health model to support personalized treatment paradigms to reduce mortality and healthcare costs of chronic and co-morbid diseases in the elderly population of Japan.

A clinician view and experience of proteomic studies in the light of lung cancer in Japanese healthcare.

In Japan, rising costs have impacted the framework of maintaining an efficient and effective healthcare system. Today, urgent implementation of programs to address this need have led to a rebuilding of the entire approach of medical evaluation and clinical care. Recent developments in clinical proteomics based on mass spectrometry (MS) for identifying, sequencing, and quantifying disease-relevant protein biomarkers is a promising means for optimal drug prescription using biomarker diagnosis. We illustrate in this report our experience with lung cancer cases with various drug therapies evaluated with proteomics studies.

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (1) Stage-related protein candidates upon non-metastatic lung adenocarcinoma.

We used formalin-fixed paraffin-embedded (FFPE) materials for biomarker discovery in cases of lung cancer using proteomic analysis. We conducted a retrospective global proteomic study in order to characterize protein expression reflecting clinical stages of individual patients with stage I lung adenocarcinoma without lymph node involvement (n=7). In addition, we studied more advanced stage IIIA with spread to lymph nodes (n=6), because the degree of lymph node involvement is the most important factor for staging. FFPE sections of cancerous lesions resected surgically from patients with well-characterized clinical history were subjected to laser microdissection (LMD) followed by Liquid Tissue solubilization and digestion trypsin. Spectral counting was used to measure the amounts of proteins identified by shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS). More than 500 proteins were identified from IA and IIIA cases, and non-parametric statistics showed that 81 proteins correlated significantly with stage IA or IIIA. A subset of those proteins were verified by multiple-reaction monitoring mass spectrometric quantitation (MRM assay), described in other paper in this issue. These results demonstrated the technical feasibility of a global proteomic study using clinically well documented FFPE sections, and its possible utility for detailed retrospective disease analyses in order to improve therapeutic strategy.

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma.

A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues.

Obesity causes a shift in metabolic flow of gangliosides in adipose tissues.

Obesity is associated with insulin resistance and a mild chronic inflammation in adipose tissues. Recent studies suggested that GM3 ganglioside mediates dysfunction in insulin signaling. However, it has not been determined the ganglioside profiling in adipose tissues of obese animals. Here, we for the first time examined semi-quantitative ganglioside profiles in the adipose tissues of high fat- and high sucrose-induced obese, diabetic C57BL/6J mice by TLC and HPLC/mass spectrometry. In control adipose tissues GM3 dominated with traces of GM1 and GD1a; obesity led to a dramatic increase in GM2, GM1, and GD1a with the GM3 content unchanged. Similar results were obtained in KK and KKAy mice. Adipocytes separated from stromal vascular cells including macrophages contained more of those gangliosides in KKAy mice than in KK mice. These results underscore those gangliosides in the pathophysiology of obesity-related diseases.

Impaired TCR signaling through dysfunction of lipid rafts in sphingomyelin synthase 1 (SMS1)-knockdown T cells.

During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.

2-Hydroxy-ceramide synthesis by ceramide synthase family: enzymatic basis for the preference of FA chain length.

Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function. Ceramides in epidermis also comprise an unusual variety, including 2-hydroxy (alpha-hydroxy)-ceramide. Six mammalian ceramide synthase/longevity assurance homologue (CerS/LASS) family members have been identified as synthases responsible for ceramide (CER) production. We reveal here that of the six, CerS3/LASS3 mRNA is the most predominantly expressed in keratinocytes. Moreover, its expression is increased upon differentiation. CerS family members have known substrate specificities for fatty acyl-CoA chain length and saturation, yet their abilities to produce 2-hydroxy-CER have not been examined. In the present study, we demonstrate that all CerS members can produce 2-hydroxy-CER when overproduced in HEK 293T cells. Each produced a 2-hydroxy-CER with a chain length similar to that of the respective nonhydroxy-CER produced. In HeLa cells overproducing the FA 2-hydroxylase FA2H, knock-down of CerS2 resulted in a reduction in total long-chain 2-hydroxy-CERs, confirming enzyme substrate specificity for chain length. In vitro CerS assays confirmed the ability of CerS1 to utilize 2-hydroxy-stearoyl-CoA as a substrate. These results suggest that all CerS members can synthesize 2-hydroxy-CER with specificity for 2-hydroxy-fatty acyl-CoA chain length and that CerS3 may be important in CER and 2-hydroxy-CER synthesis in epidermis.

Molecular characterization of membrane-associated soluble serine palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii.

Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5'-phosphate (PLP)-dependent alpha-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an approximately 30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 microM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT.

Identification and analysis of novel glycolipids in vertebrate brains by HPLC/mass spectrometry.

Glycosphingolipids are a major component of microdomains or lipid rafts in biological membranes. A new member of raft glycolipids, phosphatidylglucoside (PtdGlc), as well as 6-O-Ac-PtdGlc, a form of PtdGlc O-acetylated at position 6 of its glucopyranose ring, is present in central nervous system tissues. Because the glycolipids represent a minor constituent of lipid rafts and because their mass numbers are the same as that of phosphatidylinositol (PI), the glycolipids are difficult to detect and purify. Here we describe methods to purify and identify glycolipids from rodent brain and methods to discriminate PtdGlc from PI in chick spinal cord using HPLC/electrospray ionization ion-trap mass spectrometry.

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen.

The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained.