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1. Determination of the lipid composition of the GPI anchor

Author

Ana Maria Perez-Linero, Sofia Rodriguez-Gallardo, Susana Sabido-Bozo, Sergio López, Manuel Muñiz, Alejandro Cortes-Gomez, Auxiliadora Aguilera-Romero, Isabelle Riezman, Howard Riezman, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), European Commission, Universidad de Sevilla, Swiss National Science Foundation, Ministerio de Educación, Cultura y Deporte (España), Universidad de Sevilla. Departamento de Biología Celular, Ministerio de Ciencia, Innovación y Universidades (MICINN). España, European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER), Swiss National Science Foundation (SNFS), [Aguilera-Romero,A, Sabido-Bozo,S, Lopez,S, Cortes-Gomez,A, Rodriguez-Gallardo,S, Perez-Linero,AM, Muñiz,M] Departamento de Biología Celular, Facultad de Biología, Universidad de Sevilla e Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain. [Riezman,I, Riezman,H] Department of Biochemistry, NCCR Chemical Biology, University of Geneva, Geneva, Switzerland., and This research was funded by the FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación/BFU2017-89700-P to Manuel Muñiz, 'VI Own Research Plan' of the University of Seville VIPPIT-2020-I.5 to Manuel Muñiz, by the Incentivo al Grupo de Investigación BIO-271 (2019/BIO-271) and the NCCR Chemical Biology and the Swiss National Science Foundation (51NF40-185898 and 310030_184949) to Howard Riezman. ‘V Own Research Plan’ of the University of Seville (VPPIUS) contract (cofounded by the European Social Fund) to Sergio Lopez, University of Seville fellowships to Sofia Rodriguez-Gallardo and Ana Maria Perez-Linero, Ministry of Education, Culture and Sport (MECD) fellowship to Susana Sabido Bozo and contract from the University of Seville by the Youth Employment Initiative to Alejandro Cortes-Gomez.

Subjects
Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins::GPI-Linked Proteins [Medical Subject Headings], Glycosylphosphatidylinositols, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Fungal Proteins::Saccharomyces cerevisiae Proteins [Medical Subject Headings], Cell Membranes, Yeast and Fungal Models, Protein lipidation, Tandem mass spectrometry, Endoplasmic Reticulum, Biochemistry, Mass Spectrometry, Chemicals and Drugs::Organic Chemicals::Amides::Ceramides [Medical Subject Headings], Lab Protocol, Genes, Reporter, Macromolecular Structure Analysis, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Glycoproteins::Membrane Glycoproteins [Medical Subject Headings], Staining, Multidisciplinary, Membrane Glycoproteins, Secretory Pathway, Lipid Analysis, biology, Chemistry, Eukaryota, Genes, reporter, Lipids, Blot, Membrane Staining, Chemicals and Drugs::Carbohydrates::Glycoconjugates::Glycolipids::Glycosylphosphatidylinositols [Medical Subject Headings], Experimental Organism Systems, Cell Processes, Lípidos, Medicine, lipids (amino acids, peptides, and proteins), Cellular Structures and Organelles, Saccharomyces cerevisiae Proteins, Immunoprecipitation, Science, Saccharomyces cerevisiae, Green Fluorescent Proteins, Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, Reporter [Medical Subject Headings], Ceramides, GPI-Linked Proteins, Research and Analysis Methods, Saccharomyces, Glycolipid, Model Organisms, Organisms::Eukaryota::Fungi::Ascomycota::Saccharomycetales::Saccharomyces [Medical Subject Headings], Integral Membrane Proteins, Molecular Biology, Anatomy::Cells::Cellular Structures::Cell Membrane [Medical Subject Headings], Membranes, Glicosilfosfatidilinositoles, Cell Membrane, Genes informadores, Organisms, Fungi, Biology and Life Sciences, Membrane Proteins, Cell Biology, biology.organism_classification, Yeast, Membranas, Ceramidas, carbohydrates (lipids), Membrane protein, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Luminescent Proteins::Green Fluorescent Proteins [Medical Subject Headings], Specimen Preparation and Treatment, Animal Studies, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry [Medical Subject Headings]
Abstract

In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present a protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release and extraction of the GPI-lipid moiety and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation., This research was funded by the FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación/BFU2017-89700-P to Manuel Muñiz, "VI Own Research Plan" of the University of Seville VIPPIT-2020-I.5 to Manuel Muñiz, by the Incentivo al Grupo de Investigación BIO-271 (2019/BIO-271) and the NCCR Chemical Biology and the Swiss National Science Foundation (51NF40-185898 and 310030_184949) to Howard Riezman. ‘V Own Research Plan’ of the University of Seville (VPPI-US) contract (cofounded by the European Social Fund) to Sergio Lopez, University of Seville fellowships to Sofia Rodriguez-Gallardo and Ana Maria Perez-Linero, Ministry of Education, Culture and Sport (MECD) fellowship to Susana Sabido-Bozo and contract from the University of Seville by the Youth Employment Initiative to Alejandro Cortes-Gomez.

Published
2021

2. The nutrigenetic influence of the interaction between dietary vitamin E and TXN and COMT gene polymorphisms on waist circumference: a case control study

Author

Raúl López-Izquierdo, Sonsoles Morcillo, Maria L. Mansego, Carmen Ivorra, Juan Carlos Martín-Escudero, Veronica Gonzalez-Albert, Gemma Rojo-Martínez, Josep Redon, Fernando Martinez, Griselda de Marco, F. Javier Chaves, Federico Soriguer, [Mansego,ML, De Marco,G, Ivorra,C, Martínez,F, Redon,J] CIBER Physiopathology of Obesity and Nutrition CIBERobn, CB06/03, Institute of Health Carlos III , Madrid, Spain. [Mansego,ML, González-Albert,V, Chaves,FJ] Genotyping and Genetic Diagnosis Unit, Fundación de Investigación del Hospital Clínico de Valencia-INCLIVA, Valencia, Spain. [De Marco,G, Morcillo,S, Rojo-Martínez,G, Soriguer,F, Chaves,FJ] CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain. [Iborra,C] Cardiovascular Risk Unit, Consorcio, Hospital General, University of Valencia, Valencia, Spain. [Lopez-Izquierdo,R, Martín-Escudero,JC] Internal Medicine Unit, Rio Hortega Hospital, Valladolid, Spain. [Morcillo,S, Soriguer,F] Endocrinology and Nutrition Department, Carlos Haya University Hospital and Instituto de Investigación Biomédica de Málaga (IBIMA), Málaga, Spain. [Martinez,F, Rendon,J] Hypertension Clinic, Hospital Clínico Universitario de Valencia, Valencia, Spain., This work was co-funded with European Funds for Regional Development (FEDER), the Spanish Science and Technology Ministry [SAF2005-02883], and the health research fund from the Carlos III Health Institute [PI070497], CIBER Fisiopatología Obesidad y Nutrición (CIBERobn) [CB06/03], and CIBER de Diabetes y Enfermedades Metabólicas Relacionadas (CIBERDEM). CIBEROB and CIBERDEM are initiatives by the Carlos III Health Institute in Madrid and the Spanish Health Ministry. Funding also came from GRUPOS 03/101, PROMETEO/2009/029 and 2005/027, AMP07/075, and ACOMP/2009/201 from the Valencian Government and European Network of Excellence InGenious HyperCare (EPSS-037093) from the European Commission.

Subjects
Male, Antioxidant, Polimorphism, medicine.medical_treatment, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Thioredoxins [Medical Subject Headings], Antioxidantes, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Statistics as Topic::Probability::Risk::Risk Factors [Medical Subject Headings], Antioxidants, Vitamin E intake, Obesidad abdominal, chemistry.chemical_compound, Nutrigenomics, Thioredoxins, Polymorphism (computer science), Anatomy::Cells::Blood Cells::Leukocytes [Medical Subject Headings], Risk Factors, Genotype, Vitamin E, Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleosides::Deoxyribonucleosides::Deoxyguanosine [Medical Subject Headings], Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Physiological Effects of Drugs::Growth Substances::Micronutrients::Vitamins [Medical Subject Headings], Phenomena and Processes::Metabolic Phenomena::Metabolism::Oxidative Stress [Medical Subject Headings], Abdominal obesity, Nutrigenómica, Medicine(all), Anthropometry, Age Factors, General Medicine, Phenomena and Processes::Physiological Phenomena::Body Constitution::Body Weights and Measures::Body Size::Body Weight::Overweight::Obesity::Obesity, Abdominal [Medical Subject Headings], Middle Aged, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Statistics as Topic::Models, Statistical::Logistic Models [Medical Subject Headings], Waist circumference, Dieta, Female, Phenomena and Processes::Genetic Phenomena::Genotype [Medical Subject Headings], medicine.symptom, Factores de riesgo, Vitamin, Adult, medicine.medical_specialty, Vitamina E, Catechol-O-methyltransferase, Biology, Phenomena and Processes::Physiological Phenomena::Nutritional Physiological Phenomena::Diet [Medical Subject Headings], Phenomena and Processes::Genetic Phenomena::Genetic Structures::Genome::Genome Components::Genes::Genes, Reporter [Medical Subject Headings], Catechol O-Methyltransferase, Chemicals and Drugs::Biological Factors::Pigments, Biological::Carotenoids::Retinoids::Vitamin A [Medical Subject Headings], Polymorphism, Single Nucleotide, General Biochemistry, Genetics and Molecular Biology, Sex Factors, Internal medicine, Chemicals and Drugs::Inorganic Chemicals::Oxygen Compounds::Reactive Oxygen Species [Medical Subject Headings], Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Physiological Effects of Drugs::Protective Agents::Antioxidants [Medical Subject Headings], medicine, Perímetro abdominal, Phenomena and Processes::Physiological Phenomena::Body Constitution::Body Weights and Measures::Body Size::Waist Circumference [Medical Subject Headings], Chemicals and Drugs::Heterocyclic Compounds::Heterocyclic Compounds, 2-Ring::Benzopyrans::Vitamin E [Medical Subject Headings], Humans, Obesity, Polymorphism, Thioredoxin, Aged, Catechol-O-methyl transferase, Biochemistry, Genetics and Molecular Biology(all), Research, Case-control study, Genes informadores, Disciplines and Occupations::Natural Science Disciplines::Biological Science Disciplines::Biology::Genetics::Genomics::Nutrigenomics [Medical Subject Headings], Diet, Oxidative Stress, Endocrinology, chemistry, Spain, Oxidative stress, Case-Control Studies, Chemicals and Drugs::Enzymes and Coenzymes::Enzymes::Transferases::One-Carbon Group Transferases::Methyltransferases::Catechol O-Methyltransferase [Medical Subject Headings], Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA [Medical Subject Headings], Genotipo
Abstract

Background Abdominal obesity (AO) is a common modifiable risk factor for certain non-communicable diseases associated with enhanced oxidative stress (OS). The objective of this work was to investigate whether the interaction between antioxidant vitamin intake and OS-related polymorphisms modulates gene-associated anthropometry in a Spanish population. Methods A total of 246 subjects with AO, and 492 age and gender matched non-AO subjects were included in the study. Anthropometric, biochemical, and OS parameters, and antioxidant dietary intake data were assessed using validated procedures. DNA from white blood cells was isolated and the genotype of seven polymorphisms from genes involved in OS (pro-oxidant and antioxidant) were analyzed using the SNPlex system. The effects of the c.-793T > C polymorphism on promoter activity and thus thioredoxin (TXN) activity were examined using reporter assays. Results The AO group had higher 8-Oxo-2′-deoxyguanosine levels and took in less vitamin A and vitamin E compared to the non-AO group. Logistic regression analysis revealed that the rs2301241 polymorphism in TXN and rs740603 in catechol-O-methyltransferase (COMT) were associated with waist circumference (WC) and AO. Moreover, these polymorphisms were more strongly associated with variations in WC in subjects with low vitamin E intakes. A promoter assay revealed that the T to C conversion at c.-793 (rs2301241) induced a more than two fold increase in reporter gene expression. Conclusions WC is associated both with dietary vitamin E intake and genetic variants of TXN and COMT suggesting that existence of a complex nutrigenetic pathway that involves regulation of AO. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0652-4) contains supplementary material, which is available to authorized users.

Published
2015

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