Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation

Autor: Jean-Marc Nicaud, Pascale Jolivet, Alain Marty, Chantal Burghoffer, Franck Fudalej, Miguel Cancino, Caroline Vignaud, Thierry Chardot, Florence Bordes, Valérie Dossat
Přispěvatelé: Chimie Biologique (UCB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Laboratoire de microbiologie et génétique moléculaires (LMGM), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI)
Jazyk: angličtina
Rok vydání: 2007
Předmět:
0106 biological sciences
Yarrowia lipolytica
Spectrometry
Mass
Electrospray Ionization
Glycosylation
mutant forms
Tributyrin
extracellular lipase Lip2p
Molecular Sequence Data
Mutation
Missense
Yarrowia
Mannose
Biology
01 natural sciences
Applied Microbiology and Biotechnology
Microbiology
Substrate Specificity
Fungal Proteins
03 medical and health sciences
chemistry.chemical_compound
N-linked glycosylation
010608 biotechnology
Amino Acid Sequence
Lipase
Peptide sequence
Triglycerides
030304 developmental biology
Gel electrophoresis
0303 health sciences
[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry
Molecular Biology/Molecular biology
General Medicine
MS
biology.organism_classification
Molecular biology
enzymatic deglycosylation
N -linked glycosylation
Amino Acid Substitution
chemistry
Biochemistry
Mutagenesis
Site-Directed
biology.protein
Electrophoresis
Polyacrylamide Gel
Triolein
Chromatography
Liquid
Zdroj: FEMS Yeast Research
FEMS Yeast Research, 2007, 7 (8), epub ahead of print. ⟨10.1111/j.1567-1364.2007.00293.x⟩
FEMS Yeast Research, Oxford University Press (OUP), 2007, 7 (8), epub ahead of print. ⟨10.1111/j.1567-1364.2007.00293.x⟩
ISSN: 1567-1356
1567-1364
Popis: International audience; Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.
Databáze: OpenAIRE
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