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Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation.
- Source :
-
FEMS yeast research [FEMS Yeast Res] 2007 Dec; Vol. 7 (8), pp. 1317-27. Date of Electronic Publication: 2007 Sep 04. - Publication Year :
- 2007
-
Abstract
- 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.
- Subjects :
- Amino Acid Sequence
Amino Acid Substitution genetics
Chromatography, Liquid
Electrophoresis, Polyacrylamide Gel
Fungal Proteins analysis
Fungal Proteins chemistry
Glycosylation
Lipase chemistry
Mannose analysis
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation, Missense
Spectrometry, Mass, Electrospray Ionization
Substrate Specificity genetics
Triglycerides metabolism
Triolein metabolism
Yarrowia genetics
Fungal Proteins metabolism
Lipase metabolism
Yarrowia enzymology
Yarrowia metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1567-1356
- Volume :
- 7
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- FEMS yeast research
- Publication Type :
- Academic Journal
- Accession number :
- 17784853
- Full Text :
- https://doi.org/10.1111/j.1567-1364.2007.00293.x