Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation.
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.
Jolivet
Pascale
P
UMR206, Laboratoire de Chimie Biologique, Agro Paris Tech, INRA, Centre de Biotechnologie Agro-Industrielle, Thiverval-Grignon, France.
Bordes
Florence
F
Fudalej
Franck
F
Cancino
Miguel
M
Vignaud
Caroline
C
Dossat
Valérie
V
Burghoffer
Chantal
C
Marty
Alain
A
Chardot
Thierry
T
Nicaud
Jean Marc
JM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity / Q000235:genetics; D014280:Triglycerides / Q000378:metabolism; D014304:Triolein / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*