title: Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics.
abstract: An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis.
authors.affiliation: Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, CZ-783 71 Olomouc, Czech Republic. marek.sebela@upol.cz
authors.forename: Marek
authors.initials: M
authors.lastname: Sebela
authors.forename: Pavel
authors.initials: P
authors.lastname: Rehulka
authors.forename: Jaromír
authors.initials: J
authors.lastname: Kábrt
authors.forename: Helena
authors.initials: H
authors.lastname: Rehulková
authors.forename: Tomás
authors.initials: T
authors.lastname: Ozdian
authors.forename: Martin
authors.initials: M
authors.lastname: Raus
authors.forename: Vojtech
authors.initials: V
authors.lastname: Franc
authors.forename: Josef
authors.initials: J
authors.lastname: Chmelík
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D001329:Autolysis; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D010446:Peptide Fragments; D011134:Polysaccharides; D011506:Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases