title: Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry.
abstract: Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His₆ tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His₆ tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.
authors.affiliation: Faculty of Science and Technology, Dept. of Molecular Biology and Genetics, Aarhus University, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Dionisio
authors.forename: Malene
authors.initials: M
authors.lastname: Jørgensen
authors.forename: Karen Gjesing
authors.initials: KG
authors.lastname: Welinder
authors.forename: Henrik
authors.initials: H
authors.lastname: Brinch-Pedersen
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D002523:Edible Grain / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D001467:Hordeum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014908:Triticum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010832:6-Phytase