title: Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator.
abstract: The oligosaccharide structures present on Asn5 of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator [(r)-[K2tPA]] have been determined by a combination of techniques, including HPLC, FPLC, gel filtration, endoglycosidase digestions and mass spectrometry. The major oligosaccharides identified after their liberation by either hydrazinolysis or by the enzyme peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase, were in the oligomer range of (mannose)8(N-acetylglucosamine)2 (Man8GN2) to Man18GN2. The preponderance of these glycans spanned Man9GN2 to Man12GN2, and the major overall product was Man10GN2. An additional (less than 5%) amount of the polypeptide was hyperglycosylated. In contrast with glycoproteins produced in Saccharomyces cerevisiae, our results with specific mannosidase digestions were consistent with previous studies showing that (alpha 1,3)-linked mannose residues were not present in extensions of the core Man8GN2 unit. The results show that the N-linked glycosylation pathways in P. pastoris are substantially different from those found in S. cerevisiae, with shorter Man(alpha 1,6) extensions to the core Man8GN2 and the apparent lack of significant Man(alpha 1,3) additions representing the major processing modality of N-linked glycans in P. pastoris.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Miele
authors.forename: S L
authors.initials: SL
authors.lastname: Nilsen
authors.forename: T
authors.initials: T
authors.lastname: Brito
authors.forename: R K
authors.initials: RK
authors.lastname: Bretthauer
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D018082:Kringles; D010843:Pichia / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator