GLYCOSYLATION PROPERTIES OF THE PICHIA-PASTORIS-EXPRESSED RECOMBINANTKRINGLE-2 DOMAIN OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR

The oligosaccharide structures present on Asn(5) of the Pichia pastori s-expressed recombinant kringle 2 domain of tissue-type plasminogen ac tivator {(r)-[K2(tPA)]} have been determined by a combination of techn iques, including HPLC, FPLC, gel filtration, endoglycosidase digestion s and mass spectrometry, The major oligosaccharides identified after t heir liberation by either hydrazinolysis or by the enzyme peptide: N-4 -(N-acetyl-beta-glucosaminyl)asparaginyl amidase, were in the oligomer range of (mannose)(8)(N-acetylglucosamine)(2) (Man(8)GN(2)) to Man(18 )GN(2). The preponderance of these glycans spanned Man(9)GN(2) to Man( 12)GN(2), and the major overall product was Man(10)GN(2). An additiona l (less than 5%) amount of the polypeptide was hyperglycosylated. In c ontrast with glycoproteins produced in Saccharomyces cerevisiae, our r esults with specific mannosidase digestions were consistent with previ ous studies showing that (alpha 1,3)-linked mannose residues were not present in extensions of the core Man(8)GN(2) unit, The results show t hat the N-linked glycosylation pathways in P. pastoris are substantial ly different from those found in S. cerevisiae, with shorter Man(alpha 1,6) extensions to the cove Man(8)GN(2) and the apparent lack of sign ificant Man(alpha 1,3) additions representing the major processing mod ality of N-linked glycans in P. pastoris.