Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity.

The properties of recombinant staphylokinase (SakSTAR) expressed in Pichia pastoris cells have been determined. The single consensus N-linked oligosaccharide linkage site in SakSTAR (at Asn28 of the mature protein) was occupied in approximately 50% of the expressed protein with high-mannose-type oligosaccharides. The majority of these glycans ranged in polymerization state from Man8GlcNAc2 to Man14GlcNAc2, with the predominant species being Man10GlcNAc2 and Man11GlcNAc2. Glycosylated SakSTAR (SakSTARg) did not differ from its aglycosyl form in its aggregation state in solution, its thermal denaturation properties, its ability to form a complex with human plasmin (hPm), the amidolytic properties of the respective SakSTAR-hPm complexes, or its ability to liberate the amino-terminal decapeptide required for formation of a functional SakSTAR-hPm plasminogen activator complex. However, this latter complex with SakSTARg showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same complex with the aglycosyl form of SakSTAR. We conclude that glycosylation at Asn28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but it is detrimental to the ability of the SakSTAR-hPm complex to serve as a hPg activator. This is likely due to restricted access of hPg to the active site of the SakSTARg-hPm complex.

O-Mannosylation of Pichia pastoris cellular and recombinant proteins.

O-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan-protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (alpha1-2)-linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1-4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides. Only a very small quantity of N-linked oligosaccharides was present on the Asn289 consensus site. The major products were O-linked (alpha1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O-linked saccharides of mannose containing (alpha1-2) glycosidic linkages are present in P. pastoris cells and expressed proteins.

Characterization of the acidic oligosaccharides assembled on the Pichia pastoris-expressed recombinant kringle 2 domain of human tissue-type plasminogen activator.

The N-linked glycans assembled in Pichia pastoris on the recombinant kringle 2 domain of human tissue-type plasminogen activator (r-[K2tPA]) are composed of approx. 80% neutral and 20% charged species. After peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase-catalysed liberation of the oligosaccharides from the purified glycopeptide, the glycan mixture was resolved by HPLC on amino-silica-based resin. Oligosaccharide mapping of the resulting mixture by HPLC, gel filtration and time-of-flight matrix-assisted laser-desorption-ionization-with-delayed-extraction mass spectrometry (TOF-MALDI DE-MS) revealed that > 90% of the charged species consisted of a series of oligosaccharides possessing molecular masses that were consistent with a range of saccharides comprising phospho-Man10GlcNAc2-phospho-Man14GlcNAc2, with phospho-Man11GlcNAc2 representing the major species. The remaining material in the charged fraction contained identifiable phosphorylated glycans that were one or two mannose units shorter, and one to four mannose units longer, than those present in the above range of oligosaccharides. Treatment of the native charged glycan pool with alkaline phosphatase did not result in molecular-size alterations, showing that phosphomonoesters are not present. Mild acid hydrolysis of the glycans led to a decrease in the size of all charged glycans by one mannose residue, providing phospho-Man9GlcNAc2-phospho-Man13GlcNAc2. Following this procedure, treatment with alkaline phosphatase resulted in size decreases that were equivalent to the loss of one phosphate group from each glycan. This demonstrates that all charged glycans isolated contained phosphate in phosphodiester bonds to two mannose units. The present study shows that P. pastoris cells possess the capability of assembling phosphorylated glycans having the phosphate moiety present in phosphodiester linkages with two mannose units. These saccharides, like the neutral oligosaccharides, contain considerably smaller amounts of mannose than glycans present in other strains of yeast.

Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator.

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.