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 oligosac charide linkage site in SakSTAR (at Asn(28) of the mature protein) was occu pied in approximately 50% of the expressed protein with high-mannose-type o ligosaccharides. The majority of these glycans ranged in polymerization sta te from Man(8)GlcNAc(2) to Man(14)GlcNAc(2), with the predominant species b eing Man(10)GlcNAc(2) and Man(11)GlcNAc(2). Glycosylated SakSTAR (SakSTAR(g )) did not differ from its aglycosyl form in its aggregation state in solut ion, its thermal denaturation properties, its ability to form a complex wit h human plasmin (hPm), the amidolytic properties of the respective SakSTAR- hPm complexes, or its ability to liberate the amino-terminal decapeptide re quired for formation of a functional SakSTAR-hPm plasminogen activator comp lex. However, this latter complex with SakSTAR(g) showed a greatly reduced ability to activate human plasminogen (hPg) as compared with the same compl ex with the aglycosyl form of SakSTAR, We conclude that glycosylation at As n28 does not affect the structural properties of SakSTAR or its ability to participate in the formation of an active enzymatic complex with hPm, but i t is detrimental to the ability of the SakSTAR-hPm complex to serve as a hP g activator. This is likely due to restricted access of hPg to the active s ite of the SakSTAR(g)-hPm complex.