Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity
Rg. Miele et al., Glycosylation of asparagine-28 of recombinant staphylokinase with high-mannose-type oligosaccharides results in a protein with highly attenuated plasminogen activator activity, J BIOL CHEM, 274(12), 1999, pp. 7769-7776
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.