Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2

Bat plasminogen activators alpha 2 (DSPA alpha 2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPA alpha 2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPA alpha 2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to similar to 40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by similar to 10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPA alpha 2. This study confirms that N-glycosylation affects the biochemical function of DSPA alpha 2, which provides a reference for subsequent applications of DSPA alpha 2.

Automated summary

This study investigates the effect of N-glycosylation on the biochemical function of DSPA alpha 2. Mutating the N-glycosylation sites in DSPA alpha 2 resulted in reduced plasminogen activation and fibrin sensitivity. N-glycosylation did not affect the pH resistance or heat resistance of DSPA alpha 2.