Acutobin has been isolated from Deinagkistrodon acutus venom and used to prevent or treat stroke for Chinese patients over the last three decades.This 38 kDa α-fibrinogenase contains disialyl-capped complex type glycans at four Asn sites.The functional roles of these glycans remain to be clarified.Desialylated (DS-) acutobin,prepared by sialidase treatment,cleaved fibrinogen Aα chains as efficiently as native acutobin in vitro.However,the fibrinogen degradation product levels in DS-acutobin treated mice (i.p.) were significantly lower than those in native acutobin treated mice in 4 h,indicating that the disialyl moieties may facilitate vascular homing and prolong the half-life of acutobin in vivo.We prepared recombinant acutobins (designated as HEK-ATB and SW-ATB,respectively) from HEK293T and SW1353 cells and activated and purified them.Mass profiling showed that the glycans of HEK-ATB were smaller and less sialylated,while those of SW-ATB were larger than acutobins glycans.In spite of the fact that both recombinants showed similar reactivities toward chromogenic substrate as the native acutobin,HEK-ATB effectively cleaved the Aα,Bβ and γ chains,while SW-ATB had lower α-fibrinogenolytic and clotting activities.Partial deglycosylation by PNGase F under non-denaturing condition did not affect the activities of native or recombinant acutobins toward chromogenic substrate,but reduced their fibrinogenolytic activity,clotting activity and their thermal stability.Results of site directed mutagenesis of HEK-ATB further showed that only the Asn glycosylation site at the C-terminal region was dispensable,as mutation of the other three Asn-glycosylation sites prevented normal folding and secretion of the recombinants.Taken together,our results revealed that the terminal disialyl-capped glycans of acutobin are important for its stability,fibrinogen specificity and pharmacokinetics,and that the glycosylation pathway of venom gland tissue appears to be important for the evolution of pitviper thrombin-like enzymes.