Abstract
Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bβ and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.
Highlights
Viperid snake venoms usually contain serine protease isoforms with different specificities toward plasma proteins
The major Snake venom thrombin-like enzymes (SVTLEs) isolated from the venom of Deinagkistrodon acutus [7,11] is a specific a-fibrinogenase
We used two mammalian cell lines (HEK293T and SW1353) to prepare active ATBs which were designated as HKATB and SWATB, respectively, and analyzed their N-glycans by MALDITOF mass spectrometry. Besides comparing their in vitro fibrinogenase activities, we studied the in vivo effects of acutobin and ATBs on the plasma levels of fibrinogen and fibrinogen degradation products (FDP) in mice
Summary
Viperid snake venoms usually contain serine protease isoforms with different specificities toward plasma proteins. Snake venom thrombin-like enzymes (SVTLEs) exhibit specific fibrinogenolytic activities but do not activate factor XIII, plasminogen or platelets. These enzymes generate abnormal fibrin polymers and are useful for lowering fibrinogen concentration and blood viscosity in patients [1,2,3]. The major SVTLE isolated from the venom of Deinagkistrodon acutus (formerly named Agkistrodon acutus) [7,11] is a specific a-fibrinogenase. It has been commercialized under the names of Defibrase or Acutin, and has been applied on many Chinese patients over the past three decades. We cloned the cDNA and solved the acutobin sequence [7], and showed that its complex type N-glycan structures were capped almost exclusively by terminal NeuAca2-
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