Correlation between the Glycan Variations and Defibrinogenating Activities of Acutobin and Its Recombinant Glycoforms

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.

Structures of N-Glycans of Bothrops Venoms Revealed as Molecular Signatures that Contribute to Venom Phenotype in Viperid Snakes

“…Despite the fact that glycosylation of proteins is a major PTM in viperid and crotalid snake venoms, we still poorly understand its impact regarding variation and stability of venom proteomes. Nevertheless, N-glycan composition studies of isolated toxins and venoms from Elapidae and Viperidae families indicate significant variability (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). On the other hand, we have shown that the Bothrops jararaca N-glycan compositions from newborn and adult venoms are similar suggesting that differences in utilization might explain the variable glycosylation levels that were indicated by differential electrophoretic profiles previously reported for B. jararaca newborn and adult venoms (47).…”

“…For identification of composition, the Glycomod results were filtered according to the following specifications: Nglycans belonging to the paucimannose class or containing more than three N-glycolylneuraminic acid/deoxyhexoses residues or deoxyhexoses were excluded. The paucimannose class of glycans were excluded considering that this type of N-glycan occurs only in invertebrates; and previous studies showed that snake glycoproteins usually do not contain multiple units of N-glycolylneuraminic acid or deoxyhexoses (33)(34)(35)(36)(37)(38)(39)(40)(42)(43)(44)(45).…”

“…3) showed that the most abundant ion has a molecular mass of 3330.2 Da and is present in two charge states (ϩ2 with m/z 1676. 44 and ϩ3 with m/z 1125.32), which is generated by the neutral loss of 375 Da corresponding to the b-ion of permethylated NeuAc. Moreover, other intense ions in this spectrum are the m/z 1495.88 ϩ2 , corresponding to the loss of a sialic acid dimer (neutral loss of 736 Da), and the m/z 1630.36 ϩ2 , which is the result of the loss of a reduced reducing end with a fucose unit.…”

“…This is the second report of the NeuAc-2,8-NeuAc epitope from snake venom glycoproteins. The first observation was made by Lin et al (43) in the analysis of Deinagkistrodon acutus venom and of the serine proteinase acutobin, isolated from this same venom by Wang et al (44). For the linkage assignment of this sialyl acid dimer, the b-ion m/z 759 ϩ1 (putative NeuAc-2,8-NeuAc-1-ene) was selected for MS 3 fragmentation ( Fig.…”

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation

“…Another extensive study on the glycosylation patterns of the venoms from Deinagkistrodon acutus indicates that acutobin, a snake venom serine protease, carries a terminal disialylated multi-antennary complex type N-glycan [25], which may prolong the longevity of acutobin after i.p. injection [26]. Thus, accumulating evidence indicates that the glycan moieties in snake venom proteins appear to be formed by a common mammalian N-glycan core with biologically important carbohydrate terminals [24].…”