Glycan structures and intrageneric variations of venom acidic phospholipases A₂ from Tropidolaemus pitvipers

Abstract

Most of the phospholipases A 2 (PLA 2 ; EC 3.1.1.4) variants isolated so far from snake venoms are nonglycosylated enzymes. In the present study, we purified an active glycosylated PLA 2 and an inactive nonglycosylated Lys49-like PLA 2 from two geographical venom samples of Tropidolaemus. The PLA 2 variants from the two samples have rather different N-terminal sequences, implying that the samples were probably derived from two species (Tropidolaemus subannulatus and Tropidolaemus wagleri). The active PLA 2 s from Sulawesi and Sumatra venoms were designated as Tsu-E6 and Twa-E6, respectively, as a result of the presence of their conserved Glu6 residue. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets. Twa-E6 stimulated the aggregation of mouse platelets but inhibited the aggregation of human platelets. Both PLA 2 s were found to be glycosylated at Asn14. Using MALDI-TOF analysis, the released glycans were shown to comprise complex type oligosaccharides without sialylation. This is the first glycan structure of the snake venom PLA 2 to be solved. Furthermore, the enzymatic removal of glycans from both PLA 2 s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of glycosylated Twa-E6 was also found to be as good as that of other homologous PLA 2 s. The presence of these oligosaccharides in PLA 2 s warrants further analyses, which may provide useful insights into the functional regulation of these biomolecules.

Proteomic Characterization and Comparison of Malaysian Tropidolaemus wagleri and Cryptelytrops purpureomaculatus Venom Using Shotgun-Proteomics

“…PLA 2 detected from both species were identified as basic and acidic isoforms of PLA 2 (Table 1 and Table 2). The presence of PLA 2 isoforms in T. wagleri was consistent with the discovery of acidic and basic variants from Sulawesi and Sumatran origin Tropidolaemus genus [42]. …”

Venomics of Tropidolaemus wagleri, the sexually dimorphic temple pit viper: Unveiling a deeply conserved atypical toxin arsenal

“…The current quantitative proteome of T. wagleri venom verified the abundance of waglerins in the venom (close to 40% total venom proteins), which has not been clearly elucidated hitherto in a global profile, although based on toxin-isolation studies the estimated abundance could be, rather inconsistent, ranging from less than 1% 27 to 8–13% 26 of total venom proteins. The inconsistency was presumably due to the different methods adopted for protein purification.…”

“…Unlike most other pit vipers, T. wagleri venom does not possess hemorrhagic or anticoagulant activity 24 25 . The venom consists of unique glycosylated phospholipase A 2 and exhibits a range of common enzymatic activities: hyaluronidase, L-amino acid oxidase and phospholipase A 2 , but these are non-lethal in mice 24 26 . The lethal components were shown to be the low molecular mass proteins of 3–8 kDa in the venom, with a median lethal dose of approximately 0.2 μg/g in mice 24 .…”

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping

“…For most members of the PLA 2 family this is not an issue as they rarely have post-translational modifications [67]. However, PII and PIII SVMPs, and especially SVSPs are commonly N-glycosylated.…”