Structures of Azemiops feae venom phospholipases and cys-rich-secretory protein and implications for taxonomy and toxinology.

abstract The Azemiops snakes are pit-less and phylogenetically located at the Crotalinae and Viperinae divergence. cDNAs encoding five Azemiops venom phospholipase (sPLA 2 ) molecules were cloned and sequenced; their signal-peptides were similar to those of crotalid sPLA 2 s. Based on their calculated pI-values and residue-49 substitutions, they were designated as Af-E6, Af-N49a, Af-N49a 1 , Af-N49a 2 , and Af-N49b, respectively. The first three isoforms, comprising 3–4% of the venom proteins, were purified by reversed-phase HPLC. Af-E6 is catalytically active and has >80% sequence-similarity to other Glu 6 -PLA 2 (a pitviper venom-marker). Results of phylogenetic analyses reveal that acidic Af-N49a and Af-N49a 1 are rather unique and loosely linked with crotalid PLA 2 s, while Af-N49b is related to the viperid PLA 2 s with Ser 1 substitution. Notably, the Asn 49 -substitutions in these molecules imply catalytic-independent mechanisms. The 3D-models of Af-E6 and Af-N49a have surface electropotential maps similar to each other and to those of antiplatelet PLA 2 s, while the Af-N49b model is similar to basic and myotoxic sPLA 2 molecules. From Azemiops feae and four other Viperidae, we cloned five novel Cys-rich secretory proteins (CRISPs). Azemiops CRISP and natriuretic-peptide precursors share more sequence similarities with those of crotalid venoms than with viperid venoms, further supporting the theory that Azemiops are sister taxons to pit vipers, especially Tropedolaemus .