cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from Kenyan Echis carinatus venom.

The complete amino acid sequence of ecarin is deduced from the nucleotide sequence of a cDNA clone isolated by screening a venomous gland cDNA library of Kenyan Echis carinatus. The cDNA sequence with 2379 base pairs encodes an open reading frame of 616 amino acids with a remarkable sequence homology to the putative precursor protein of trigramin from Trimeresurus gramineus venom (61% identity) and a large hemorrhagin, jararhagin, from the pit viper Bothrops jararaca venom (62% identity). Thus, ecarin, as well as jararhagin and trigramin, is translated as a precursor protein, which may be processed posttranslationally. The ecarin proprotein has a "cysteine switch" motif (-Pro-Lys-Met-Cys-Gly-Val-) similar to that involved in the activation of matrix metalloproteinase zymogens. The processed mature protein consists of 426 amino acid residues (residues 191-616), showing the strongest sequence similarity with that of Russell's viper venom factor X activator (RVV-X) heavy chain (64% identity). Like RVV-X heavy chain, ecarin contains metalloproteinase, disintegrin, and cysteine-rich domains. The metalloproteinase domain has a typical zinc-chelating sequence (-His-Glu-Xaa-Xaa-His-Xaa-Xaa-Gly-Xaa-Xaa-His-), as found in crayfish astacin. In the disintegrin domain of ecarin, the Arg-Gly-Asp sequence is replaced by Arg-Asp-Asp, as found in the disintegrin domains of RVV-X heavy chain (Arg-Asp-Glu) and a guinea pig sperm fusion protein, PH-30 beta (Thr-Asp-Glu). These findings show that while there are structural and evolutionary relationships among these proteins, each has a unique functional activity.