**3.3 PCNS and FV from** *Pseudonaja textilis* **(PFV)**

266 Gene Duplication

not in the liver, while TrFX is expressed in the liver but not in the venom gland. Further, the expression of trocarin D is ~30 times higher in the venom gland than TrFX in the liver (Reza et al. 2007). Such differential expression patterns of trocarin D and TrFX strongly support

To understand the evolution of group C prothrombin activators, we also determined the cDNA sequence of the *P. textilis* FX (PFX) from the liver. Interestingly, two PFX isoforms (PFX1 and PFX2) were detected in the liver, and their cDNA sequences are ~85% similar (Reza et al. 2006). The domain architecture and cysteine residues of these two isoforms are also conserved compared to group D prothrombin activators. Amino acid sequence comparison shows that PFX1 is more similar to TrFX (~94%), while PFX2 is more similar PCCS and trocarin D (~90%) (Figure 5). Further, PFX1 has a longer activation peptide, similar to plasma FXs, whereas PFX2 has a shorter activation peptide, similar to PCCS and trocarin D. Also, PFX2 has a 9-residue insert, which is not present in PFX1. These structural differences suggest that PFX1, PFX2 and PCCS are encoded by three independent genes and that PFX2 is an evolutionary intermediate between PFX1 and PCCS (Reza et al. 2006) (Figure 5). This similarly confirms the presence of a parallel prothrombin activator system. The expression profiles of PFX1, PFX2 and PCCS were determined in liver and venom gland tissues by RT-PCR (Reza et al. 2006). The results show that PFX1 and PFX2 are expressed only in the liver, while PCCS is expressed only in the venom gland. PFX1 is also found to be expressed ~55,000 times higher than PFX2 in the liver, and PCCS is expressed ~80 times higher in the venom gland than is PFX1 in the liver (Reza et al. 2006). In summary, the sequence comparisons and expression profiles indicate that PCCS has evolved from PFX1 by gene duplication and PFX2 is an intermediary product of this "recruitment" process (Figure 6).

Fig. 6. Schematic diagram showing the probable evolutionary path in the recruitment of FX

protein as toxin in the venom (Reza et al. 2006).

the distinct physiological roles of these two proteins.

**3.2 PCCS and FX from** *Pseudonaja textilis* **(PFX)** 

The cDNA sequence of *P. textilis* FV (PFV) was determined from its liver (Minh et al. 2005). The deduced amino acid sequence of PFV shows similarities to other mammalian and nonmammalian FVs (~50%) and PCNS (~96%) and shares identical domain architecture (Minh et al. 2005). Like the FVs of other species, PFV and PCNS comprise A1, A2, B, A3, C1 and C2 domains (Figure 3A). Functionally important domains A and C are highly conserved in both PFV and PCNS, whereas domain B is the most variable (Minh et al. 2005). The domain B (126 residues) of PFV is one residue shorter than that of PCNS (127 residues), and is much shorter than that of mammalian and non-mammalian FVs. A more detailed comparison shows that all the FXa and thrombin proteolytic cleavage sites (which are important for activation of these nonenzymatic proteins) are conserved in PFV and PCNS (Figure 3A). However, PFV has an additional FXa proteolytic cleavage site at Arg1765 (Minh et al. 2005; Rao et al. 2003b). This cleavage site also exists in mammalian FV but not in FVs of teleosts (Minh et al. 2005). This is evolutionarily interesting as this additional cleavage site may be a characteristic found only in tetrapod FVs. However, the functional implication of this cleavage with regards to procoagulant activity of FV is not yet known.

As mentioned previously, PCNS has evolved to be resistant to inactivation by activated protein C (APC), which is crucial to its function as a toxin. On the other hand, PFV is similar to other FV, as it can still be inactivated by APC. PFV can be inactivated by APC by cleavage at Arg316, a primitive inactive site (van der Neut et al. 2004a), and at Arg506 (Minh et al. 2005) (Figure 3B). The expression profiles of PFV and PCNS in the liver and the venom gland were determined using RT-PCR. As with other venom prothrombin activator genes, PCNS is expressed only in the venom gland, while PFV is expressed only in the liver. It was found that PCNS is expressed ~280 times higher in the venom gland than is PFV in the liver (Minh et al. 2005). Thus, PCNS and PFV are have differential expressions (Minh et al. 2005).

Based on sequence comparisons, we confirmed the presence of parallel prothrombin activator systems in Australian elapid snakes and showed for the first time that groups C and D prothrombin activators in snake venom and their plasma coagulation factor counterparts are closely related. We also proposed that these venom prothrombin activators evolved from their plasma coagulation factor counterparts by gene duplication and were subsequently modified to function efficiently as toxins.
