cDNA sequence of FX of P. textilis
Blood coagulation FX in the plasma is primarily synthesized in the liver. To obtain the full-length FX cDNA sequence, RT-PCR followed by RACE was performed using the total RNA extracted from the liver tissues of P. textilis. RT-PCR using the sense and antisense primers (Ps_FXF and Ps_FXR) yielded a product of ∼1.2 kb size. The PCR products were purified, cloned and sequenced. Interestingly, sequencing data indicated the presence of two distinct types of clones. To complete their sequences, both 5′- and 3′ RACE libraries were amplified using gene-specific RACE primers (5′Ps_GSP1 and 3′Ps_GSP1) and UPM (see Materials and methods for details). The resultant PCR products were reamplified using nested gene-specific primers (5′Ps_GSP2 and 3′Ps_GSP2) and the nested UPM to reduce the non-specific background amplification. After reamplification, clear bands of ∼600 bp and ∼800 bp were obtained in 5′- and 3′-RACE, respectively. Resultant PCR products were purified, cloned into pGEMT vector and sequenced. The cDNA and the deduced amino acid sequences, as expected, showed 100% identity in the overlapping regions obtained from the sequence of the RT-PCR product. Assembled sequencing data for RT-PCR and RACE confirmed the existence of two types of FX cDNA in P. textilis liver; we named the isoforms as PFX1 (1452 bp) and PFX2 (1392 bp). Both PFX1 and PFX2 cDNA sequences have been submitted to GenBank (DQ017705 and DQ017706, respectively).
PFX1 and PFX2 share high percentage of identity in cDNA sequence (∼85%) as well as at protein level (∼81%). The deduced amino acid sequence also shows ∼40–50% identity with other known mammalian and piscine FX, as well as with the vPAs with identical domain architecture (Gla – EGF-I – EGF-II – SP). Interestingly, cDNA sequence of PFX1 shows much higher identity (∼94%) to the FX of another Australian elapid, T. carinatus, compared with PFX2 (∼79%). On the contrary, PFX2 cDNA sequence shows comparatively higher identity to its homologs in the venom (∼90% and ∼81% identity with PCCS and trocarin D, respectively) compared with PFX1 (∼76% identity with both PCCS and trocarin D). Thus there are two distinct molecular forms of FX present in the liver of P. textilis, one similar to the blood coagulation FX and the other similar to its vPA. As FX has the same domain architecture and sequence similarity with blood coagulation FIX and VII [8–10], we also compared the level of identity of PFX1 and PFX2 with them. Both P. textilis FX isoforms show only ∼30–35% identity with both FIX and FVII. Therefore, it is quite likely that PFX1 and PFX2 are not snake FIX or FVII.
Mammalian FX has a pre–pro leader sequence [20,21]. The leader sequences in both PFX1 and PFX2 are identical and consist of 40 amino acid residues each. The predicted processing site of the signal peptide occurs between the residues Ala20 and Glu21 (SignalP 3.0: http://www.cbs.dtu.dk/services/SignalP/), which is the same as in T. carinatus FX. The propeptide region and a short internal tripeptide are excised during proteolytic maturation to yield a two-chain mature zymogen. The propeptide region plays a critical role in the γ-carboxylation of Glu residues in the Gla domain . The motifs for enzyme recognition for proteolytic cleavage at both the propeptide and the internal peptide are conserved in PFX1 and PFX2 cDNA sequence and are therefore expected to be similarly processed.
During injury, FX is activated to FXa by TF-VIIa [23,24] or IXa–VIIIa [23,25] complexes through a proteolytic cleavage at Arg194-Ile195 (human FX numbering) and removal of the activation peptide. The activation sites in PFX1 and PFX2 cDNA are conserved. However, activation peptides of PFX1 and PFX2 show interesting differences. PFX1 has a longer activation peptide (57 residues), which is nearly identical (single-residue difference) to that of T. carinatus FX . In contrast, PFX2 has a comparatively shorter activation peptide (27 residues)  which is almost identical (same single-residue difference) to the PCCS.
Like all other mammalian FX, as well as group D prothrombin activators, both PFX1 and PFX2 deduced sequence have 24 conserved cysteine residues. Based on homology with mammalian and T. carinatus FX, all 12 disulphide bonds are expected to be conserved in both the PFX1 and PFX2 of P. textilis.
One crucial post-translational modification for vitamin K-dependent blood clotting factors is γ-carboxylation [26–28]. The recognition site for γ-carboxylase is located in the propeptide region of FX. Two critical residues (Phe16 and Ala10) for the recognition of carboxylase  are conserved in both FX isoforms. There are 12 Glu residues in the Gla domain of both types of FX of P. textilis; all are at identical positions compared with their venom counterpart PCCS [12,18], FX of T. carinatus  and group D prothrombin activators trocarin D and hopsarin D [11,13]. We have shown that all except Glu38 residues are carboxylated in trocarin D  and hopsarin . Based on the homology (Fig. 1), we propose that only the first 11 Glu residues are carboxylated in both isoforms of P. textilis FX. Unlike mammalian FX [30–32], group D prothrombin activators (such as trocarin D) are not hydroxylated at Asp63 . Although this residue is conserved in both PFX1 and PFX2, similar to T. carinatus FX, β-hydroxylation at this position is yet to be determined.
Figure 1. Alignment of amino acid sequence of PFX1 and PFX2 with Tropidechis carinatus factor (F) X and group C and group D venom prothrombin activators (vPA). Identical amino acid residues are shaded in black. Residues showing >70% and >50% identity are shaded in dark gray and light gray, respectively. Regions showing maximum variation are boxed. The mature protein starts with Ala (+1) at the amino terminus of the light chain of the mature zymogen. Boundaries of each domain are marked by arrows. The putative signal peptide and the propeptide are shown by double line (from −40 to −21) and dotted line (from −20 to −1) above the sequence. Putative γ-carboxylated Glu residues, O- and N-glycosylation sites are marked by rectangle, circle and triangle, respectively.
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We have shown that unlike mammalian FX [33,34], the vPAs, such as trocarin D from T. carinatus and hopsarin D from Hoplocephalus stephensi, are glycosylated in their light and heavy chains [11,13]. These prothrombin activators have O-glycosylation at Ser52 position of the light chain and N-glycosylation on the Asn254 residue of the heavy chain. Similarly, N- and O-glycosylation are also found in PCCS from P. textilis [12,18]. These sites are conserved in T. carinatus FX, PFX1 and PFX2 protein sequences. In addition, PFX1 has two potential N-glycosylation sites within the activation peptide, at identical positions compared with T. carinatus FX, whereas PFX2 has only one. However, the existence and the functional role of glycosylation at these positions in snake plasma FX are yet to be determined experimentally.