**Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators**

Shiyang Kwong1 and R. Manjunatha Kini1,2

*1Department of Biological Sciences, Faculty of Science National University of Singapore, Singapore 2Department of Biochemistry, Medical College of Virginia Virginia Commonwealth University, Richmond,Virginia 1Singapore 2USA* 

## **1. Introduction**

20 Will-be-set-by-IN-TECH

256 Gene Duplication

Sigrell, J. A., Cameron, A. D., Jones, T. A. & Mowbray, S. L. (1998). Structure of *Escherichia coli*

Torres, J. C., Guixé, V. & Babul, J. (1997). A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in *Escherichia coli*., *Biochem. J.* 327: 675–684. Torres, N. V., Mateo, F. & Meléndez-Hevia, E. (1988). Shift in rat liver glycolysis

Tsuge, H., Sakuraba, H., Kobe, T., Kujime, A., Katunuma, N. & Ohshima, T. (2002). Crystal

Tuininga, J. E., Verhees, C. H., van der Oost, J., Kengen, S. W., Stams, A. J. & de Vos,

van Rooijen, R. J., van Schalkwijk, S. & de Vos, W. M. (1991). Molecular cloning,

Verhees, C. H., Tuininga, J. E., Kengen, S. W., Stams, A. J., van der Oost, J. & de Vos,

Woese, C. R. & Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: the

Woese, C. R., Kandler, O. & Wheelis, M. L. (1990). Towards a natural system of organisms:

Wu, L. F., Reizer, A., Reizer, J., Cai, B., Tomich, J. M. & Saier, M. H. (1991). Nucleotide sequence

Zhang, Y., Dougherty, M., Downs, D. M. & Ealick, S. E. (2004). Crystal structure of

evolution of the ribokinase superfamily., *Structure* 12(10): 1809–1821.

methanogenic archaea., *J. Bacteriol.* 183(24): 7145–7153.

primary kingdoms., *Proc. Natl. Acad. Sci. USA* 74(11): 5088–5090.

insights into a new family of kinase structures., *Structure* 6(2): 183–193. Sigrell, J. A., Cameron, A. D. & Mowbray, S. L. (1999). Induced fit on sugar binding activates

ribokinase., *J. Mol. Biol.* 290(5): 1009–1018.

phosphofructokinase., *FEBS Lett.* 233(1): 83–86.

*Sci.* 11(10): 2456–2463.

*Biol. Chem.* 274(30): 21023–21028.

*Biochem. J.* 375(2): 231–246.

87(12): 4576–4579.

*coli*., *J. Bacteriol.* 173(10): 3117–3127.

ribokinase in complex with ribose and dinucleotide determined to 1.8 Å resolution:

control from fed to starved conditions. flux control coefficients of glucokinase and

structure of the ADP-dependent glucokinase from *Pyrococcus horikoshii* at 2.0-Å resolution: a large conformational change in ADP-dependent glucokinase., *Protein*

W. M. (1999). Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon *Pyrococcus furiosus*., *J.*

characterization, and nucleotide sequence of the tagatose 6-phosphate pathway gene cluster of the lactose operon of *Lactococcus lactis*., *J. Biol. Chem.* 266(11): 7176–7181. Verhees, C. H., Kengen, S. W. M., Tuininga, J. E., Schut, G. J., Adams, M. W. W., De Vos, W. M.

& Van Der Oost, J. (2003). The unique features of glycolytic pathways in Archaea.,

W. M. (2001). ADP-dependent phosphofructokinases in mesophilic and thermophilic

proposal for the domains Archaea, Bacteria, and Eucarya., *Proc. Natl. Acad. Sci. USA*

of the *Rhodobacter capsulatus* fruK gene, which encodes fructose-1-phosphate kinase: evidence for a kinase superfamily including both phosphofructokinases of *Escherichia*

an aminoimidazole riboside kinase from *Salmonella enterica*: implications for the

Snake venom is a complex mixture of pharmacologically active molecules which are responsible for immobilization, paralysis, death and digestion of prey organisms. This armory of toxins has evolved to target two key systems, namely the neuromuscular and circulatory systems, in order to induce rapid immobilization and death. So far, several hundreds of protein toxins from snake venoms have been purified and characterized. Most of these toxins have been documented to be structurally, and at times functionally, similar to proteins expressed in different tissues of the body. For example, elapid phospholipase A2 toxins are structurally and catalytically similar to mammalian pancreatic phospholipase A2 enzymes (Robin Doley et al. 2009). Similarly, sarafotoxins are structurally and functionally similar to endothelins produced primarily in endothelium (Landan et al. 1991a). Based on such structural and functional similarities, it is hypothesized that toxin proteins are "recruited" from body proteins by gene duplication (Fry 2005). Accordingly, the genes of body proteins are duplicated and modified to have differential and specific expression in venom glands. This phenomenon is broadly termed as "recruitment". This "recruitment" process of body proteins has not only been observed in snakes but also in various other venomous animals, such as cone snails, spiders, scorpions and sea anemones as well as hematophagous animals (Fry et al. 2009). Although this overarching concept existed in the field of snake venom toxins for decades, there is not much direct molecular evidence for this process of "recruitment".

Our laboratory has extensively characterized prothrombin activators from Australian elapid snake venoms and documented their structural and functional similarity with mammalian plasma coagulation factors. Through systematic, detailed studies, we provided the molecular details of the "recruitment" of venom prothrombin activators from plasma coagulation factors after gene duplication. We also identified several key structural changes that make these prothrombin activators better toxins. In this chapter, we will describe the first molecular evidence for the "recruitment" process and the evolution of prothrombin activators in venoms of Australian elapid snakes.

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 259

Group D prothrombin activators are found exclusively in the venom of Australian elapid snakes (Rosing and Tans 1991). Notecarin from *Notechis scutatus scutatus* venom was the first member of this group to be isolated and characterized (Tans et al. 1985). Since then, similar prothrombin activators have been characterized from several other snake venoms. They are glycoproteins with a molecular weight of ~50 kDa (Table 1). As a group of proteins, they share striking resemblances and requirements for optimal activity with activated mammalian plasma coagulation factor X (FXa) (Table 1) (Joseph et al. 1999; Marsh et al.

Venom of the Australian elapid *Tropidechis carinatus* (rough-scaled snake) was documented to have procoagulant properties 29 years ago (Chester and Crawford 1982). A prothrombin activator was isolated using gel filtration and benzamidine-based affinity chromatography and was partially characterized (Marsh et al. 1997). Our laboratory purified a prothrombin activator, trocarin D, from *T. carinatus* venom to homogeneity using a series of high performance liquid chromatography techniques including gel filtration, ion-exchange and reverse-phase chromatographies (Joseph et al. 1999). This purification procedure was refined to a single-step reverse-phase chromatographic method and was subsequently used for the purification of several other group D prothrombin activators such as notanarin D from *N. ater niger* venom, notecarin D from *N. scutatus* venom and hopsarin D from *Hoplocephalus stephensi* venom (Rao et al. 2003a). Our laboratory characterized trocarin D for its functional and structural properties in detail as a representative of group D prothrombin

Functionally, trocarin D has properties which are similar to mammalian plasma coagulation FXa. They both promote blood coagulation by activating prothrombin to thrombin (Joseph et al. 1999). Trocarin D and FXa achieve activation of prothrombin by cleaving the same peptide bonds (Arg274-Thr275 and Arg323-Ile324). Both proteins have identical co-factor requirements of Ca2+ ions, phospholipids and activated factor V (FVa) for their optimal activities (Joseph et al. 1999). We determined the amino acid sequence of trocarin D and its precursor using Edman degradation (Joseph et al. 1999) and cDNA sequencing (Reza et al. 2005a), respectively. Trocarin D and mammalian FXa share significant sequence identity (~53-60%) and exhibit identical domain architecture (Joseph et al. 1999; Rao et al. 2003a) (Figure 1). Both proteins comprise two chains: a heavy chain, which has a serine proteinase with the characteristic catalytic triad (His42, Asp88 and Ser185), and a light chain, which has a Gla domain followed by two epidermal-growth factor-like domains (EGF-I and EGF-II). These two chains are held together by a single inter-chain disulfide bond (Joseph et al. 1999) (Figure 1). The differences between trocarin D and mammalian FXa reside in an insertion in the heavy chain, the size of the activation peptide and post-translational modifications. Firstly, there is a 12-residue insert in the heavy chain of trocarin D (Reza et al. 2005a). However, the functional importance of this insertion is not clear. Secondly, the activation peptide of trocarin D precursor is only 27 residues long (Reza et al. 2005a) compared to the activation peptides of mammalian FXs which ranges from 48 to 52 residues (Figure 1). Lastly, post-translational modifications show that trocarin D is glycosylated, but mammalian FXa is not. In addition, trocarin D also contains a *O*-linked carbohydrate at Ser52 of the light chain and a *N*-linked carbohydrate at Asn45 of the heavy chain (Joseph et al. 1999) (Figure 1). Interestingly, the *O*-linked carbohydrate moiety has a *N*-acetylglucosamine moiety, which is found commonly in nuclear and cytoplasmic proteins but rarely in secreted proteins (Hanover et al. 1987; Holt et al. 1987; Holt and Hart 1986; Snow et al. 1987). The

**2.1 Group D prothrombin activators** 

activators.

1997; Rao and Kini 2002; Stocker et al. 1994; Tans et al. 1985).
