A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Abstract

Summary. Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as antihaemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis

“…Masci et al (9) identified and characterized serine protease inhibitors from the venom, named them textilinins, and showed that they significantly reduce bleeding in an animal model. More recently, six isoforms of textilinin have been identified in P. textilis venom gland-derived cDNA (10). A prothrombin-activating complex was identified by Masci et al (11) and was found to comprise a large proportion of the venom.…”

“…These include postsynaptic ␣-neurotoxins (2-4), the presynaptic neurotoxin textilotoxin (5-7), phospholipase A 2 s (PLA 2 s) 1 (6,8); serine protease inhibitors (9,10), and a prothrombin activator (11,12). Fry (13) has written a comprehensive review on the properties of venom components from Australian elapids.…”

Purification, characterization and molecular cloning of chymotrypsin inhibitor peptides from the venom of Burmese Daboia russelii siamensis

“…To date a number of BPTI-like superfamily serine protease inhibitors from Viperidae and Elapidae venoms have been purified or characterized [3,4,12,13,15,16,18,19,25,26,[29][30][31][32][33][34]35,36,39]. In this paper, one chymotrypsin inhibitor, BBPTI-1, was purified to homogeneity from the venom of Burmese Daboia russelli siamensis by gel filtration, cation exchange and reversed phase chromatography.…”

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 fromPseudonaja textilis textilis

“…The 24-amino-acid propeptide sequence is hydrophobic, which suggests that it facilitates secretion of the protein prior to cleavage. Of the six textilinins that have been characterized, only Txln-1 and Txln-2 are effective in reducing blood loss in the mouse-tail model (Filippovich et al, 2002). A sequence alignment of the textilinins shows that arginine in position 17 (the residue that binds in the P1 pocket in serine proteases) is common to Txln-1 and Txln-2, while the other four textilinins have an asparagine, lysine, glutamate or aspartate in this position.…”

“…Txln-1 is an acidic molecule with a pI of 4.4 (Filippovich et al, 2002), while aprotinin is basic with a pI of 8.9 (Gebhard et al, 1986). The reason for this difference is that there are 11 negatively charged and seven positively charged amino acids in Txln-1, while in aprotinin there are only four negatively charged and ten positively charged amino acids.…”