Gene regulation of snake venom prothrombin activators

Abstract

Snake venom prothrombin activators are functional and structural homologues of blood coagulation factors. It is hypothesized that the venom prothrombin activators evolved from blood coagulation factors by gene duplication. The copy gene of the blood coagulation factor is genetically modified which causes changes to its expression pattern. The modified copy gene becomes differentially expressed than its ancestral gene to become a toxin gene, i.e. it expression becomes elevated and exclusive in the venom gland. This whole process is broadly termed as ?venom recruitment?.

Trocarin D, a group D venom prothrombin activator, resembles activated mammalian blood coagulation factor X (FXa). Both trocarin D and FXa activate prothrombin to thrombin by targeting the same cleavage sites and have identical domain architectures. Despite such similarities, trocarin D and FXa have different expression patterns which facilitate their respective functions. While FX functions as a haemostatic factor and has low level of liver-specific expression, trocarin D functions as a toxin and has high level of venom gland-specific expression. A snake FX-like (TrFX) cDNA sequence was determined from the liver of Tropidechis carinatus. Comparison of trocarin D and TrFX gene organizations confirmed that trocarin D was ?recruited? from TrFX. Both genes have identical architectures and significant sequence identities, except for insertions/deletions in their promoter and intron 1 regions. Compared to TrFX, the trocarin D promoter has a 264 bp insertion termed as the Venom Recruitment/Switch Element (VERSE). Compared to TrFX, the trocarin D intron 1 has three insertions and two deletions. These insertions/deletions in the promoter and intron 1 regions are hypothesized to facilitate the ?venom recruitment? process of TrFX into the venom gland transcriptome for elevated and specific expression as a toxin. However, more importantly, these insertions/deletions are hypothesized to regulate the expression of trocarin D. Using trocarin D as a representative, this thesis aims to characterize the promoter and intron 1 insertion/deletion segments to investigate the gene regulation of snake venom prothrombin activators.

The trocarin D VERSE promoter segment was characterized using luciferase assays in primary snake venom gland cells and mammalian cell lines. VERSE is capable of driving luciferase expression and its up-regulatory effect is comparable to the full trocarin D promoter. Hence, this indicates that VERSE is the main segment responsible for the trocarin D elevated expression in the venom gland. Subsequent characterization confirms the presence of TATA-, GATA- and Y-box cis-elements. Three other novel cis-elements (Sup, Up1 and Up2) were also identified and characterized. The transcription factor which interacts with the Sup cis-element was purified and identified as a class 6 POU transcription factor. Although expression of trocarin D is venom gland-specific, VERSE is still able to drive luciferase expression in mammalian cell lines. Hence, the trocarin D intron 1 insertion/deletion segments were characterized for their roles in tissue-specific expression.

The insertion/deletion segments within the trocarin D intron 1 region were characterized using luciferase assays in mammalian cell lines. A scaffold matrix attachment region which was previously predicted using bioinformatics tools was found to be nonfunctional. The characterization of the trocarin D intron 1 insertion 2 segment pinpoints a 26 bp segment as a silencer cis-element which probably functions to ?turn off? trocarin D expression in other tissues. The results of this thesis have elucidated how trocarin D is regulated for its elevated and tissue-specific expression. Finally, it has significantly contributed towards our understanding of gene regulation of snake venom prothrombin activators.