The Gene Structure and Evolution of ku-wap-fusin (Kunitz Waprin Fusion Protein), a Novel Evolutionary Intermediate of the Kunitz Serine Protease Inhibitors and Waprins from Sistrurus catenatus (Massasauga Rattle- snake) Venom Glands

Snake venom proteins belong to various families which differ in their primary and secondary structures. Recently, we found a novel transcript in the venom gland of Sistrurus catenatus edwardsii which was named “Ku-wapfusin” because it encodes Kunitz-type serine protease inhibitors (Kunitz SPIs) and whey acidic protein (WAP) domains in tandem. We have now determined the gene structure of this unique transcript (from S. c. edwardsii and S. c. tergeminus) and analyzed its evolutionary relationships with genes encoding Kunitz-type serine protease inhibitors and waprins. Structurally, Ku-wap-fusin has four exons and three introns. Comparison of all gene structures (Kunitz SPI, WAP and fusin) shows that exon I in all of these genes is highly conserved and codes for the signal peptide. However, in the Ku-wap-fusin gene, there is an insertion of an exon, which codes for the WAP domain, in the intron II region of the Kunitz SPI gene. In the waprin gene, the exon II, encoding the Kunitz domain, has been lost. Ku-wap-fusin and waprin (whey acidic protein related proteins) genes therefore appear to have evolved via insertion/deletion of an exon. We propose a model for the evolution of these genes (using their gene structures) in which Kunitz, Ku-wap-fusin and waprin genes have evolved from a common ancestor. During the evolution of this protein family complex, the Ku-wap-fusin gene is likely an intermediate between the ancestral and the waprin genes.