Evolution of the immune pore-forming protein perforin

The granule-exocytosis pathway is used by cytotoxic lymphocytes to kill virally infected, malignant and foreign cells. The pore-forming protein perforin is central to this pathway and organisms deficient in perforin cannot control viral infection. Although this mechanism of killing is conserved in bony vertebrates, cytotoxic cells are present in other chordates and invertebrates, and their cytotoxic mechanism has not been elucidated. In order to understand the evolution of this pathway, here the origins and evolution of perforin has been investigated. Perforin genes are present in the genomes of all cartilaginous and bony vertebrates, but not in more primitive chordates. In placental mammals perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Comparisons of these paralogues show that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. Perforin is a member of a superfamily of proteins that share the membrane attack complex/perforin (MACPF) domain. A model for the evolution of perforin via the duplication of another MACPF family member, the ancient MPEG1 gene, is presented. This model also includes a relationship to the functionally related complement proteins C6, C7, C8 and C9, and to the more distantly related neural MACPF proteins. Only partially covered (incomplete) perforin genes were found in bird genomes, so the full-length chicken perforin gene was cloned and characterised. This encodes a protein with an extended C-terminus that is 90 amino acids longer than any mammalian perforin. The pore-forming ability of perforins from flounder and chicken was compared to that of the well characterised mouse perforin. These proteins did not display any activity, suggesting a model in which most perforin proteins (other than mouse perforin) are synthesised as inactive precursors.