Cytotoxic lymphocytes (CLs) is a collective term for natural killer (NK) and cytotoxic T lymphocytes (CTL). As the name suggests, these cells are cytotoxic towards virally infected, neoplastic or foreign cells. The two major mechanisms they use to elicit apoptosis in target cells involve (1) cell surface death receptors and their ligands (e.g., Fas/Fas-ligand) and (2) the granule-exocytosis pathway . The latter involves the targeted secretion of specialised secretory lysosomes (granules) from CLs into the immunological synapse, a cleft formed at the site of CL-target cell contact . The granules contain the granzyme family of serine proteases, effectors that cleave cytoplasmic proteins to induce apoptosis, and perforin, a membrane pore forming protein that is required for entry of the granzymes into target cells [3, 4].
CTL form part of the adaptive immune system in all jawed vertebrates but not in earlier chordates [5, 6]. The lamprey, a jawless vertebrate, has an unconventional adaptive immune system which does not use the major histocompatibility complex (MHC) or T cell receptor (TCR) recognition system, but a more primitive leucine rich repeat-containing antigen receptor . This species appears to have CTL-like leukocytes but whether they are armed with granule mediated cytotoxic machinery is unknown. NK cells, by contrast, are more difficult to define than CTL, but appear to have evolved earlier. There is evidence for cells with NK properties in the tunicate Botryllus schlosseri, and the Ciona intestinalis genome includes homologs of some NK cell receptors [5, 8]. More basic cytotoxic NK-like cells have been described in earlier divergent invertebrates such as earthworms . How similar these cells are to conventional mammalian NKs, including their mechanisms of killing, remains to be seen.
Perforin (gene symbol PRF1) is essential and central to the granule-exocytosis pathway in mammals. Effective CL induction of apoptosis requires both granzymes and perforin, although at high concentrations perforin alone can kill cells by causing necrosis, whereas granzymes are ineffective without perforin to translocate them into the target cell cytoplasm. This is highlighted by the human autosomal recessive disease familial hemophagocytic lymphohistiocytosis type 2 (FHL2), caused by mutations in the perforin gene . CTL from these patients cannot kill Fas-deficient target cells and so do not have an active granule-exocytosis pathway .
Perforin forms circular pores in the plasma membrane of target cells by a mechanism involving at least three steps: (1) perforin monomers bind to the membrane via their C2 domains in a calcium dependent manner; (2) monomers polymerise into a ring, mediated in part by salt bridging between residues in adjacent N-terminal membrane attack complex/perforin (MACPF) domains; (3) two clusters of α-helices within each MACPF domain rearrange into anti-parallel β-strands that puncture and span the membrane, creating an aqueous pore [11–16]. The mechanism of (3) and the order of (2) and (3) are inferred from structural similarity to the well-studied cholesterol-dependent cytolysin family of proteins as well as experimental observations of the perforin pore [16, 17].
The MACPF domain has been identified in 12 human proteins and is named after the six best characterised members found in the immune system: five of the terminal complement components (C6, C7, C8α, C8β and C9) that form the membrane attack complex (MAC), and perforin [18, 19]. The MAC is formed when C5b, C6, C7, C8 (a complex of C8α, C8β and C8γ) assemble on foreign cell membranes, which then recruits multiple C9 monomers to polymerise and insert into the membrane [20, 21]. Perforin has long been compared to C9 as they are both able to polymerise and insert into membranes and the pores formed look similar by transmission electron microscopy [22–24].
The only other MACPF domain-containing protein known to be involved in the human immune system is macrophage expressed gene 1 protein (also referred to as mps1 and mpg-1, here-in the gene and protein is abbreviated as MPEG1), produced by macrophages . Besides the MACPF domain MPEG1 contains one or more additional domains with no identified relationship to known protein folds, and a C-terminal transmembrane anchor . MPEG1 is an ancient gene with homologs in species from one of the earliest metazoan lineages, the phylum Porifera (sponges), Amphimedon queenslandica and Suberites domuncula[26, 27]. The homolog from S. domuncula is the best studied MPEG1 gene and is part of an ancient toll-like receptor pathway that is upregulated by lipopolysaccharide . This role in innate immunity, along with its expression in macrophages, has led to the hypothesis that MPEG1 clears phagocytosed Gram-negative bacteria . Indeed, recent evidence shows that the isolated MACPF domain from MPEG1 of the Pacific oyster Crassostrea gigas has anti-microbial activity against both Gram-positive and Gram-negative bacteria .
Here we trace the origins and evolution of the perforin gene to gain insight into the evolution of the granule-exocytosis pathway. Using a variety of approaches including linked gene comparisons, BLAST searches and protein phylogenetic trees we have catalogued all of the available perforin homologs. These data suggest that the perforin-dependent granule-exocytosis pathway originated in jawed vertebrates (Gnathostomata), at around the same time as true CTLs. In addition, we present evidence that MPEG1 is the precursor of perforin.