Perforin evolved from a gene duplication of MPEG1, followed by a complex pattern of gene gain and loss within Euteleostomi

Abstract

Background: The pore-forming protein perforin is central to the granule-exocytosis pathway used by cytotoxic lymphocytes to kill abnormal cells. 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 we characterize the origins and evolution of perforin.

Results: We identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and present evidence for an earlier ortholog in Gnathostomata 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. Our comparisons of these many-to-one homologs of human perforin show that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. We also present evidence that perforin arose from duplication of the ancient MPEG1 gene, and that it shares a common ancestor with the functionally related complement proteins.

Conclusions: The evolution of perforin in vertebrates involved a complex pattern of gene, as well as intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism.

Figure 1

Human perforin protein domains, transcript structure and genetic locus. The protein domains are shown along with the amino acids numbers they encompass as inferred from the mouse perforin structure [PBD:3NSJ]. Signal, secretion signal peptide; MACPF, membrane attack complex/perforin domain; EGF, epidermal growth factor-like domain; C2, C2 domain; CTE, c-terminal extension. The transcript [Refseq:NM_005041.4] is represented as a line where 1 cm = 500 bp, the black region represents the coding sequence (CDS), the yellow region represents the untranslated regions and the red ‘V’ shapes indicated positions where introns have been spliced out. The phasing of the CDS intron is indicated to left of the marker. The genes on Homo sapiens chromosome (Chr) 10 q22.1 (drawn as a black line, not to scale) are shown as arrowheads, with their gene symbols above. The direction of the arrowhead indicates the relative transcriptional orientation.

Figure 2

Conservation of the human perforin locus extends to platypus but not more divergent vertebrates. Genome scaffolds/contigs are drawn as a black lines (not to scale), and genes are shown as arrowheads, with their gene symbols above. Syntenic genes are color coded. The direction of the arrowhead indicates the relative transcriptional orientation, and the relevant chromosome (Chr) coordinates are indicated on the right.

Figure 3

A roadmap of perforin gene evolution created by tracking the perforin gene locus through extant vertebrate genomes. Scaffolds from the last common ancestors of vertebrate lineages are shown as pink lines (not to scale), with a grey background. Genome scaffolds from representative extant species are drawn as a black lines (not to scale), with a white background. Predicted scaffolds (where direct evidence is not available) are shown as dotted lines. Genes are shown as arrowheads, with their gene symbols above. Syntenic genes are color coded; genes that are not conserved are white with no label. The direction of the arrowhead indicates the relative transcriptional orientation and the relevant genome coordinates indicated on the right.

Figure 4

Perforin gene distribution inChordata. The phylogenetic tree shows the relationship between the species. The numbers of genes per species is shown on the right, in the format: (# full length genes, # partial genes, # pseudogenes) # total genes. Line colour reflects total gene number as per the key.

Figure 5

Multiple perforin genes were present at distinct loci in the LCA of teleost fish. The Bayesian inference phylogenetic tree of fish perforin homologs was made using the alignment in Additional file 4: Figure S3. Posterior probabilities for major clades are shown in italics. Sequences with evidence of expression are denoted by the letter ‘e’ to the right of relevant tip labels. The clades formed reflected the loci to which genes belong, as shown by a representative scaffold to the right of each clade. Related branches and scaffolds are color coded. Proteins are labelled as the three letter genus/species abbreviation followed by the relevant perforin gene number. Genus/species abbreviation are: Homo sapiens, Hsa; Takifugu rubripes, Tru; Tetraodon nigroviridis, Tni; Danio rerio, Dre; Carassius auratus langsdorfii, Cau; Oryzias latipes, Ola; Gasterosteus aculeatus, Gac; Salmo salar, Ssa; Ctenopharyngodon idella, Cid; Oncorhynchus mykiss, Omy; Paralichthys olivaceus, Pol.

Figure 6

Perforin genes have acquired introns in multiple lineages. The coding sequences (CDS) of perforin genes are drawn as black lines to the scale shown. Positions of introns are indicated by red ‘V’ shapes, with the intron phasing numbered at the left of the markers. Conserved introns are linked with dotted vertical lines. Introns in the 5’ untranslated region (UTR) are not shown as they require EST evidence and therefore cannot be traced consistently.

Figure 7

Genomic evidence shows that perforin originated from a duplication of MPEG1, but the MACPF domain of perforin is more similar to C6. A. Genome scaffolds from the five fish species with assembled genomes that contain both MPEG1 and perforin genes are shown, along with the inferred scaffold of the last common ancestor (LCA) of Teleostei. B. Bayesian inference phylogenetic tree generated from an alignment of the MACPF domains of perforin, MPEG1 and C6 from human (Hsa), mouse (Mmu), anole lizard (Aca) and fugu (Tru). The tree is rooted at the midpoint. Node labels are posterior probabilities (italicised). Important branch lengths are labelled to indicate the degree of divergence between the three proteins.

Figure 8

Predicted events in the evolution of perforin: A. Locus, transcript and protein domain architectures of: i. the gene cluster of MPEG1 and a hypothetical perforin/C6 common ancestor (P/C6); ii. the MPEG1, perforin gene cluster and a C6-like gene from early chordates. Domains are abbreviated as follows: secretion signal peptide, SP; membrane attack complex/perforin, MACPF; transmembrane anchor, TM; epidermal growth factor-like, EGF; C2 domain, C2; thrombospondin, TSP; low-density lipoprotein-receptor class A, LR; unknown region, ?. B. Phylogenetic tree showing the relationship of species from major branches of Metazoa. The loci for MPEG1, perforin and C6 are shown and loci not found in some genomes are crossed out. Genus/species abbreviations are: Amphimedon queenslandica, A. queenslandica; Crassostrea gigas, C. gigas; Patiria miniata, P. miniata; Strongylocentrotus purpuratus, S. purpuratus; Branchiostoma floridae, B. floridae; Ciona intestinalis, C. intestinalis.