New insights into the evolutionary dynamic and lineage divergence of gasdermin E in metazoa

Abstract

Gasdermin (GSDM) is a family of pore-forming proteins that induce pyroptosis. To date, the origin and evolution of GSDM in Metazoa remain elusive. Here, we found that GSDM emerged early in Placozoa but is absent in a large number of invertebrates. In the lower vertebrate, fish, three types of GSDME, i.e., GSDMEa, GSDMEb, and a previously unreported type (designated GSDMEc), were idenitied. Evolutionarily, the three GSDMEs are distinctly separated: GSDMEa is closely related to tetrapod GSDME; GSDMEb exists exclusively in fish; GSDMEc forms the lineage root of tetrapod GSDMA/B/C/D. GSDMEc shares conserved genomic features with and is probably the prototype of GSDMA, which we found existing in all tetrapod classes. GSDMEc displays fast evolutionary dynamics, likely as a result of genomic transposition. A cross-metazoan analysis of GSDME revealed that GSDMEa shares a conserved caspase recognition motif with the GSDME of tetrapods and cnidarians, whereas GSDMEb has a unique caspase recognition motif similar to that of mammalian GSDMD, and GSDMEc exhibits no apparent caspase recognition motif. Through functional test, four highly conserved residues in vertebrate GSDME proved to be essential to auto-inhibition. Together our results provide new insights into the origin, evolution, and function of metazoan GSDMs.

Keywords: auto-inhibition; evolution; fish; gasdermin; metazoan; recognition motif.

FIGURE 1

The identification and phylogenetic analysis of invertebrate GSDM. (A) The phylogenetic tree of the major metazoan phyla with GSDM distribution. Orange and black icons indicate GSDM-positive and negative phyla, respectively. (B) The phylogenetic analysis of GSDM in invertebrate and vertebrate. The different categories of GSDM and metazoan clades are presented in different colors. The vertebrate and invertebrate GSDM are shaded with yellow and blue, respectively. (C) A micrograph of Trichoplax adhaerens (phylum: Placozoa). (D) The violin plot showing the Ka/Ks ratios of GSDM_in in Cnidaria and Mollusca. (E) The genetic distance between invertebrate GSDM_in and vertebrate GSDMA/B/C/D/E/F. The color from blue to red indicates decreasing genetic distance.

FIGURE 2

The phylogeny and evolutionary dynamics of GSDM in Actinopterygii. (A) The phylogenetic relationship between the GSDM of Actinopterygii and other vertebrate were conducted via JTT + F + R7 method with bootstrap of 1,000. Different clades are indicated by different colors. (B) The Ka/Ks ratios of GSDMEa, GSDMEb and GSDMEc in Actinopterygii. The black line represents Ka/Ks = 1. (C) The locations of the two GSDMEc copies and the density of the adjacent transposons in the Poecilia formosa reference genome scaffold 1,076 in a 1,000 bp bin. The Y-axis represents the percentage of repetitive elements.

FIGURE 3

Sequence and synteny analysis of Actinopterygii GSDME in comparison with other vertebrate GSDM. (A–C). Heatmap showing the sequence similarity between Actinopterygii GSDMEa (A), GSDMEb (B), and GSDMEc (C) and the GSDM from other vertebrates. (D). Schematic diagrams showing the conserved neighbor genes of a representative Actinopterygii (Anguilla) GSDMEc in comparison with the conserved neighbor genes of GSDMA in Mammalia (Homo sapiens), Aves (Gallus), Reptilia (Anolis carolinensis), and Amphibia (Geotrypetes seraphini).

FIGURE 4

The identification and analysis of conserved GSDME motifs in vertebrate. (A) The 11 most conserved amino acid residues in Mammalia, Aves, Reptilia, Amphibia and Actinopterygii. The X-axis represents the position of the residue corresponding to that in human GSDME. The Y-axis represents probability. (B) The three dimensional structural model of human GSDME predicted by AlphaFold2. The 11 conserved residues, as well as Phe2, in the N and C terminal domains are labeled. (C) Pyroptosis analysis of full length GSDME (FL) with/without mutation at the 11 conserved residues. Images were captured with a confocal microscope under red (mCherry), green (Sytox Green), and bright field. (D) The analysis of the pyroptosis-inducing ability of GSDME N-terminal (NT) with/without mutation at seven conserved residues. Images were captured as above. (E,F) LDH release from the cells of (C) and (D) was measured and shown in (E) and (F), respectively. Data are the means ± standard deviation of triplicate experiments. ***, p < 0.001. (G) The caspase consensus tetrapeptide motifs in the linker region of vertebrate GSDME. The motifs are represented by the amino acids in the P4–P1 and P1′ positions, with Asp set as P1.