Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

Abstract

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles but MACPF but pore-forming toxic function are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 15 Å resolution determined by negative stain electron microscopy (NS-EM) and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. Tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin ..B.. delivery subunit would bind to target membranes, and then its MACPF ..A.. toxic subunit disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions give rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg MACPF-Toxin

“…Comparative genomic analysis together with expression patterns and proteomic validation showed that although these lectin and MACPF are present in the genomes of four species of the family, as well as in the genomes of other Mollusks, only in Pomacea these two proteins experienced extensive gene expansion by tandem duplication and neofunctionalization into the PV2 complex, which is expressed as such only in an accessory gland of females and transferred to eggs (30). Although the immune role of these two proteins are largely unexplored in snails, a PV2-67-like protein found in the kidney of the snail Littorina littorea showed overexpression when infected with a trematode parasite (31), indicating a putative immune function in the common ancestor of mollusks MACPF (12). In addition to their immune role, another prominent role of animal MACPFs is in the embryonic development of several organisms, ranging from sea urchins to mammals (32).…”

“…Similar to those proteins PmPV2 is maternally transferred to the eggs, where it is massively accumulated during the early developing stages, before the embryo consumes it (33). However, PV2 structure lacks some key structural features described in developmental MACPFs, such as absence of ancillary domains and shorter TMH1 (12,32). Finally, a less-extended group of animal MACPFs also act as toxins such as those from some cnidarians and the stone fish, where they play a role in prey capture (22,34).…”

“…This binary structure is unique among animals and resembles those of bacterial and plant AB toxins, where a¨B¨-moiety acts as a delivery unit of a toxic¨A¨-moiety (10,11). Unlike AB toxins from bacteria or plants, snail PV2 contains a unique arrangement of two AB toxins in a head-to-tail fashion (12). Interestingly, many of these AB toxins, such as the cholera toxin (CT), heat labile toxin (LT), and shiga toxins (Stxs) from bacteria and the type-2 ribosome inactivating proteins (RIPs) from plants, act as enterotoxins (11), an unexplored function in PV2.…”

Ancient but Not Forgotten: New Insights Into MPEG1, a Macrophage Perforin-Like Immune Effector

“…PRF appears to only form ordered ring-like structures upon transition to the final pore form ( 94 ). Deviations of the common mechanism also are evident in pleurotolysin and perivitellin-2, which are two-component toxins ( 12 , 13 ).…”

“…; SPECT, MAOP) ( 7 , 8 ), as venom components ( e.g. sea anemone toxin PsTX-60B, stonustoxin, perivitellin-2) ( 9 – 12 ), and nutrition ( e.g. fungi; pleurotolysin) ( 13 , 14 ).…”