Perivitellin-2 (PV2) is a pore-forming toxin present in the egg

perivitelline fluid The perivitelline fluid is an extracellular fluid found in the eggs of most gastropods and constitutes the main source of nutrition and defense for their embryos. It replaces the egg yolk of other animals, which in snail eggs is reduced to non-nutri ...

of the apple snails ''

Pomacea maculata ''Pomacea maculata'' is a species of large freshwater snail with an operculum, an aquatic gastropod mollusk in the family Ampullariidae, the apple snails. Common name of its synonymous name ''Pomacea insularum'' is the island applesnail. Toge ...

'' (PmPV2) and ''

Pomacea canaliculata ''Pomacea canaliculata'', commonly known as the golden apple snail or the channeled apple snail, is a species of large freshwater snail with gills and an operculum, an aquatic gastropod mollusc in the family Ampullariidae, the apple snails. S ...

'' (PcPV2). This

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

, called perivitellin, is massively acumulated in the eggs (~20 % total

. As a toxin PV2 protects eggs from predators, but it also nourishes the developing snail embryos.

Structure and stability

These ~172-kDa proteins are dimers of

AB toxin The AB toxins are two-component protein complexes secreted by a number of pathogenicity, pathogenic bacteria, though there is a pore-forming AB toxin found the eggs of a snail. They can be classified as Exotoxin#Type III: intracellular, Type III t ...

s, each composed of a carbohydrate-binding protein of the tachy

lectin Lectins are carbohydrate-binding proteins that are highly specific for sugar groups that are part of other molecules, so cause agglutination of particular cells or precipitation of glycoconjugates and polysaccharides. Lectins have a role in rec ...

family (targeting module) disulfide-linked to a pore-forming protein of the Membrane Attack Complex and Perforin (

MACPF The Membrane Attack Complex/Perforin (MACPF) superfamily, sometimes referred to as the MACPF/CDC superfamily, is named after a domain that is common to the membrane attack complex (MAC) proteins of the complement system (C6, C7, C8α, C8β and ...

) family (toxic unit). Like most other studied

perivitellins Perivitellins are egg proteins found in the perivitelline fluid of many gastropods. They are multifunctional complexes providing the developing embryo with nutrition, protection from the environment, and defense against predators. Despite the cent ...

from ''

Pomacea ''Pomacea'' is a genus of freshwater snails with gills and an operculum, aquatic gastropod mollusks in the family Ampullariidae, the apple snails. The genus is native to the Americas; most species in this genus are restricted to South America. ...

'' snails, PV2s are highly stable in a wide range of pH values and withstand gastrointestinal digestion, characteristics associated with an antinutritive defense system that deters predation by lowering the nutritional value of the eggs.

Functions

As part of the

, perivitellin-2 constitutes a nutrient source for the developing embryo, notably in the last stages where it is probably used as an endogenous source of energy and structural molecules during the transition to the free life. PV2s also play a role in a complex defense system that protects the embryos against predation. PV2s have both

and perforin activities, associated to the two subunits of their particular structures. As a

, PV2s can agglutinate rabbit

red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

s and bind to the

plasma membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (t ...

of intestinal cells both ''in vitro'' and ''in vivo''. As a perforin, PV2s are able to disrupt intestinal cells altering the

conductance and to form large pores in artificial lipid bilayers. An interesting issue with these

is that the combination of two immune proteins (

and perforin) gave rise to a new toxic entity, an excellent example of protein

exaptation Exaptation and the related term co-option describe a shift in the function of a trait during evolution. For example, a trait can evolve because it served one particular function, but subsequently it may come to serve another. Exaptations are common ...

. This binary structure includes PV2s within “ AB-toxins”, a group of toxins mostly described in

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...

and

plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclud ...

s. In PV2

toxin A toxin is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. Toxins occur especially as a protein or conjugated protein. The term toxin was first used by organic chemist Ludwig Brieger (1849– ...

s, the

would bind to target membranes through the recognition of specific

glycan The terms glycans and polysaccharides are defined by IUPAC as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". However, in practice the term glycan may also be used to refer to the carbohydrate p ...

s, acting as a delivery “B” subunit, and then the pore-forming “A” subunit would disrupt lipid bilayers forming large pores and leading to cell death, therefore constituting a true pore-forming toxin.

Toxicity toward mammals

PV2 toxins proved to be highly toxic to mice when it enters the bloodstream ( LD50, 96 h 0.25 mg/kg, i.p.) and those receiving sublethal doses displayed neurological signs including weakness and lethargy, low head and bent down position (ortopneic), half-closed eyes, taquipnea, hirsute hair, extreme abduction of the rear limbs, paresia and were not able to support their body weight (

tetraplegic Tetraplegia, also known as quadriplegia, is defined as the dysfunction or loss of motor and/or sensory function in the cervical area of the spinal cord. A loss of motor function can present as either weakness or paralysis leading to partial or ...

), among others. Histopathological analyses of affected mice showed that PV2 toxins affect the dorsal horn of the

spinal cord The spinal cord is a long, thin, tubular structure made up of nervous tissue, which extends from the medulla oblongata in the brainstem to the lumbar region of the vertebral column (backbone). The backbone encloses the central canal of the spi ...

, particularly on the 2nd and 3rd gray matter laminas, where alters the calcium metabolism and causes

neuron A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...

apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...

. Apart from its neurotoxicity, it has been recently shown that PV2s are also enterotoxic to mice when ingested, a function that had never been ascribed to animal proteins. At the cellular level, PV2 is cytotoxic to intestinal cells, on which it causes changes in their surface morphology increasing the membrane roughness. At the system level, oral administration of PV2 induces large morphological changes on mice intestine mucosa, reducing its absorptive surface. Additionally, PV2 reaches the Peyer's patches where it activates

lymphoid follicles A lymph node, or lymph gland, is a kidney-shaped organ of the lymphatic system and the adaptive immune system. A large number of lymph nodes are linked throughout the body by the lymphatic vessels. They are major sites of lymphocytes that include ...

and triggers

Evolution of a pore-forming toxin

Evolution of Perivitellin-2 PV2 pore-forming toxin

Proteomic analysis indicates that the

and the Tachy lectins are among the most abundant proteins in ''Pomacea'' eggs but are minor proteins in the genera laying eggs below the water. According to the fossil record, some 3 MYA, when ''Pomacea'' diverged from ''Marisa'' and began laying eggs above the water, these two genes were subjected to extensive duplication and these unrelated proteins were combined by a covalent bond resulting in the dimerization into PV2

that co-opted to new roles. This new structure rendered a novel toxin that is non-digestible, enterotoxic and neurotoxic.

References

{{Reflist Proteins Storage proteins Neurotoxins Toxins