A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions

Abstract

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular target of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example for a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore- forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and elimination of its biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity on an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

Animal secretory endolysosome channel discovery

“…Regulated assembly of βγ-CAT complex relies on environmental cues: BmALP1 and BmTFF3 are products of two genes with distinct secretory pathways ( Liu et al, 2008 ; Wang et al, 2020 ). For the assembly and dissociation of complexes in toads, positive and negative control methods are required.…”

“…For the assembly and dissociation of complexes in toads, positive and negative control methods are required. Indeed, as a novel af-PFP, BmALP3 is characterized by a lack of membrane pore formation capacity ( Wang et al, 2020 ). BmALP3 senses environmental oxygen tension and acts as a negative regulator of the βγ-CAT complex.…”

“…BmALP3 binds to a homodimer to oxidize BmALP1 through disulfide bond exchange, thereby forming a BmALP1 homodimer and polymer. This effect causes the βγ-CAT complex to dissociate and lose its biological activity ( Wang et al, 2020 ).…”

Immunoreactivity and neutralization study of Chinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxin antisera towards purified bungarotoxins and snake venoms

“…The mass spectrometry analysis was performed according to the reported methods [ 18 – 19 ]. Briefly, the molecular weights of purified bungarotoxins were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (AUTOFLEX III MALDI-TOF, Bruker Corporation, Germany).…”

“…Then, the target bands were cut and digested by trypsin at 37˚C. The detailed sample handling was the same as our recently reported methods [18]. Operation was carried out on positive ion mode PLOS NEGLECTED TROPICAL DISEASES using α-Cyano-4-hydroxycinnamic acid as matrix.…”