Binding and sequestration of poison frog alkaloids by a plasma globulin

Abstract

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid binding molecule in blood from poison frogs. Proteomic and biochemical studies establish this plasma protein to be liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid binding activity, ABG sequesters and regulates the bioavailability of "free" plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin or albumin, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin binding proteins. Alkaloid-binding globulin (ABG) represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points towards serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

Passive accumulation of alkaloids in inconspicuously colored frogs refines the evolutionary paradigm of acquired chemical defenses

“…Little is known regarding the mechanisms of toxin sequestration in poison frogs or in other toxin-sequestering animals. An alkaloid-binding globulin was recently characterized in the poison frog O. sylvatica [19]. While plasma assays demonstrated that the aposematic species O. sylvatica, E. tricolor , and D. tinctorius can bind and sequester a PTX-like photoprobe, plasma from the undefended Allobates femoralis showed no binding activity.…”

“…Target-site resistance to some alkaloids appears to have evolved in several defended clades and in some undefended species [20,21]. Some defended species also appear to have alternative target mechanisms including binding proteins like alpha-binding globulin [19] and saxiphillin [40] that might prevent alkaloids from accessing their molecular targets. Accumulation of alkaloids in skin glands could help to prevent alkaloids from reaching their targets.…”

“…Here we propose a combination of these hypotheses, i.e., that passive accumulation, alkaloid consumption, and some level of alkaloid resistance was present in an early dendrobatid lineage; enhanced resistance and active sequestration mechanisms then arose later, resulting in the chemical defense phenotype. This hypothesis places less emphasis on dietary changes and more strongly emphasizes novel molecular mechanisms (e.g., binding proteins and target-site insensitivity [19][20][21]). Phylogenies in each subpanel highlight how increasing resolution impacted our understanding of phenotypic diversification in Dendrobatidae.…”

A toxic environment selects for specialist microbiome in poison frogs

“…Skins were processed for alkaloid extractions as described in Alvarez-Buylla et al, 2023 46 . Briefly, the methanol in which the skin was stored was filtered and spiked with (-)-nicotine (Sigma Aldrich, N3876-100ML), then stored at −80 °C for 24 hours to precipitate lipids and proteins.…”

The Diverse Mechanisms that Animals Use to Resist Toxins

“…Documenting toxin diffusion kinetics and tissue-specific concentrations ( Jeckel et al 2020, Melnikova & Magarlamov 2022 can help generate new hypotheses regarding toxin sequestration (Malykin et al 2021, Zhang et al 2020. New tools are also advancing our understanding of sequestration, for example, through the identification of a new binding protein in poison frogs using photo-labeled toxins (Alvarez-Buylla et al 2022) and the generation of a pufferfish model with a knocked-out toxin-binding protein (Kato-Unoki et al 2018). The role of xenosensors, which are toxin-sensing transcription factors that activate the expression of metabolic genes (Nakata et al 2006), is largely unexamined in toxin-sequestering taxa, yet they may play an important role in the origin of sequestration.…”