A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation

Abstract

Ricin is a potent plant cytotoxin composed of an A-chain [RTA (ricin A-chain)] connected by a disulfide bond to a cell binding lectin B-chain [RTB (ricin B-chain)]. After endocytic uptake, the toxin is transported retrogradely to the ER (endoplasmic reticulum) from where enzymatically active RTA is translocated to the cytosol. This transport is promoted by the EDEM1 (ER degradation-enhancing α-mannosidase I-like protein 1), which is also responsible for directing aberrant proteins for ERAD (ER-associated protein degradation). RTA contains a 12-residue hydrophobic C-terminal region that becomes exposed after reduction of ricin in the ER. This region, especially Pro250, plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro250 with alanine)] in the hydrophobic region of RTA to study the intracellular transport of the modified toxin. The introduced mutation alters the secondary structure of RTA into a more helical structure. Mutation P250A increases endosomal-lysosomal degradation of the toxin, as well as reducing its transport from the ER to the cytosol. Transport of modified RTA to the cytosol, in contrast to wild-type RTA, appears to be EDEM1-independent. Importantly, the interaction between EDEM1 and RTA(P250A) is reduced. This is the first reported evidence that EDEM1 protein recognition might be determined by the structure of the ERAD substrate.

Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro

“…The ER is the intracellular site for correct folding and post-translational processing of secretory as well as membrane proteins. It possesses a specialized set of chaperones and enzymes that ensure proper folding proteins as well as provide effective quality control system of this process [16]. The UPR is signalled by three transmembrane proteins with luminal domains that sense the changes in the ER environment and cytosolically disposed effector domains: RNA-dependent protein kinase like ER kinase (PERK); inositol-requiring ER to nucleus signal kinase-1 (IRE1) and activating transcription factor-6 (ATF6).…”

“…This toxin can serve as a membrane marker and as a probe in investigations of endocytosis and various intracellular pathways [12,13]. Among these intracellular pathways is retrotranslocation of proteins from the endoplasmic reticulum (ER) to the cytosol [14][15][16], a process that has been intensively studied in recent years as an important part of the glycoprotein folding quality control system operating in the ER [17]. In medicine ricin can be used to bring into cells different biologically active constituents as well as epitopes for vaccination purposes.…”

Immunity to Ricin: Fundamental Insights into Toxin–Antibody Interactions

“…RTB also mediates ricin endocytosis and delivery of RTA via retrograde transport to the endoplasmic reticulum (ER) (Rapak et al 1997; Spooner and Lord 2011; van Deurs et al 1986). In the ER, RTA and RTB separate via a process involving protein disulfide isomerase (PDI) and ER degradation-enhancing α -mannosidase I-like protein 1 (EDEM1) (Slominska-Wojewodzka et al 2006; Sokolowska et al 2011; Spooner et al 2004). Liberated RTA partially unfolds and is retrotranslocated across the ER membrane into the cytoplasm where refolding is facilitated by cytoplasmic chaperons (e.g., Hsc70), and possibly ribosomes themselves (Argent et al 2000; Spooner et al 2008, 2011).…”

Toxin Instability and Its Role in Toxin Translocation from the Endoplasmic Reticulum to the Cytosol

“…The importance of RtxA/phospholipid interaction for Rtx intoxication is highlighted by studies documenting a loss of in vivo (but not in vitro ) activity for RtxA variants with either point mutations in the C -terminal domain or charged residues extending from the C -terminus [83,84,85,86,87]. In some cases, the loss of in vivo activity was directly linked to an inhibition of toxin translocation to the cytosol [85,87].…”

“…In some cases, the loss of in vivo activity was directly linked to an inhibition of toxin translocation to the cytosol [85,87]. No studies have directly examined the predicted loss of toxin-phospholipid interactions for these RtxA variants, but the available data strongly suggest the unfolding resulting from ER membrane interaction with the C -terminus of RtxA is a pre-requisite for toxin delivery to the cytosol.…”