Binding and intracellular routing of the plant-toxic lectins, lanceolin and stenodactylin

doi:10.1016/j.bbagen.2010.09.006

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 1800, Issue 12, December 2010, Pages 1276-1282

https://doi.org/10.1016/j.bbagen.2010.09.006 Get rights and content

Abstract

Background

The present research studied the interaction of two ribosome-inactivating proteins (RIPs) from Adenia genus with HeLa cells. Namely, lanceolin and stenodactylin were examined in comparison to volkensin, another toxic two-chain RIP from Adenia genus.

Methods

The binding, endocytosis, intracellular routing, degradation and exocytosis were investigated by measuring the distribution of radiolabelled RIP and by determining its cytotoxicity.

Results

Stenodactylin was the most toxic, resulting in the greater inhibition of protein synthesis and cell death. Lanceolin and stenodactylin bound to cells with comparable affinity and have a similar number of binding sites (10⁵/cell). The uptake of lanceolin and stenodactylin was 13 and 36 times greater, respectively, than that reported for volkensin. The two toxins bound to cell membrane receptors via their lectin B chain, were endocytosed through a clathrin-independent pathway, were internalised in a manner independent from endosomal acidification, and required routing through the Golgi apparatus, as reported for modeccin and volkensin. Stenodactylin showed greater uptake, exocytosis and re-uptake of non-degraded RIP than lanceolin and volkensin, whereas volkensin had the highest residual activity after being released from the cell.

Conclusions

The high cytotoxicity of RIPs from the Adenia genus may depend on the following: high affinity binding to the cell and efficient endocytosis, intracellular routing that appears similar to that of other ricin-like toxic RIPs, partial resistance to proteolysis, and, regarding stenodactylin, high accumulation in cell.

General significance

The data provide a model that could lead to new strategies for anti-cancer therapy and neuroscience studies.

Introduction

Ribosome-inactivating proteins (RIPs) (reviewed in [1], [2], [3]) are expressed by a large number of plants. They are characterised by an N-glycosidase activity, which removes a specific adenine residue (A₄₃₂₄ in rat liver rRNA) from eukaryotic rRNA. This depurination results in an irreversible alteration of the ribosome and consequently causes inhibition of protein synthesis [4]. RIPs also depurinate DNA and other polynucleotides. Thus, it has been proposed that RIP also be referred to as an adenine polynucleotide glycosylase [5].

RIPs are classified as type 1, single-chain proteins with enzymatic activity, and type 2, two-chain proteins consisting of an A chain similar to type 1 RIP and a B chain with galactoside-specific lectin properties. The presence of B chain allows for the binding of type 2 RIPs to glycosylated receptors present on cell membrane [6]. The toxicity of type 2 RIP for the mouse is highly variable, with LD₅₀ ranging from 1 μg/kg to 40 mg/kg body weight. It has been reported that this difference in toxicity could be attributed to a different intracellular routing (reviewed in [7]). In contrast, the LD₅₀ of type 1 RIP ranges between 1 and 44 mg/kg (reviewed in [8]). Toxic type 2 RIPs, like other lectins, are transported in a retrograde manner along axons and have been used to produce selective lesions in the nervous system by a molecular-neurosurgery technique. RIPs from the Adenia genus, namely modeccin, volkensin (reviewed in [8]), lanceolin and stenodactylin [9], [10], are amongst the more toxic lectins and are the only type 2 RIPs transported in a retrograde manner along peripheral nerves and also in the central nervous system [11], [12]. This property could be exploited to selectively lesion specific neurons in neuroscience research as one of the possible biomedical applications of RIP (reviewed in [13]).

The mechanism of entry and the intracellular routing of type 2 RIPs has been extensively studied, particularly those of ricin (reviewed in [14], [15]). After binding the glycoproteins and glycolipids of the cell surface through the lectin chain, ricin is taken up by cells either via a clathrin-dependent or clathrin-independent pathway (reviewed in [16]). The internalised ricin is routed through the endosomal compartment to the multi-vesicular body and from there, is sorted to the trans-Golgi network. The involvement of the Golgi apparatus has also been suggested during cell toxicity by modeccin [17] and volkensin [18]. Ricin reaches the endoplasmic reticulum (ER) by using the retrograde transport system that mediates the routing of misfolded proteins from the trans-Golgi network to the ER compartment (reviewed in [19]). The translocation of type 2 RIPs from the ER to the cytosol requires a reduction of the disulphide bridge between the A and B chains, followed by the entry of the A chain into the cytosol using the quality control pathway that leads to ER-associated protein degradation (reviewed in [20]).

Present research was undertaken to investigate the binding, endocytosis, intracellular routing, degradation and exocytosis of lanceolin and stenodactylin in relation to their cytotoxicity. HeLa cells have been used to compare the results to those previously obtained with the same cell line about the intracellular routing and cytotoxicity of volkensin, the best-known type 2 RIP from the Adenia genus.

Section snippets

Materials and methods

Lanceolin, stenodactylin and volkensin were prepared as described [9], [10], [21]. The RIPs were labelled with ¹²⁵I using the Iodogen reagent, as described in [22].

¹²⁵Iodine, sodium salt and L-[4,5-³H]leucine (2,33 TBq/mmol) were supplied by GE Healthcare (Buckinghamshire, UK). Culture media and supplements, as well as dynasore and brefeldin A, were supplied by Sigma-Aldrich (St. Louis, MO, USA). Proteasome inhibitor II was provided by Calbiochem (San Diego, CA, USA). All other reagents were

Inhibition induced by RIP to HeLa cell protein synthesis and viability

The concentration-response curves of protein synthesis and cell viability after 24 h exposure to RIPs indicated similar cytotoxicities for lanceolin and volkensin, whereas stenodactylin was two orders of magnitude more cytotoxic (Fig. 1A). Although for each RIP, the IC₅₀ was almost coincident with the LC₅₀, a low percentage of viability remained after the complete inhibition of protein synthesis, which occurred at 10^− 12 M stenodactylin and 10^− 10 M lanceolin or volkensin. According to the

Discussion

The present work assesses the binding of lanceolin and stenodactylin to HeLa cells, including their uptake, intracellular routing, degradation and exocytosis, and compares these parameters with those reported for volkensin. HeLa cells were chosen because this cell line has been the most utilized to assess RIP cytotoxicity. Moreover, HeLa cells have been used in many investigations on RIP/cell interaction since 1974, when it was reported that they express glycosylated receptors recognized by

Acknowledgments

This study was supported by grants from the University of Bologna, Funds for Selected Research Topics, by the Cornelia and Roberto Pallotti Legacies for Cancer Research and by the Carisbo Foundation, Bologna.

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Binding and intracellular routing of the plant-toxic lectins, lanceolin and stenodactylin

Abstract

Background

Methods

Results

Conclusions

General significance

Introduction

Section snippets

Materials and methods

Inhibition induced by RIP to HeLa cell protein synthesis and viability

Discussion

Acknowledgments

Biochim. Biophys. Acta

J. Biol. Chem.

Toxicon

Toxicon

J. Neurosci. Methods

Neurotoxicology

Biochim. Biophys. Acta

Exp. Cell Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochimie

Phytochemistry

FEBS Lett.

Cell