Tài liệu Báo cáo khoa học: Isolation and characterization of four type 2 ribosome inactivating

Isolation and characterization of four type 2 ribosome

inactivating pulchellin isoforms from Abrus pulchellus

seeds

Priscila V. Castilho1,2, Leandro S. Goto2

, Lynne M. Roberts3 and Ana Paula U. Arau´ jo1,2

1 Programa de Po´ s-graduac¸a˜o em Gene´tica e Evoluc¸a˜o, Universidade Federal de Sa˜o Carlos, Brazil

2 Instituto de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, Brazil

3 Department of Biological Sciences, University of Warwick, Coventry, UK

Ribosome-inactivating proteins (RIPs; rRNA N-glyco￾sidases; EC 3.2.2.22) are found predominantly in

plants but they may also occur in fungi and bacteria

[1]. Collectively, unless mutated, they are all rRNA￾specific N-glycosidases capable of selectively cleaving a

glycosidic bond to release an adenine within the uni￾versally conserved sarcin ⁄ricin loop of the large rRNA

in 60S ribosomal subunits [2]. This modification pre￾vents the binding of elongation factors and thereby

irreversibly inhibits protein synthesis in eukaryotic

cells. Despite this common activity, RIPs can vary in

their physical properties and cellular effects [3].

Currently, RIPs are divided into three groups.

Type 1 RIPs comprise a single catalytically active sub￾unit of 25 ± 30 kDa, whereas type 3 RIPs consist of

an amino-terminal domain, resembling type 1 RIPs,

linked to a carboxyl-terminal domain with unknown

function [4]. In contrast, type 2 RIPs contain at least

one ribosome inactivating A-chain and a correspond￾ing number of carbohydrate-binding B-chains, with the

latter generally showing a preference for b1-4 linked

galactosides [3]. It follows that, although type 1 and

type 2 RIPs are active against ribosomes in vitro, only

the type 2 proteins are cytotoxic due to the presence of

a B-chain that mediates surface binding and entry of

holotoxin into susceptible cells. From studies of the

biosynthesis of type 2 RIPs in their producing tissues,

it is apparent that both polypeptides are made in cor￾rect stoichiometry by being derived from a single pre￾cursor through the excision of a intervening peptide

sequence [5]. The two polypeptides remain covalently

joined, however, by a disulfide bridge between cysteine

Keywords

Abrus pulchellus; characterization; cloning;

isoforms; ribosome-inactivating protein

Correspondence

A. P. U. Arau´jo, Grupo de Biofı´sica

Molecular, IFSC, PO Box 369,

Zip 13560-970, Sa˜o Carlos, Brazil

Fax: +55 16 33715381

Tel: +55 16 33739834

E-mail: [email protected]

(Received 14 November 2007, revised 11

December 2007, accepted 20 December

2007)

doi:10.1111/j.1742-4658.2008.06258.x

Abrus pulchellus seeds contain at least seven closely related and highly toxic

type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain

linked to a sugar binding B-chain. In the present study, four pulchellin

isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion

exchange and chromatofocusing chromatographies, and investigated with

respect to toxicity and sugar binding specificity. Half maximal inhibitory

concentration and median lethal dose values indicate that P I and P II have

similar toxicities and that both are more toxic to cultured HeLa cells and

mice than P III and P IV. Interestingly, the secondary structural character￾istics and sugar binding properties of the respective pairs of isoforms corre￾late well with the two toxicity levels, in that P I⁄P II and P III⁄ P IV form

two specific subgroups. From the deduced amino acids sequences of the

four isoforms, it is clear that the highest similarity within each subgroup is

found to occur within domain 2 of the B-chains, suggesting that the

disparity in toxicity levels might be attributed to subtle differences in

B-chain-mediated cell surface interactions that precede and determine toxin

uptake pathways.

Abbreviations

GalNAc, N-acetylgalactosamine; IC50, half maximal inhibitory concentration; LD50, median lethal dose; P I, pulchellin isoform I; P II, pulchellin

isoform II; P III, pulchellin isoform III; P IV, pulchellin isoform IV; RIP, ribosome-inactivating protein.

948 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS