Ribosome-inactivating proteins from plants

Biochimica et Biophysica Acta, 1154 (1993) 237-282 237 Elsevier Science Publishers B.V.

BBAREV 85427

Ribosome-inactivating proteins from plants

Luigi Barbieri, Maria Giulia Battelli and Fiorenzo Stirpe * Dipartimento di Patologia Sperimentale, Uniuersith di Bologna, Via San Giacomo 14, 1-40126 Bologna (Italy)

(Received 25 January 1993) (Revised manuscript received 14 July 1993)

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 A. Historical notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

II. General properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 A. Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 B. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 C. Purification procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 D. Physicochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

1. Molecular mass and isoelectric point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 2. Amino acid and sugar composition and sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 3. Three-dimensional structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 4. Chemical modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

E. Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 1. Plant tissue localisation and endogenous biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 2. Biosynthesis in heterologous systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

III. Biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 A. Effects on ribosomes: the enzymatic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 B. Interaction of ribosome-inactivating proteins with cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1. Internalisation of type 2 ribosome-inactivating proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 254 2. Internalisation of type 1 ribosome-inactivating proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 257 3. Effects on mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

C. Effects on laboratory animals and man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 1. Toxicity and lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 2, Axonal transport and effect on the nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 3. Immunosuppressive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 4. Abortifacient activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

D. Effects on other organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 E. Antiviral activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

1. Plant viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 2. Animal viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

IV. Targeting of ribosome-inactivating proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 A. lmmunotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

1. Immunotoxins with type 2 ribosome-inactivating proteins . . . . . . . . . . . . . . . . . . . . . . . . . 264 2. Immunotoxins with type 1 ribosome-inactivating proteins . . . . . . . . . . . . . . . . . . . . . . . . . 265

* Corresponding author Fax: +39 51 251315.

Abbreviations: RIP(s), ribosome-inactivating protein(s); PCR, polymerase chain reaction; EF, elongation factor; TGN, trans Golgi network; PEC, peritoneal exudate cells; HIV, human immunodeficiency virus; GVHD, graft versus host disease; ALL, acute lymphoblastic leukaemia. For abbreviations of ribosome-inactivating proteins names see Tables I and II.

23s

B. F a r g e t i n g , a i l h hifunctional ant ibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !~>o

( ' . ( o n j u g a t e s with l lOn-Il l lnlUIl¢ carr iers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2~0

V. Possible uses m expcr imenta l and clinical medic ine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2~7

A. A n t i - t u m o u r l h c r a p y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2~7

1. Ex vivo purging of bone mar row . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .'1~7

2. In viw) local t r e a lme n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21~S

3. In viw~ syslemic t r e a tme n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2~'i,';

B. I m m u n e disordcrs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

1. A u t o i m m u n c diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2oS

2. Prevent ion and t r e a t m e n t of graf t -versus-host disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 20t)

3. A I D S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27O

VI. Appl icat ions in agr icul ture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

VII . Biological rolc and perspect ives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

A c k n o w l e d g e m e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Refe rences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

1. Introduction

The ribosome-inactivating proteins (RIPs) from plants are RNA N-glycosidases that depurinate the major rRNA, thus damaging ribosomes, and arresting protein synthesis [1-8]. They can be divided into two groups: single-chain proteins, type 1 RIPs, and two- chain proteins, type 2 RIPs. Evidence from structural and genetic studies indicates that proteins of both types evolved from a common ancestor, suggesting that type 2 RIPs evolved from type 1 RIPs [9].

Type 1 ribosome-inactivating proteins are more common and have been identified and purified from more than 30 plants. Type 2 RIPs consist of two polypeptide chains, both necessary for the toxin molecule to act: an A chain which damages ribosomes through its enzymatic activity, and a B chain, with lectin properties, which binds to galactosyl-terminated receptors on the cell surface, allowing the entry of the A chain.

Two chain type 2 RIPs may be very potent toxins, two of which, namely ricin, from the seeds of Ricinus commtmis (castor beans) and abrin, from the seeds of Abrus precatorius 0equirity beans) are known since the end of last century (for an historical review see Ref. 10 and following section). These toxins act in an apparently identical manner and have a similar structure. Recently, a sub-class of type 2 RIPs has been proposed Ik~r proteins that possess RIP enzymic activity and lcctin activity but nevertheless are scarcely toxic or non toxic [11]. To this sub-class may belong Ricinus agglutinin [12], ebulin-I [11] and possibly other lectins with translational inhibitory activity [13]. The lectin moiety is also responsible for the agglutinating activity, which is considered beyond the scope of the present review and therefore will not be discussed.

Type 1 RIPs and the A chains of type 2 RIPs have

the same enzymatic activity: RNA N-glycosidase at a specific site, A4324 in the case of rat liver 28S rRNA. There is confusion concerning the exact number of the position in the sequence of nucleotides in 28S rRNA of the ricin-sensitive adenosine. In accordance to Ref. 14 we retain the designation A4324 a s suggested by Ref. 15.

The same enzymatic activity has been demonstrated as the mechanism of action of Shigella dysenteriae type 1 toxin and Escherichia coli Shiga-like toxins SLT I and SLT II [16]. The protein sequence data available at present show that these prokaryotic proteins share considerable homology with plant RIPs.

In some cases different names have been used to designate the same RIP (see Tables I and II below). In an effort to unify the nomenclature, the following criteria have been used in this review: (i) when no name bad been assigned, the proteins have been desig- nated with the name of the plant (e.g., barley RIP); (ii) when more than one name was present in the literature, the first assigned name or, when appropriate, its most frequently used abbreviation has been used; (iii) different RIPs from the same plant tissue are distin- guished by a number or letter, usually found in the first description of the protein; (iv) RIPs from different part from the same plants are indicated by adding the appropriate suffix to the name: S for seeds, L for leaves, R for roots, C for cultured cells (e.g., saporin 6 becomes saporin-S6). As a partial exception to these rules we retain the name of viscumin for mistletoe lectin I, since viscumin was the name given to the lectin when its RIP properties were demonstrated.

I-A. Historical notes

The ribosome-inactivating proteins first known were the toxins ricin and abrin. The toxicity of the seeds of

239

R. communis and A. precatorius had been known since ancient times. Dixon in 1887 [17] was the first to suspect that the toxic principle of castor beans was a protein. Shortly afterwards, Stillmark [18,19] in Kobert's laboratory in Dorpat purified the protein, for which he proposed the name ricin, and attributed its toxicity to the property of agglutinating erythrocytes. Hellin [20], also in Kobert's laboratory, discovered the agglutinating properties of abrin. Ehrlich [21,22] induced immunity to abrin and ricin by feeding rabbits with small amounts of seeds, a fundamental observation in the history of immunology. The pathology of ricin and abrin poisoning was admirably described by Flexner [231.

Interest in these toxins was revived as late as 1960, when Lin et al. [24] reported that they were more toxic to tumour (Ehrlich ascites) than to normal cells. This observation was followed by a considerable amount of work, which led to the definition of the two-chain structure and of the function of the chains of both abrin and ricin and of their inhibitory activity on protein synthesis (reviewed in Ref. 10). Modeccin, the toxin of Adenia (Modecca) digitata, already suspected to be similar to ricin [25], was independently identified as a type 2 RIP in two laboratories [26,27]. Few other type 2 RIPs were found (see Table II below). The first type 1 RIP to be identified was the pokeweed antiviral protein (PAP). This was isolated as the active principle of the extracts of Phytolacca americana leaves which prevented transmission of tobacco mosaic virus infection [28]. Numerous other type 1 RIPs were and are still being described (see Table I below).

The last and very important advancement was the elucidation of the mechanism of action of RIPs by Endo and colleagues, who discovered the RNA N-glycosidase activity of these proteins (reviewed by Endo [29]).

II. General properties

H-A. Distribution

Studies on the distribution in the plant kingdom of the species containing RIPs are still scarce (some screening data have been reported [30-36]). Most species examined belong to the class of Angiospermae: RIPs have been identified and purified from both monocotyledonae and dicotyledonae. No RIP has yet been purified from the class of Gymnospermae. Trans- lational protein inhibitory activity has been detected in 18 species out of 52 tested belonging to the monocotyledonae and in 122 out of 292 dicotyledonae tested. A few considerations should be kept in mind in inter- preting these raw data.

(i) Screenings were for translational inhibitory activity, which is not a definite proof of RIP presence,

although RIPs could always be identified as responsible of the activity when the latter was high enough to allow the purification.

(ii) Usually only one particular part of the plant was tested (mostly seeds), whilst RIPs have been detected in different parts of the same plant: RIPs are present in the latex but not in the seeds of Hura crepitans [37].

(iii) Some families were explored more extensively with the aim of finding new RIPs, rather than of studying their distribution.

(iv) Since ribosomes from different organisms have various patterns of sensitivity to different RIPs, the possibility exists that some materials containing RIPs very active in certain systems were discarded, being scarcely or not active on the ribosomes used in the assay (most frequently from rabbit reticulocytes).

The vast majority of the RIPs so far purified are single chain proteins (type 1, see section II-B). Type 2 RIPs were found only in seven plant species, members of five different families: Euphorbiaceae, Fabaceae, Passifloraceae, Sambucaceae and Viscaceae (as classi- fied by Ref. 38). The family of Euphorbiaceae is the only one in which RIPs of both types were found.

The lectin moiety of the type 2 RIPs is always galactose-specific, and in some type 1 RIP containing plants a galactose-specific lectin is also present as a separate molecule (e.g., in Trichosanthes kirilowii seeds [391).

The first type 1 RIP to be identified and isolated was the pokeweed antiviral protein [40], which was studied for its inhibitory activity on plant viruses. Only after its purification, the effect on protein synthesis and on ribosomes was discovered. It is possible that other antiviral proteins identified from various plants (e.g., Yucca recureifolia [41]) or fungi ( Lentinus edodes [41,42]) are RIPs, as is suggested also by their physicochemical characteristics. Since all type 1 RIPs tested showed abortifacient activity (see section III-C.4), RIPs may well be the active principle of a group of abortifacient herbal remedies known since centuries in China. The activity of some of these remedies has been indeed attributed to proteins which have been purified and identified as proper type 1 RIPs.

RIPs, similar, but not identical to each other (possibly isoenzymes), may be present in different parts of the same plant, or even in the same anatomic structure. Thus, more than one type 1 RIPs were identified and purified from the leaves, roots and seeds of pokeweed (Phytolacca americana) [43-46], bryony (Bryonia dioica ) [31,35,47] and soapworth (Saponaria officinalis ) [48]. The type 2 RIPs modeccin [49] and volkensin [50] were present at least in the roots and seeds of the respective plants, whereas ricin is present only in the seeds of the castor bean plant [2]. The presence of RIPs in plant cells cultured in vitro was ascertained (Refs. 46,51-53; and Barbieri, L., Minghetti, A. and

24(t

Stirpe, F., unpublished results). Out of 43 species of 25 families tested, some translational inhibitory activity was found in callus cultures of 26 species belonging to 17 different families. Cells from RIP-producing plants did not always produce RIPs in culture, and actually differences were observed among cultures from the same species (Phytolacca americana) [46]. Similar differences were observed in the production of the Mirabilis antiviral protein by Mirabilis jalapa cultured cells, from which high-producing colonies could be selected [54-57].

H-B. Structure

As mentioned above, RIPs can be divided into two categories, depending on the presence or absence of a least one polypeptide chain with lectin activity (Fig. 1): type 1, composed of a single chain polypeptide and type 2, two chain proteins.

Single-chain type 1 RIPs are vcry similar to each other (see following sections) with the notable exception of b-32, a RIP from Zea mays. The active form of this grain RIP is generated by internal proteolysis of the precursor form: the final structure is composed of two chains of 16.5 and 8.5 kDa linked together by non peptidic bonds [63].

Two-chain type 2 RIPs are composed of two or four polypeptide chains, have a molecular mass of roughly 60000 or 12000[) respectively. At least one of thc chains is an A chain (A, active) with RNA N-glycosidase activity and one is a B chain (B, binding), a galactose-specific lectin. The two polypeptide chains are linked by disulphide bonds and other non covalent bonds [64]. Abrin, cbulin 1, modcccin, ricin and volkensin are heterodimers composed of one A chain and one B chain [2,11]. Viscumin has similar composition, but in concentrated solutions two molecules associate to form a tetramer [65,66]. The tetrameric struc-

TABLE 1

Purified type 1 ribosome-inactivating proteins

A list of synonyms and abbreviations is reported at the end of the table (see also the section Introduction).

Family, genus and species Plant tissue Name Yield Refs. ( rag/100 g)

Asparagaceae Asparagus officinalis seeds

Caryophyllaceae Agrosternma githago seeds

Cucurbitaceae

Dianthus barbatus leaves Dianthus caryophyllus leaves

Lychnis chalcedonica Petrocoptis glaucifolia Saponaria officinalis

Bryonia dioica

Citrullus colocynthis

Cucumis melo Luffa acutangola Luffa cylindrica

Momordica charantia

Momordica cochinchinensis Tricchosanthes kirilowii

seeds plant leaves leaves roots roots roots seeds seeds seeds seeds leaves roots seeds seeds seeds seeds seeds

seeds

seeds roots roots roots seeds

asparin 1 asparin 2 agrostin 2 agrostin 5 agrostin 6 dianthin 29 dianthin 30 dianthin 32 lychnin petroglaucin saporin-Ll saporin-L2 saporin-Rl saporin-R2 saporin-R3 saporin-S5 saporin-S6 saporin-S8 saporin-S9 bryodin-L bryodin-R colocin 1 cotocin 2 melonin luffaculin luffin a luffin b momordin I momordin I Ib momorcochin-S trichosanthin a-tr ichosanthin TAP 29 trichokirin

5.4 47 4.9 47 8.4 37

34.2 37 18.4 37

77 2.0 78 2.0 7S

160 47 0.3 7~

35 48 13 48 2O 48

5 48 9 48

63 48 268-414 37,

15 71 24 71

0.2 47 0.5 35

10 47 16 47

80 12.11 81 20.0 81 12.0 81

150-180 83 84

61.7 85 140 86,

88, 2 9O 5.7 91

87 89

241

TABLE I (continued)

Family, genus and species Plant tissue Name Yield Refs. (mg/100 g)

Euphorbiaceae Croton tiglium seeds crotin 2 2.9 ,1 crotin 3 0.2 a

Gelonium multiflorum seeds gelonin 250-300 92 Hura crepitans latex Hura crepitans RIP 146 37 ~' Jatropha curcas seeds curcin 2 1.4 Manihot palmata seeds mapalmin 16 47 Manihot utilissima seeds manutin 1 20

seeds manutin 2 23 a

Nyctaginaceae Mirabilis jalapa tissue culture MAP 3 52 roots MAP 90 93

Phytolaccaceae Phytolacca americana leaves PAP 9.2 43 PAP II 3.6 44

seeds PAP-S 100-180 45 tissue culture PAP-C 17.5 46 roots PAP-R 7.0 47

Phytolacca dioica seeds PD-S1 10 J94 PD-S2 37 94 PD-S3 2 94

Phytolacca dodecandra leaves dodecandrin 3.4 95 tissue culture dodecandrin-C 1.0 53

Poaceae Hordeum vulgare seeds barley RIP 139 33, 96 Secale cereale seeds Secale cereale RIP 200 33 Triticum aesticum germ tritin 3.0 33, 97

seeds tritin 32 98 Zea mays seeds maize RIP 12-20 63

a Ferreras, J.M., Barbieri, L., Girb6s, T., Battelli, M.G., Roio, M.A., Arias, F.J., Rocher, M.A., Soriano, F., Mendez, E. and Stirpe, F. (unpublished results).

b The same term was used to designate a RIP from M. balsamina (see below). List of synonyms and abbreviations: asparin 1, Asparagus officinalis RIP peak 2; asparin 2, Asparagus officinalis RIP peak 5; barley RIP, barley inhibitor, barley RIP 30; dianthin 30, DAP 30; dianthin 32, DAP 32; gelonin, GAP 31; Hura crepitans RIP, H. crepitans peak 5, H. crepitans inhibitor; luffin, Luffa aegyptiaca RIP; luffin-a, a-luffin; luffin-b, /3-1uffinn; maize RIP, corn inhibitor, its preproform: b-32; MAP, Mirabilis antiviral protein; momordin I, M. charantia inhibitor, M. charantia isoRIP 3, a-momorcharin, momordin a, momordin (major isoform); momordin II, M. charantia isoRIP 4, fl-momorcharin, MAP 30; PAP, pokeweed antiviral protein, pokeweed antiviral peptide, phytolaccin; PD, Phytolacca dioica RIP; saporin-L1, saporin-S6 4; trichosanthin, TAP (Trichosanthes antiviral protein); a-trichosanthin, karasurin; tritin, wheat germ inhibitor.

ture is typical of the Ricinus agglutinin, which is composed of two identical heterodimers, very similar to ricin, held together by non covalent bonds [12].

Polypeptide Type 1 Type 2 with

N-glycosidase activity

i I - ~, ~ Polypeptidewith lectin

t S --S- activity

O A chain B chain

Fig. 1. Structure of type 1 and 2 ribosome-inactivating proteins. Only one carbohydrate binding site is shown in the model of the B chain because the presence of two carbohydrate binding sites was deter-

mined only for ricin [58-61] and abrin [62].

With respect to structure and inhibitory activity on protein synthesis at the elongation step, the type 2 RIPs from plants show surprising similarities with toxins produced by some bacteria such as Corynebac- terium diphteriae [67] and Pseudomonas aeruginosa [68], which associate a lectin and an EF2 ADP ribosylase, and E. coli (strain 0157:47) [16] and S. dysenteriae [16,69,70] which associate a lectin and a RIP-like rRNA N-glycosidase.

II-C. Purification procedure

Two different procedures are currently followed to purify type 1 and type 2 RIPs. Type 1 RIPs are purified essentially by cation-exchange chromatography on car- boxymethyl or sulfopropyl-derivatized matrices [37,71], taking advantage of their p I in the extreme alkaline region. This general procedure was scaled up for pilot scale preparations with minor adaptation for each RIP [711.

242

Affinity chromatography on Sepharose, acid-treated Sepharose or other galactose-containing stationary phases followed by elution with galactose or lactose is the method of choice for the purification of type 2 RIPs [2], exploiting the lectin propert ies of their B chains. In the case of ricin and abrin, fur ther steps are required to separate the toxins from two ga[actose-binding agglutinins present in the same seeds. Several methods have been devised to obtain highly purified A chains of type 2 RiPs (e.g., Refs. 5{),72-76).

Lists and yields of the RIPs purified so far with the respective references are given in Tables I and 11.

H-D. Physicochemical properties

l l-D. 1. Molecular mass and isoelectric po in t

The molecular mass of RIPs has usually been determined by polyacrylamide-gel-electrophoresis in the presence of sodium dodecylsulphate ( S D S - P A G E ) [109]. The values obtained by gel filtration are generally smaller due to some interaction with the matrix (type 1 RIPS are highly basic) or to a non globular shape of the protein [110]. Values obtained by complete sequencing are in good agreement with those obtained with S D S - P A G E (within 10%). The molecular masses of RIPs are repor ted in Tables l i t and IV. They are a round 30000 Da, both for type 1 RIPs and the A chains of type two RIPs, and slightly higher for the B chains of type 2 RIPs.

The p l of all type 1 RIPs is invariably basic, usually >_ 9.5, and cannot be measured accurately with conven-

tional methods [129]. Consistently, al though the pro- fl~rm of maize b-32 has a p / of 0.5, thc processed active form is highly basic (p l > 9) [63]. The p l of the A-chains of type 2 RIPs span from 4.6 in Ihc case of abrin A chain to 8.6 of some ricin A chains. Isoelectric points of type 2 RIPs arc repor ted in Tablc IV.

II-D.2. A m i n o acid and sugar composi t ion and sequence

The amino acid composit ion is known for a number of RIPs of both types (see Ref. 112 and the specific references reported in the tables). As shown in Tables III and IV, the majority of RIPs arc glycoproteins. The carbohydrate componen t though, does not seem to play any major role in the enzymatic activity of RIPs, since (i) some RIPs of either type do not contain carbohydrates (e.g., t r ichosanthin and abrin A chain), (ii) the sugar composit ion of different RIPs wtries in respect of quantity and quality, (iii) gelonin and ricin A chain could be partially deglycosylated without affecting their capability of inhibiting protein synthesis in a rabbit reticulocytes lysate, and (iv) recombinant ricin A chain produced in E. coli is not glycosylated and yet fully functional. The sugars present are glucose, galactose, mannose, fucose, xylose, and N-acetyl-l)-glucosamine.

A complete amino acid sequence has been reported for various RIPs (Table V) and partial amino-terminal sequences are reported for most RIPs. The sequence homologies vary from 17 to 75% identical primary structure. Three regions involved in enzymatic activity have been identified by site-directed mutagenesis [154- 160] and three-dimensional structure [60,161,162] al-

TABLE 11

Purified type 2 ribosome-inactil,ating proteins

A list of synonyms and abbreviations is reported at the end of the table (see also the section Introduction).

Family, genus and species Plant Name Yield Refs. tissue (mg/100 g)

Euphorbiaceae Ricinus communis seeds ricin D 120 73, 99 ricin E I00 Ricinus agglutinin 12

Fabaceae Abrus precatorius seeds abrin a 10 I abrin c 75 102

seeds ~' abrin-a 100 103 abrin-b 25 103 abrin-c 35 103 abrin-d 1 103

Passifloraceae Adenia digitata roots modeccin 20-180 49, 104 modeccin 6B 7 74

Adenia colkensii roots volkensin 0.2 105, 5~1 Sambucaceae Sambucus ebulus leaves ebulin 1 3.2 11 Viscaceae Phoradendron californicum leaves P. califbrn, lectin 106. 1l}7

Viscum album leaves viscumin 7 65, 108

~' These abrins were purified from a probably different variety of seeds and do not appear to correspond to the other forms described in the literature by other authors.

List of synonyms and abbreviations: abrin c, abrin (major form); Phoradendron californicum lectin, PCL; ricin D, ricin (major form), ricin 60, Ricinus toxin (RT); Ricinus agglutinin, Ricinus communis agglutinin (RCA) 120, ricin 120; viscumin, Viscum album lectin I (major form), VAL 1. mistletoe lectin I, ML I.

TABLE III

Physico-chernical characteristics of type l ribosome-inactivating proteins

RIP Molecular Neutral Extinction Refs. mass a sugar coefficient (Da) content at 280 nm

(%) (M)

Agrostin 2 30600 6.68 37 Agrostin 5 29500 6.87 37 Agrostin 6 29 600 7.17 37 Asparin 1 30500 traces 30500 47 Asparin 2 29 800 traces 29 800 47 Barley RIP 29976 b 27 600 33, 111 Bryodin-L 28800 2.72 23 000 47 Bryodin-R 30000 6.3 26400 35 Colocin I 26300 0.40 16600 47 Colocin 2 26300 1.59 18400 47 Crotin 2 30 200 0.41 29300 " Curcin 2 28100 c Dianthin 30 29500 1.56 78, 112 Dianthin 32 31 700 2.34 78, 112 Dodecandrin 29000 0 95 Dodecandrin-C 31-32000 4.46 53 Gelonin 30000 21000 37, 112 Hura crepitans RIP 27500 0.94 25200 37 c Luffaculin 28 000 present 81 Luffin a 28242 b 0 d 82, 113 Luffin b 27275 b 0 d 82, 114 Lychnin 26600 0.31 19400 c Maize RIP 25 000 0 26500 63 Manutin 1 30700 4.0 22400 c Mapalmin 32300 5.99 22900 47

though conflicting results have been repor ted [156,163].

Several residues appear impor tan t for the catalytic action of ricin A-chain: thus, var iants Tyr8°-Phe, Tyr123-Phe, Glu177-Gln [158,160] and ArglS°-His [158]

had a substant ia l reduct ion in activity (7- to 1000-fold). Var iants Tyr72-Phe and Tyr118-Phe of M A P have sub-

stantially reduced inhibi tory activity only against the

r ibosomes from E. coli [164], while the cor responding variants of the ricin A chain have reduced activity against mammal i an r ibosomes [160]. Sequence homologies in proposed active site regions are part icularly high (Fig. 2).

F rom the results repor ted above it appears evident

that (i) RIPs may exist as different isoforms in the same plant, and that (ii) the differences observed in the sequences were not due to a l ternate splicing but to different nucleot ide sequences. Moreover, three slightly different amino- te rmina l sequences were repor ted for t r ichosanthin, suggesting that there may be a sub- species var iat ion [87,88,120,165]. The few available data indicate that the B chains of type 2 RIPs have also a similar amino acid sequence [125,166,167].

The s tructures of the carbohydrate side chains of momord in I [168], of ricin D (A and B chain) [169] and of abr in a (B chain) [170] have been elucidated in detail (Fig. 3). The A1 chain of ricin contains a single

TABLE III (continued)

243

RIP Molecular Neutral Extinction Refs. m a s s a sugar coefficient (Da) content at 280 nm

(%) (M)

MAP 27833 b 0 24000 115 Momorcochin-S 30 7011 2.82 21000 85 Momordin I 29092 h 1.74 112, 116 Momordin II 300011 1.30 117 PAP 29 000 0 24 000 43 PAP II 30 000 0 26 700 44 PAP-C 29 000 traces 45 PAP-R 29 800 0 23 800 47 PAP-S 29 167 b 0 d 22500 45, 118 PD-S 1 30 900 0.88 22 600 94 PD-S2 32000 2.22 24700 94 PD-S3 32000 0.49 21 800 94 Secale cereale RIP 30000 33 Saporin-L1 31 600 29 400 48 Saporin-L2 31600 26 400 48 Saporin-R 1 30 200 23 200 48 Saporin-R3 30 900 24 700 48 Saporin-S5 29 500 23 800 37, 48, 119 Saporin-S6 29500 0 24800 37, 119 Saporin-S8 29 500 37 Saporin-S9 29 500 0 37 Trichokirin 27 000 1.27 23 330 91 Trichosanthin 24-29000 0 86, 87, 91,120 a-Trichosanthin 27215 b 88, 89, 121 Tritin 30 000 97

~' From SDS-PAGE. b From amino acid sequence. c Ferreras, J.M., Barbieri, L., Girb~s, T., Battelli, M.G., Roio, M.A.,

Arias, F.J., Rocher, M.A., Soriano, F., Mendez, E. and Stirpe, F. (unpublished results).

d Luffin a has six Asn-linked N-acetylglucosamine residues, luffin b two residues, and PAP-S three residues [122].

complex oligosaccharide unit , whilst the A2 chain con-

tains a high mannose oligosaccharide in addi t ion to the complex unit. The B chain contains two high mannose

type oligosaccharides. Six asparagine residues of luffin a, two of luffin b and three of PAP-S are glycosylated

with only N-acetyl-D-glucosamine. This is a unique glycosylation pa t te rn and it has been a t t r ibuted to the presence in the respective tissue of endo-N-acetylglu-

cosaminidase [122]. Glycosylation sites have been iden-

tified also in the pro-saporin C-terminal extension sequence, cleaved to form the mature protein [119], which is devoid of neut ra l sugars.

II-D.3. T h r e e - d i m e n s i o n a l s t ruc ture

Ricin has been crystallised and the result ing struc-

ture examined at 2.8 and 2.5 A resolut ion [60,161]. The A chain is a globular protein. It has an extensive secondary s t ructure and a well def ined cleft. This cleft is assumed to be the enzymatically active site on the basis of its s t ructure and because the activity of the recombinan t ricin A chain (see previous section and Ref. 155) was abolished by modif icat ions in t roduced in

244

the n u c l e o t i d e s e q u e n c e , in pos i t ions c o r r e s p o n d i n g to

a m i n o acids invo lved in this cleft . T h e B cha in is a g e n e

dup l i ca t ion p r o d u c t [171] showing 32C/c a m i n o acid

ident i ty b e t w e e n the two halves. Acco rd ing ly , X- ray

d i f f rac t ion da t a show tha t the B cha in is d iv ided into

two domains , s imi la r to e a c h o the r , c o n t a i n i n g o n e

b ind ing si te for l ac tose in a sha l low cleft . T h e e p i m e r i c

specif ic i ty (D-ga lac tose ) o f the b ind ing is g iven by a

h y d r o g e n b o n d b e t w e e n a g l u t a m i n e r e s i d u e and the

hydroxyl g r o u p in pos i t ion 4 o f t he ga lac tos id i c r e s idue .

T h e d a t a o b t a i n e d f r o m X- ray d i f f r ac t ion s tud ies con-

f i rm also the b i o c h e m i c a l e v i d e n c e tha t the two cha ins

a re he ld t o g e t h e r by a d i s u l p h i d e b r i d g e and by hy-

d r o p h o b i c bonds . Crys ta l l i s a t ion o f s o m e o t h e r R I P s

has b e e n r e p o r t e d [172-176] . P r e l im ina ry dat:i a rc

ava i lab le also on the t h r e e - d i m e n s i o n a l s t ruc tu re of

t r i c h o s a n t h i n (at a r e so lu t ion o1"4 , ~ ) [ 1 7 7 - 1 7 9 ] and

M A P [933]. In p a r t i c u l a r the s t ruc tu re of t r i chosan th in

a p p e a r s to be very s imi la r to tha t of ricin A cha in

[178-18o].

ll-D. 4. Chemical modifications D e t a i l e d s tud ies on the e f fec t o f c h e m i c a l mod i f i ca -

t ions have b e e n r e p o r t e d for r icin ( r e v i e w e d in Refs .

2,126), P A P [181] and ge lon in [182]. C h e m i c a l modi f i -

ca t ions o f R I P s w e r e u n d e r t a k e n ma in ly in o r d e r to (i)

e l u c i d a t e t he essen t i a l a m i n o acid r e s idues which may

p r o v i d e s o m e ins ight c o n c e r n i n g the e n z y m a t i c si te and

TABLE IV

Physico-chemical characteristics of type 2 ribosome-inacticating proteins

RlP Molecular mass ~' Neutral sugar lsoelectric Extinction (Da) content point coefficient

(~4) (pl) at 280 nm (M)

Refs.

Abrin a 60 100 b 5.5 70700 Ai chain 31 000 present Aii chain 32 500 present B chain 35 500 present

Abrin c <" 63800 t, 7.4 6.1 82500 A chain 30 000 0 4.6 23 60(I B chain 36 000 7.4 7.2 63 700

Ebulin I 56000 A chain 26 000 B chain 30000

Modeccin 60000 2.7 7.1 8.5 A chain 27800 5.8-6.7 B chain 31000 7.3-8.2

P. californicum l ectin 69 000 14.1 A chain 31000 B chain 38000

Ricin Da d 63-66000 5.5-6.2 7.1 77900 AI chain ~ 30000 4.5 7.6 23000 A2 chain 33 000 6.0 7.6 B chain 34 000 6.4 5.5 50 700

Ricin Db ' 63 0(10 present 8.3 A1 chain 30000 4.5 7.6 B chain 33 000 present 8.8

Ricin E 63000 Ricinus agglutinin 120 000 4.5 7.8 140 400

A chain 31000 B chain 34 000

Viscumin 60000 I 1.8 A chain 29000 B chain 32000

Volkensin 62 000 5.7 7.5-8.5 A chain 29000 B chain 36000

t01, 123

101, 102, 124. 125

I1

49.74

107

126

126

1()¢1, 127. 128 12

65, 66

50, 105

From SDS-PAGE. i, By sedimentation equilibrium. c Abrins purified from a probably different variety of seeds which do not appear to correspond to the other forms described in the literature by

other authors [103]. ,i The first value is for an A1-B composition the second for an A2-B composition.

A1 and A2 chains differ only for the carbohydrate content. r The A chain of ricin Db is identical to the AI chain of ricin Da: the two ricins differ only for the B chain.

245

TABLE V

Complete sequences of type 1 and type 2 RIPs

References.

RIP Genome a cDNAb Protein c

Abrin 130 Abrin-a 131,132 0

Abrin c 125 b-32/maize RIP 133 63, 134 Barley RIP 111 135 Dianthin 30 136 Luffin a 137 113 Luffin b 138 114 MAP 139 140 115 Momordiea balsamina RIP 141 Momordin I 116 PAP-S 118 PAP 142 143 Ricin D 144 145 99, 146 Ricinus agglutinin 147 Saporin-S6 148, 149 150 151 Trichosanthin 152 153 120 a-Trichosanthin 88, 121

a Deduced from sequencing of a genomic clone, b Deduced from sequencing of a cDNA clone, c Direct sequencing of the purified protein, d A-chain [131], B chain [132].

action, (ii) identify amino acid residues which can be modified without affecting the enzymatic activity of RIPs to be used in the preparation of conjugates and (iii) modify in vivo kinetics and tissue distribution.

This last mentioned objective was pursued particu- larly with ricin. Ricin is a glycoprotein and its clearance from the blood stream depends not only on the lectin activity of the B chain but also on the recognition of its carbohydrate moiety by cell-surface lectins. The high mannose oligosaccharide present on the B chain and on the A2 chain contains a trimannosidic core structure which is avidly bound by rat non-parenchymal liver ceils. The complex oligosaccharide present on the A1 and A2 chains lacks the trimannosidic core but may be recognised by the hepatocytes through a fucose residue. Thus, immunotoxins containing ricin or ricin A chain have an in vivo fate strongly influenced by their glycoprotein nature. This problem has been partially overcome by chemical deglycosylation or by the use of recombinant non-glycosylated products (reviewed in Ref. 183).

II-E. Molecular biology

The cloning of DNA sequences coding for several RIPs has been reported (Table VI). Several strategies have been employed: (i) cDNA expression library, screened with antibodies; (ii) cDNA library screened with oligonucleotides constructed on known protein sequences; (iii) synthetic genes; (iv) genomic DNA se-

quences obtained by direct PCR (polymerase chain reaction) amplification or by screening with cDNA probes.

No introns were found in genomic sequences with the notable exception of a M. jalapa clone [139]. This genomic sequence is composed of two exons separated by a short intron. The position of the intron was mapped between Lys 178 and Ile 179.

Maize b-32 is synthesised as a 34 kDa precursor during kernel development. With germination, the precursor form is proteolytically processed with removal of a N-terminal peptide, a C-terminal peptide and an internal sequence. This internal sequence is apparently located near or at the active site (by comparison with structure-activity correlation data obtained with ricin). Thus, only the processed protein has high RIP activity [63].

The genomic structure of MAP gene [139] and of maize b-32 [63] is highly suggestive of a two domain composition of the ancestral RIP. This evolutionary scheme may be useful to learn more about structure- funtion relationships in RIPs.

In most cases mRNA sequences code for N-terminal and C-terminal peptides, which are post-translationally cleaved to yield the mature form (Fig. 4). The structures of prepro-forms of three type 2 RIPs (ricin, Ricinus agglutinin and abrin) have been elucidated [125,144,147] (Fig. 4): they are notably similar even though the proteins come from plants belonging to unrelated families (Fabaceae and Euphorbiaceae). Most of the amino acid residues composing the N- terminal signal sequences are hydrophobic (Fig. 5), a feature common to signal sequences in eukaryotes and prokaryotes [140].

A genomic clone that specifies a single polypeptide precursor of ricin has been isolated, sequenced and mapped [144]. The ricin gene possesses consensus sequences found in the 5' and 3' flanking regions of other eukaryotic genes.

From genome analysis and protein sequence data it can be inferred that probably all RIPs are encoded by small multigene families [143,150,152,184]. Thus, Southern blot analysis of R. communis DNA digested with restriction enzymes gives a picture compatible with the presence of 6 genes [144], and three clones of maize b-32 have been isolated and sequenced [133]. Microheterogeneity could be explained also by sub- species variations due to segregation of populations of R. communis plants in different geographic areas (e.g., Eastern Asia and Africa). This seems to be the case for ricin E, present in plants grown in Japan and not in plants grown in tropical lands [128]. From amino acid sequence data, ricin E appears to be constituted by the A chain of ricin D and a B chain which is composed of the N-terminal half of ricin D and the C-terminal half of Ricinus agglutinin [128].

240

RIP

Abrin A chain Barley RIP

Dian th in 30

Luffin a a

Luffin b b

Maize b.32

~L~.P

MB RIP c

Momordin I pApd

PAP-S

Ricin A chain e

Saporin-S6

Tr ichosanth in f

(x-Trichosanthing

Abrin A chain h

Barley RIP

Dian th in 30

Luffin a a

Luffin b b

Maize b-32 i

MAP

MB RIP

Momordin I

PAP

PAP-S

Ricin A chain e

Saporin-S6 J

Tr ichosanth in f

(~-Trichosanthin

Abrin A chain h

Barley RIP

Dian th in 30

Luffin a a

Luffin b b

Maize b-32

MAP

MB RIP

Momordin I PAP d

PAP-S

Ricin A chain

Saporin-S6 J

Tr ichosanth in f

c~-Trichosanthin

(66) (77)

(64)

(61)

(61)

(85) (63)

(61) (61)

(63)

(63) (71)

(63)

(61)

(61)

Rcfs.

(162) (162) (165) (147) (147) (195) (165) (149) (149) (164) (163) (165) (164) (148) (148)

(81)

(93) (80) (77)

(77)

(100)

(79)

(77)

(77)

(79)

(79)

(87)

(79)

(77)

(77)

(181)

(181)

(184)

(166)

(166)

(214)

(184)

(168)

(168)

(183) (182)

(184)

(183)

(167)

(167)

(192)

(2o5) (203)

(186) (186)

(234) (194)

(187)

(187)

(202)

(201)

(2o5) (202)

(186)

(186)

(206)

(220)

(217)

(201)

(201)

(249)

(208)

(201)

(2Ol)

(216)

(215)

(219)

(218)

(2OO)

(2OO)

125,130.131

111,135

136

113

114

63,133,134

115,140

141

l l 6

143

118

144,145

149-151

153

88,152

130

111,135

136

113

114

133,134

115,140

141

116

143

118

144,145

150

153

88,121,152

130,131

111,135

136

113

114

63,133,134

115,140

141

116

143

118

144-147

150

153

88,121,152

TABLE VI

Cloning strategies for RIP genes

247

Genus and species Cloning system Nucleic acid Screening system Protein product Refs. s o u r c e

Abrus precatorius genomic PCR leaves 3 primers " abrin A chain 130 genomic PCR leaves oligonucleotides abrin A chain 125

Dianthus caryophyllus cDNA expression library leaves antibodies dianthin 30 136 Hordeum culgare cDNA expression library mature seeds antibodies barley RIP 30 111 Luffa cylindrica cDNA PCR immature seeds luffin-a, -b 137, 138

b MAP 115 Mirabilis jalapa synthetic gene n.a. cDNA cell culture synthetic gene MAP 140 genomic PCR (cDNA) MAP 139

Momordica balsamina cDNA M. balsamina RIP 141 Mormordica charantia cDNA expression library fresh seeds antibodies momordin I 116 Phytolacca americana cDNA leaves oligonucleotides PAP 143

genomic 142 Ricinus communis genomic seeds cDNA probe ricin D 144

cDNAs seeds oligonucletides c ricin D 145 cDNA seeds oligonucletides d Ricinus agglutinin 147

Saponaria officinalis cDNA summer leaves 2 probes ~ saporin-S6 150 genomic young leaves DNA probes t sap2, sap3/4 g 149

Trichosanthes kirilowii cDNA radix heterologous probe h trichosanthin 153 genomic PCR leaves oligonucletides a-trichosanthin 152

Zea mays genomic cell line cDNA probe b-32 133 cDNA endosperm b-32 134 cDNA immature kernels antibodies b-32 63

Deduced from primary abrin A chain structure. b n.a.: not applicable. The sequence was constructed from the amino acid sequence. Maximumum frequency Escherichia coli codons were used. c From ricin B chain protein sequence. Degenerated sequence. d From ricin D B chain sequence. e The sequence was constructed from the amino acid sequence of two saporin-S6 peptides. Maximumum frequency codons were used. i Saporin-S6 gene probes from protein sequence data using inosine-oligonucleotides, genomic DNA and the polymerase chain reaction [148]. g Almost identical to saporin-S6. h 120-bp cDNA from momordin 1 (72.5% homology with trichosanthin in the primary amino acid sequence).

Thorough studies have been reported on the regulation of the synthesis of maize b-32 before it was known to possess RIP enzymatic activity [185]. The maize locus Opaque2 controls the expression of b-32 and other proteins in the developing endosperm. The promoter of the b-32 gene is activated in vivo by the Opaque2 gene product. Two of the five Opaque2 binding sites are inserted amongst copies of the 'endosperm box', a motif involved in the endosperm specific expression. The b-32 is a precursor with only slight RIP activity: it is processed to the more active final form during germination [63].

The reported cDNA sequence for saporin-S6 [150] indicates that the length of the signal peptide (24 amino acids) is the same as that of prepro-ricin, although there is no similarity in the predicted amino acid sequence (Fig. 5). This sequence comes from a library constructed with leaf RNA, indicating that

saporin-S6 is present also in the leaves of Saponaria officinalis. Probably, saporin-S6 is a member of a small multigene family as suggested for ricin [184] and trichosanthin [152]. Saporin-S6, ricin and trichosanthin are encoded with a C-terminal extension sequence, which is cleaved to form the mature protein [119].

II-E.1. Plant tissue localisation and endogenous biosynthesis

RIPs can be found in all plant tissues (Tables I and II), but a RIP producing plant does not necessarily express RIP activity in all its tissues (see section II-A).

The distribution in the carnation plant of dianthins 30 and 32 has been investigated with the aid of antibodies (Table VII): dianthin 30 was found throughout the plant, seeds included, whilst dianthin 32 was found only in leaves and growing shoots. Dianthins were much more concentrated in old leaves, where they

Fig. 2. Comparison of amino acid sequences of putative active site of proteins with RNA N-glycosidase activity. The number of the first and of the last aminoacid of each sequence is reported. Nearly identical sequences have been obtained for (a) luffin a [137], (b) luffin b, (c) Momordica balsamina RIP, (d) PAP [94], (e) Ricinus agglutinin A chain [147], (f) trichosanthin [120], (g) a-trichosanthin [121], (h) abrin A chain [125,131], (i)

maize RIP [63], (j) saporin-S6 [149]. For comparison between different sequences published for RIPs from Trichosanthes kirilowii see Ref. 121.

248

represent up to 1-3c/~ of the extractable protein, rather than in young leaves [186].

Significant translational inhibitory activity was found in all Saponaria officinalis tissues examined with the exception of highly immature seeds [48]. The highest activity was found in the mature seeds, 10-fold more than in roots, 50-fold more than in leaves (Fig. 6).

The biosynthesis of RIPs must be compatible with the enzymatic activity of these proteins that are capable of damaging the ribosomes. Three mechanisms appear to be involved in the 'safe' synthesis of these proteins (i) relative resistance of autologous ribosomes to the actions of RIPs; (it) synthesis of inactive pro- forms; (iii) synthesis of preproforms with N-terminal signal sequences that direct the nascent peptide chain into the lumen of the reticulum before it is finally folded in the active configuration.

Ricin and Ricinus agglutinin biosynthesis have been studied in detail [187-190]. During ricin biosynthesis in

Man let1

ii Man ]ctl

M a n j a l

i Man et i

Ricin D A chain

Xyl i[31 [ F u c j a l

(8%)

(92%)

Man lal

Momordin I

ixy, [ruc

(7%)

(75%)

1 (18%)

Fig. 3. Proposed structures of N-linked carbohydrate structures of ricin A chain [169] and momordin I [168]. The percentage of each

structure in the mature glycoprotein is reported in parentheses.

TABLE VII

77ssue distribution q/ dianthi;>

Data are from Ref. 186.

Tissue p~g of R I P / m g of protein

Dianthin 30 Dianthin 32

Old leaves 12.5 Young leaves 2.9 Shoots 3.(/ Stems 17.9 Roots 30.2 Branch roots 33.3 Seeds 10.1

3.9 1,7 2.1

Ricinus seeds, the N-terminal signal sequence of the prepro-ricin mediates the cotranslational translocation of the nascent precursor into the lumen of the endoplasmic reticulum. The signal sequence is cleaved during this step. Pro-ricin is glycosylated and the disulphide bonds established. From the endoplasmic reticulum, the pro-ricin is transported via the Golgi apparatus and Golgi vesicles to the protein bodies. Here, pro-ricin is processed by an acid proteinase to the mature form.

The biosynthethic pathway of Ricinus agglutinin appears to be similar to that of ricin, and the protein is finally housed in the endosperm together with ricin D in the soluble matrix fraction of the protein bodies, single membrane organelles [191,192]. During germination, unlike seed storage proteins, Ricinus RIPs are not degraded by proteolytic enzymes in the first stages of the biosynthetic process [191].

The two main RIPs from pokeweed leaves are synthesised with a different seasonal pattern. Only small amounts of PAP II are found in pokeweed leaves in spring and its concentration appears to be dependent upon the time of harvest, being higher in summer. The level of PAP, on the other hand, appears to be independent of the season of harvest, suggesting that PAP synthesis occurs throughout the life span of the leaves, whilst production of PAP II increases progressively with the ageing of the tissue [40].

Ready et al. [193] conclude from electron mi- croscopy studies that pokeweed antiviral protein is synthesised in the cytoplasm of leaf cells and extruded across the cellular membrane into the cell wall matrix. The protein does not seem to be attached to the wall, but to be trapped between the cell wall matrix of the mesophyll cells.

The fate of the grain RIPs following germination has been studied by Coleman and Roberts [33]. No significant net synthesis or degradation of RIPs from Triticum aestivum, Arena sativa and Hordeum culgare was observed one week after germination, as compared to the RIP content of the unsprouted seeds. Most of

leader C-terminal

Dianthin 30 23 252 18 Saporin-S6 24 253 22 MAP 28 250

RNA N-glycosidase

. ] LeclJn

Absent in the mature protein

249

leader linker C-terminal

Maize RIP 16 141 25 71 44

leader

B chain ]-C

linker

A chain

Ricin D 24 267 12 262

Ricinus agglutinin 24 266 12 262

Abrin 34 241 14 276

Abrin c 34 251 14 263

Fig. 4. Structure of prepro-form of some type 1 and 2 ribosome-inactivating proteins. See [136] for dianthin 30, [148,149] for saporin-S6, [140] for MAP, [63] for maize RIP, [144,145] for ricin D, [147] for Ricinus agglutinin, [130] for abrin, and [125] for abrin c.

the inhibitory activity remained in the residual seed material, a l though significant amounts of R I P activity could be found both in the roots and in the shoots of the seedlings. Al though no new synthesis takes places in maize during germinat ion, the processing of b-32 leads to a significant increase of R IP activity [63,194]. It remains to be seen if this is a general mechanism.

Nor thern hybridisation with barley RIP c D N A as a probe showed that the corresponding m R N A s accumu- late only in the starchy endosperm during late seed development . Fur thermore , the mature barley RIP and

maize b-32 could be found only in the starchy endosperm by in situ immunostaining [111,134]. The absence of an N-terminal extension suggests that these two grain RIPs are cytoplasmic proteins [11,134].

lI-E.2. Biosynthesis in heterologous systems One of the major problems in molecular biology is

the expression of the gene product in an active form and in quanti ty suitable for large scale exploitation. Several constructs containing sequences for RIPs could not be expressed at high level in E. coil systems (10-30

RIP

Abrin

Ricin D

Dianthin 30 PAP Saporin-S6 b MAP a-Triehosanthin Momordin I MB RIP Maize b-32

~ A R T P P V A ~

FGIS|LEDNI

S T

VNPQR T T P A E i GGSIKi G V P T K

. . . . . . . . . . . . . . . . . I E I T L E P S L

Fig. 5. Comparison of amino acid sequences of N-terminal signal sequences of ribosome-inactivating proteins. Data are: for abrin [125], ricin D [145], dianthin 30 [142], PAP [136], saporin-S6 1150], MAP [140], a-trichosanthin [152], momordin I [116], MB (Momordica balsamina) RIP [141] and maize b-32 [63]. Two grain RIPs cDNAs showed no signal N-terminal sequence [134,111]. (a) First residue of the mature protein. (b)

Identical sequence was present in sapl and sap3/4 genes [149].

250

-r.

E

| u

10,00t1,~

1,0RI,~

..~ = t ~ , ~

,~ ~ lO,O00

I,RI~

iii!iiiiiitiiiiliiiii~iiliiiiiiiii!ii

iiiiiiiiNiiiiii!i! i

ii!iii!iiliiiiiiii!iii[i!iiiiiiil ¸

i!!iiiiiiliiiiiiiiiiiilliiiiiilil; iii!!iiiiliiiiiiiiiiiiiiiiiiiiiiil

100

~ . . . . .

r~ra~ ~

Fig. 6. Tissue distribution of translational inhibitory activity in Saponaria (~fficinalis plants. One unit of activity is defined as the amount of protein necessa~ to inhibit protein synthesis by 5IF# in 1

ml of rabbit reticulocyte lysate reaction mixture [48].

/xg/1 of culture [142,153]) due to their toxicity to this translation system [115,153] (see section III-A). Better yields were obtained by the use of expression vectors under the control of the T7 promoter (trichosanthin [195]) or the tac promoter (PAP [196]). Fully active recombinant PAP variants could be purified from transformant cultures with yields varying from 1.74 to 5.55 m g / l depending upon the presence of a secretion ornpA signal peptide. Expression of fully functional ricin A chain or abrin in E. coli was obtained without toxicity problems [142,145,197]. In the case of ricin, the expressible plasmid directs the synthesis of a fusion protein containing the recombinant ricin A chain sequence and 10 extra amino-terminal residues under the control of a strong coliphage T5 promoter. This engineered product is fully active in inhibiting protein synthesis by a lysate of rabbit reticulocytes, and is capable of binding to ricin B chain to form a holotoxin whose toxicity to cells is indistinguishable from that of the native ricin. Prepro-abrin could be expressed at

high levels in E. coli (> 65i of total protein) [125] and active A chain could be isolated.

Kumagai et al. [198] developed an interesting plant viral vector derived from tobacco mosaic virus (TMVI that directs the expression of c~-trichosanthin in transfected cells. Mature hybrid virions were accumulated in transfected plants in a way which was similar to wild type TMV. Two weeks after inoculation the RIP was highly concentrated in the upper leaves (2('~ of the soluble proteins). Fully active ~-trichosanthin could bc purified to homogeneity from harvested leaves.

lntrace[lular targeting of the protein offers a different approach to the expression of RIPs. This may bc necessary with RIPs inhibiting the host cell protein synthesis, as in the case of insects [199] and yeast [155] cells which are insensitive to external ricin, but arc killed by cytoplasmic expression of ricin A chain. A chimaeric gene was constructed with ricin A chain sequence and a pre-scquence from a whcat protein. The product was translated and imported into pea chloroplast following the targeting of the pre-sequencc [2oo].

When recombinant ricin A chain transcripts are translated in a rabbit rcticulocyte lysatc, the ribosomes are rapidly inactivated, whereas protein synthesis by wheat germ ribosomes is not inhibited under the same conditions [156]. A chimerical gene was formed by fusing the promoter and 5' flanking sequences of the lens specific mouse c~A-crystallin gene with a modified ricin A chain eDNA. This construct was integrated into the germ line of transgenic mice [201]. These mice have severe development defects in the eye, primarily due to the death of cells forming the lens.

A different class of chimerical genes may be constructed to produce fusion proteins containing polypeptide sequences capable of targeting ricin A chain toward specific cell surface receptors (see section IV).

Expression of the B-chain in an active form has been obtained in different organisms: Saccharomyces cerevisiae [202], Xenopus laevis [203], mammalian cells [204]. Yeast cells expressed an active B chain [2(12] when DNA fusions were constructed in which the B chain coding sequence was preceded by a signal sequence. Pre-B chain could be expressed by microinject- ing in vitro transcripts into X. laecis oocytes. These recombinant products were processed, glycosylated and folded into a biologically active conformation [203].

III . B io log i ca l ac t iv i t i e s

The direct effect of both type 1 and of type 2 RIPs (the latter through their A chains) on cell structure and function is an irreversible damage to ribosomes, and more precisely of their larger subunit [205,206], which becomes unable to bind the elongation factors, with

TABLE VIII

Effects of type 1 and type 2 ribosome-inactivating proteins on protein synthesis by a cell-free system (lysate of rabbit reticulocytes) and by a whole cell system (HeLa ceils) and toxicity to mice

Cell-free Whole cells Mouse Refs. IC5o IC50 (nM) LDso (nM) (mg/kg)

Type 1 RIPs Agrostin 2 0.60 1.0 37 Agrostin 5 0.47 9 200 1.0 37 Agrostin 6 0.57 7 800 1.0 37 Asparin 1 0.27 > 3 300 20 47 Asparin 2 0.15 > 3300 10 47 Barley RIP 2.13 33, 96 Bryodin-L 0.09 > 3 300 > 10 47 Bryodin-R 0.12 12 35 Colocin 1 0.04 > 3300 10.7 47 Colocin 2 0.13 1410 12.6 47 Crotin 2 0.48 > 3300 a Crotin 3 0.20 > 3300 a Curcin 2 0.19 > 3 300 a Curcin 3 0.15 > 3 300 " Curcin 4 0.09 > 3 300 a Dianthin 30 0.30 14 207 Dianthin 32 0.12 44 207 Dodecandrin 0.04 95 Gelonin 0.40 > 3 300 40 92 Hura crepitans RIP 0.10 2000 37 a Luffaculin 0.12 81 Luffin a 1 82 Luffin b 4 82 Lychnin 0.17 > 3300 9.3 47

consequent arrest of protein synthesis. Proteins of either type have similar potency in cell-free systems, but have different toxicity to cells and animals (Table VIII). Type 2 RIPs may be 106-fold more potent than type 1 RIPs in inhibiting protein synthesis by cells in culture. This difference is due to the presence in type 2 RIPs of a B chain with lectin activity which binds to, and mediates the penetration of the A chain into the cell. Toxicity to animals reflects this difference. The mechanism of action of RIPs will be better discussed separat- ing their effects at the molecular and cellular level.

I l iA. Effects on ribosomes: the enzymatic activity

Ribosomes damaged by RIPs do not sustain protein synthesis. Several steps have been considered for the ricin inhibition of 80S ribosomes: GTP-dependent binding of EF-2, EF-2 dependent GTP hydrolysis, EF-1 binding, formation of the initiation complex (for a review see Olsnes and Pihl [2]). These functional damages are a consequence of modifications of rRNA as demonstrated by Endo and co-workers who found that type 1 RIPs and the A chains of type 2 RIPs possess a unique RNA N-glycosidase activity and cleave the N-glycosidic bond of adenine4324 of 28S eukaryotic mammalian rRNA [6,7,208-210]. This base is adjacent

TABLE VIII (continued)

251

Cell-free IC50 (nM)

Whole cells IC50 (nM)

Mouse LDs0 (mg/kg)

Refs.

Maize RIP 0.037 Manutin 1 0.03 Manutin 2 0.04 Mapalmin 0.05 Momorcochin-S 0.12 Momordin 1 0.06 PAP 0.24 PAP II 0.25 PAP-C 0.07 PAP-R 0.05 PAP-S 0.04 PD-S1 0.12 PD-S2 0.06 PD-S3 0.08 Saporin-L1 0.25 Saporin-L2 0.54 Saporin-R1 0.86 Saporin-R2 0.47 Saporin-R3 0.48 Saporin-S5 0.05 Saporin-S6 0.037 Saporin-S8 0.040 Saporin-S9 0.041 Secale cereale RIP 4.00 Trichokirin 0.113 Trichosanthin 0.25 Tritin 2

Type 2 RIPs Abrin c 88

A chain 0.5 Ricin D 84

A chain 0.1 Modeccin 45

A chain 2.3 Viscumin 43.3

A chain b 3.5

Volkensin 84 A chain 0.37

210 1250

> 3300 2870

> 3300

3400 > 3300

3400 2950 1620 2140

> 3300 > 3300

340 170

3200 420 610-2340

5400

1500

> 8.0 24.5

7.4

0.95 1.19 2.6

1.12

4.0

1.7

8.1

63 a

a

47 85 83 28 44 46 47 45 94 94 94 48 48 48 48 48 37,48 37,48 37 37 96 91 87 96, 97

0.0039 0.00056 5,102 > 0.4

0.0011 0.0026 5, 73 > 0.4

0.0003 0.0023 74, 104

0.008 0.0024 65, 66

0.0123 0.00138 50, 105

a See note (a) in Table I. b Reduced toxin.

to the site of cleavage of rRNA by a group of highly specific RNases from fungi: a-sarcin from the mould Aspergillus giganteus [211], restrictocin and mitogillin from Aspergillus restrictus [212-214] that cleave the phosphodiester bond between G4325 and A4326 of rat liver RNA [211] (Fig. 7).

Ribosomal RNA identity elements for ricin recognition and catalysis have been studied by Endo et al. [15]. All cleaved adenine residues are located in a loop and stem sequence of rRNA having a GAGA sequence in the loop, which suggests that RIPs recognise this specific structure (Fig. 7).

Much higher concentrations of ricin were required to depurinate purified 28S rRNA, its 3' fragment [6,216] or a synthetic oligoribonucleotide that mimics

252

the relcwmt region of the 28S rRNA [217] than those required to depurinate RNA in whole ribosomes. The Kc, t of ricin for native (e.g., in intact ribosomes) 28S rRNA is about 105-fold greater than that for naked 28S rRNA [216].

Differences in the sensitivity to RIPs seem to exist among ribosomes from metazoan organisms as is indicated (i) by the high level of resistance to some RIPs of Artemia salina [218] and of Musca domestica ribosomes [219]; and (ii) by inhibition of poly(U) translation systems involving ribosomes from a broad range of species (Refs. 37,43,44,46,66,78,83,85,91,220-224 and references reported below).

The observations reported above indicate that the RiP- r ibosome interaction is more complex than the simple recognition of a primary RNA structure.

A high variability was observed in the spectra of sensitivity to RIPs of several protozoan ribosomes [225 -228].

Ribosomes from fungi are generally sensitive to RIPs. Thus, barley RIP [224] and tritin [4] inhibited ribosomes from Neurospora crassa with high efficiency, and ribosomes from S. cerecisiae were sensitive to both type 1 and type 2 RIPs. It has been demonstrated that ricin depurinates the major (26S) ribosomal RNA of S. cerecisiae at the position corresponding to rat liver 28S A 4.~24 [229].

Plant ribosomes show a variable sensitivity to RIPs from other plant species [35,95,186,230-232]. Conflict- ing results were obtained by studies on the sensitivity of plant translation systems to RIPs produced by autologous plants. It was reported that RIPs did not affect ribosomes from their own plants [186,194,230,232,233]. Subsequent studies revealed that some RIPs act on their own (autologous) ribosomes at concentrations

much higher than those required to inhibit mammalian ribosomes (ricin [231,234]; PAP [235], various RIPs [236]). At least some RIPs are accumulated in the cell wall matrix in leaves (PAP [193]) or in protein bodies in seeds (ricin [189]), hence being separated from ribosomes, which are spared from inactivation in nature. When ribosomes are purified, they come in contact with, and are inactivated by RIPs, so that no further effect can be seen on subsequent addition of RIPs.

However, ribosomes from wheat germ [186,232,233], carnation leaves [232] and bryony leaves [35] (i) trans- late efficiently poly(U) after purification, thus being not inhibited by the RIPs contained in these tissues, (ii) are not inactivated upon addition of their own RIPs (tritin, dianthin and bryodin, respectively) and (iii) are inactivated by RIPs from other plants.

It was observed (i) that the ineffectiveness of RIPs on some metazoan ribosomes is due to lack of some A T P - d e p e n d e n t factor(s) acting on r ibosomes [233,237-239] and (ii) that some ribosomes are only partially inactivated by RIPs, as if there was a subpopu- lation of resistant ribosomes {79,232]. Thus, the variable effects of RIPs on animal and plant ribosomes, including those from their own plants, may depend upon one of these two possibilities, namely the presence or absence of some unknown factor(s) or a condi- tion of resistance to RIPs.

All type 1 RIPs tested inhibited protein synthesis by E. coli ribosomes (Refs. 77,240,241; and Arias, F.J., Barbieri, L., Rojo, M.A., Ferreras, J.M., Girbds, T. and Stirpe, F., unpublished results), although at a much higher RIP concentration, sometimes more than 10 000-fold, than that inhibiting protein synthesis in the rabbit reticulocyte lysate. There are marked differences in the inhibitory activity of various RIPs on

28 S rRNA 26 S rRNA 25 S rRNA 23 S rRNA 16 S rRNA

A C G ~ G AG GFA~G G~A~G A ~A-~ G

U G U G U G A U G A C G

G A G A U A A A C U G A A

u A

C G A U U A 1006(e) 5'3' 1023(e) C G A U

4308(r) 5' 3'4341 (r) 3000(s) 5' 3' 3033(8) G / " "~ C U [ I C G [ 3' 2676 (e)

4259 (m) 4292 (m) 3000 (o) 5' 3' 3033 (o) 2643 (e) 5' 3714 (x) 3747 (x) 4249 (h) 4582 (h)

Fig. 7. Structure of rRNAs substrates for N-glycosidase activity of RIPs. Secondary structures are as compiled by Gutell and Fox [215]. (e) E. co//. (hi Homo sapiens, (m) mouse, (o) Oryza sativa, (r) rat. (s)S. ceret,isiae, (x) X. lael'is.

protein synthesis by a cell-free E. coli system (Arias, F.J., Barbieri, L., Rojo, M.A., Ferreras, J.M., Girb6s, T. and Stirpe, F., unpublished results). These differences exist among RIPs from taxonomically related plants (e.g., crotins, very active, and gelonin, inactive, both from Euphorbiaceae) and even among RIPs from the same plant (e.g., saporins). Accordingly, expression of recombinant RIPs in E. coli systems is often limited because of toxicity to the bacterial cell due to ribosome inactivation with consequent arrest of protein synthesis (see section II-E.2). The RNA N-glycosidase activity of RIPs on E. coli ribosomes was consistent with the inhibitory effect on protein synthesis (Arias, F.J., Bar- bieri, L. Rojo, M.A., Ferreras, J.M., Girb6s, T. and Stirpe, F., unpublished results). The residue affected is the adenine2660 of 23S rRNA [77], homologous to the adenine affected in eukaryotic 28S rRNA. This residue is involved in the binding of the elongation factors EF-Tu and EF-G [242].

Amongst type 2 RIPs, ricin did not affect significantly protein synthesis by bacterial or mitochondrial ribosomes [243-248], and, consistently, ricin does not

253

depurinate rRNA in whole E. coli ribosomes. Thus, genes coding for ricin [145,249] and abrin [130] could be expressed in E. coli.

However, ricin A chain as well as type 1 RIPs have N-glycosidic activity on naked 23S rRNA purified from E. coli: cleavage of the N-glycosidic bond of adenine2660 [216,250] at concentrations similar to those effective on mammalian naked rRNA. RIPs act also on naked 16S rRNA, cleaving the N-glycosidic bond of adenine~014 [216,241], although this site is not depurinated in whole ribosomes.

Although it has been definitely demonstrated that RIPs behave differently on the various ribosomal substrates tested, it is possible that some of these differences could be due to the experimental conditions (source of enzymes, ionic conditions, cofactors, etc.). These were usually chosen for optimal poly(U) or endogenous mRNA translation by each system, rather than for optimal conditions for the enzymatic activity of each single RIP [238]. Carnicelli et al. [119], extend- ing previous results [233,237,238], could divide RIPs into two categories with respect to the need for cofac-

=1-

o l , ~ l

r./3

10,000

1,000

100

10

Gelonin

Momordin I

mPAP-S

m Saporin-S6

m Ricin D

Rabbit Musca Vicia Acanthamoeba Escherichia reticulocytes domestica sativa castellani coli

Fig. 8. Sensitivity spectra to RIPs of translation systems of various sources. The data on rabbit reticulocytes are from the same sources as in Table VIII. For Musca domestica and Acanthamoeba castellani see Refs. 219 and 251, respectively. Ricin was reduced. The data on Vicia satica and E.

coli are from Arias, F.J., Barbieri, L., Rojo, M.A., Ferreras, J.M., Girb6s, T. and Stirpe, F. (unpublished results).

254

tors required to express high levels of activity on Artemia salina ribosomes.

The spectra of sensitivity to RIPs of various endogenous translation systems may be very different (Fig. 8). This pattern suggests a double reciprocal recognition event between RIPs and ribosomes. On the ribosomal side this high specificity is not due to rRNA itself since (i) the affected region is highly conserved throughout the different species and (ii) it has been shown that ricin depurinates at the same rate purified rRNA from rat liver, from E. coli, and even an artificial 35mer. In all cases the concentrations of ricin required to depurinate purified RNA arc much higher than those effective on cukaryotic ribosomes [216,217]. Thus, the high activity on the latter ribosomes may be duc to their ribosomal proteins, which either interact with RIPs in some way or keep rRNA in a conformation which allows the RIPs to act [29,213].

The diversity in the sensitivity patterns of ribosomes to RIPs has to be related to a RIP structure different from that of the catalytic site, since all RIPs have N-glycosidic activity on a sensitive substrate. Consis- tently, Ippoliti et al. [252] demonstrated that saporin-S6 interacts with at least one ribosomal protein of the 60S ribosomal subunit of yeast ribosomes. This interaction is not present in the case of E. coli ribosomes and seems fairly specific.

Recently, it has been shown that some RIPs, at high concentrations, are capable of depurinating ribosomes at more than one site [253]. This phenomenon was not common to all RIPs: only saporins, trichokirin, and PAP-R out of all tested RIPs were capable of multiple site depurination at a concentration equimo[ar to that of ribosomes. This depurination activity was probably unspecific in the case of saporins, since the depurination reaction was linear with time, and at least 30 residues could be removed (saporin-R2). On the other hand, trichokirin removed only two residues and PAP-R only 4: increasing the amount of these RIPs 100-times did not modify the number of adenine residues removed. Multiple depurination produced acid aniline labile sites in the rRNA molecules, as demonstrated by the appearance of new bands on electrophoresis. At least in the case of saporin-S6, multiple site depurination was not species specific: multiple depurination was observed with rat liver, Musca dornestica and E. coli ribosomes.

A model has been proposed for the fine mechanism of action of ricin based on X-ray analysis of interaction with analogues of the substrate and theoretical mod- elling [162]. According to this model, an oxycarbonium ion is stabilized on ribose by GIu 177 in the transition state; Arg ~s° stabilizes anion development on the leaving adenine by protonation (N-3) and activates a trapped molecule of water that is the ultimate nucle- ophilc centre.

III-B. btteraction of ribosome-inacticating proteins with cells

The mechanism of entry into ceils of protein toxins with intracellular sites of action has been the object of many studies in recent years (reviewed in Refs. 254,255). Nevertheless, it is not yet completely under- stood. The interest in toxin internalisation is growing because this mechanism is a key point in a possible therapeutic utilisation of them or of their derivatives, and also because these studies give important insights in the intracellular transport and sorting of physiological ligands. In fact, much evidence currently suggests that toxin entry and routing inside cells are not toxin-specific and mimic pathways of physiological molecules.

Most of the observations concern type 2 RIPs and suggest that more than one mechanism of internalisation must be involved. The ce l l -RIP interaction shows common patterns: (i) protein synthesis inhibition cannot be detected in the cells before a time lag of at least 30 rain [256]; (ii) only few of the toxin molecules taken up by the cell are transferred into the cytosol and reach their target [256]; (iii) a single RIP molecule may be sufficient to induce cell death [257].

III-B.I. Internalisation of type 2 ribosome-inactit,ating proteins

Binding to the cell surface. The first step in cell-RIP interaction consists of binding of RIPs to membrane receptor sites. This process can be accomplished either at physiological temperature or at 0°C. The binding at 0°C can be reversed by addition of galactose, since no uptake occurs at this temperature [258].

The number of binding sites for a given toxin varies from one cell type to another. The number of ricin binding sites was estimated to be 0.2 to 8. 10 7 per cell [256,259-262] A similar number of abrin binding sites (0.2 to 3" 107) was found [256,263,264]. Ceils have a lower number of modeccin binding sites (1 to 2. l0 s sites per cell [262,263]), and still the cytotoxicity of this RIP is generally higher than that of ricin and abrin [265]. Viscumin binding sites per cell have the widest range (0.9 to 40. 10 6) [266]. Ceils resistant to viscumin bind approximately as much toxin as sensitive cells [265]. The latter observation and the modeccin data suggest that total binding capacity for a given toxin is not correlated with the sensitivity of cells to that toxin.

The Scatchard analysis of the abrin binding to CHO cells showed a non-linear plot, suggesting the existence of different surface receptors with dissimilar affinities [267]. Similar results were observed with modeccin [262] and ricin [268-270]. This fact can be relevant to the fate of the toxic molecule, since intracellular routing depends upon the receptor [260]. It is possible that the efficiency of each receptor in delivering toxin to

255

the cytosolic target has an important role in the cytotoxicity. Two-chain plant RIPs have galactose-specific lectin activities. They are potent toxins that are taken up by most cells, because galactose-containing glycoproteins and glycolipids are present on the surface of every cell type. Added galactose and lactose compete with the membrane receptors for the toxin, thus inhibiting the binding to the cell membrane and reducing dramatically the toxicity [271]. A panel of cell lines derived from different human and animal tissues showed a widely different sensitivity to either viscumin or modeccin or abrin, and these toxins had a different toxicity to each of the cell lines tested (Ref. 265 and Table VIII below). Thus, it is possible that different galactose-containing receptors are involved in the uptake of these toxins, readdressing them to a different intracellular routing.

Another recognition process involves the interaction of cell receptors with carbohydrate side chains in the toxins. Both the A and B chains of ricin have mannose-containing oligosaccharide groups, which are mainly responsible for the uptake and toxicity of the toxin in rat liver non-parenchymal (Kupffer and especially sinusoidal cells) [272-274]. Ricin uptake by these cells is scarcely affected by galactose, is inhibited more effectively by mannose or mannan, and is completely abolished in the presence of both galactose and man-

nan [270,275]. This indicates that ricin binds to, and is taken up by, non-parenchymal cells through two different mechanisms: (i) via the galactose-binding sites of its B-chain the toxin binds to galactosyl residues on the cell membrane, whilst (ii) the mannose residues present on both ricin chains are bound by the non- parenchymal receptors for mannose. The higher inhibitory effect of mannose on the cytotoxicity of ricin to non-parenchymal liver cells suggests that the latter mechanism is more efficient in delivering ricin to the cytoplasm. By contrast, ricin enters rat hepatocytes essentially through the galactose-recognition route [272,273].

Internalisation by endocytosis. A considerable amount of evidence suggests that type 2 RIPs do not cross directly the plasma membrane but enter the cytosol by the endocytic pathway. Electron microscopic studies show that a ricin-ferritin conjugate clusters first at the cell surface and appears in endocytic vesicles 60 min later [258]. Endocytosis is a temperature-dependent process. Thus, very little intoxication can be observed if cells are exposed to RIPs at 0°C and then washed with a competing ligand before restoring a physiological temperature [276]. Metabolic inhibitors protect the cells from toxin activity by preventing endocytosis, which is an energy-consuming process [277]. The time lag of 30 rain observed between exposition to ricin and inhibi-

ated ~ ) ~ "-;- "Jl~_pits.,~ E Uncoated ~J ,

pits

~% s S %1 S S A 4 ',

I VV A"

A

Fig. 9. Intracellular routing and transfer to the cytosol of ribosome-inactivating proteins. Dotted arrows indicate the progression of molecules in the endocytic and synthetic/exocytic pathways. Continuous arrows indicate the most likely transfer sites.

25(~

tion of protein synthesis correlates with the time required for the intracytoplasmic visualisation of the toxin linked to horseradish peroxidase [261]. Therefore, endocytosis appears to be a necessary step in the internalisation of RIPs.

Receptor-mediated endocytosis usually occurs by way of clathrin coated pits, specialised depressions on the cell surface. Clathrin is a fibrous protein conferring to the pit the mechanical strength combined with the flexibility needed when a vesicle is pinched off from the membrane [278]. This is the mechanism by which coated vesicles are produced. Then coated vesicles lose their clathrin coat to become smooth-surfaced vesicles, called endosomes. Mannose-containing RIPs can utilise this way of endocytosis, after being bound to mannose receptors localised in coated pits [270].

Ricin cytotoxicity has been observed even when the coated pits pathway was blocked, either by hypotonic shock, followed by K+-depletion [279] or by acidification of the cytosol [280]. Thus, a clathrin-independent endocytosis by uncoated pits is apparently involved in ricin internalisation. These results suggest that ricin, and possibly other type 2 RIPs, can reach the endoso- real compartment by both coated and uncoated pits pathways after binding to galactosyl residues on the cell surface.

Routing to intracellular compartments. The system for the transport and sorting of macromolecules inside the cell consists of the endocytic and the biosynthetic/ secretory pathways, representing the afferent and the efferent arm, respectively, of this system (Fig. 9). The endocytic pathway comprises early and late endosomes and lysosomes. The biosynthet ic/secretory pathway in- cludes the endoplasmic reticulum and the Golgi complex in which post-translational processing and sorting of proteins take place. In both pathways the acidity of the environment increases progressively, allowing for vectorial transport of endocytosed molecules or of newly synthesised proteins [281].

Endocytosis involves cell surface invagination of both coated and non coated pits leading to the formation of endosomal vacuoles. Thus, internalised molecules reach the endosomat compartment which consists of a system of vacuoles and tubulo-vesicular structures, sometimes appearing as multivesicular bodies. Its main functions are believed to be uncoupling, sorting and routing of ligands and receptors [282]. After uncoupling, the default pathway of the ligand is represented by lysosomes, while the receptor is recycled back to the cell membrane. Internalised toxins are directed in part to the lysosomes where they can be degraded. Abrin and ricin very slowly undergo proteolysis. This degradation can be inhibited by NHaC1, which increases the pH in lysosomes. A small part of the endocytosed abrin and ricin may be recycled back to the plasma membrane by diacytosis and released into the medium [276].

The endocytic pathway is functionally connected to the exocytic pathway represented by the Golgi complex and by the constitutive and regulated secretory vesicles and granules. The Golgi complex consists of threc groups of cisternae (cis, medial and trans) and of the trans Golgi network (TGN). In the TGN, newly synthesised proteins are sorted by signals to be routed to the lysosomes or the cell membrane, whereas the lack of targeting informations may route proteins to secretion as the default pathway. The amount of plasma membrane that enters the cell during endocytosis is sup- posed to be balanced by the membranc leaving the TGN to be inserted into the cellular membrane during exocytosis [283]. The connection between the endocytic and the biosynthetic/secretory pathways is also suggested by the fact that, after endocytosis, ricin has been mainly detected in the Golgi complex [261]. Further- more, modeccin is transported from endosomes to another compartment with acidic vesicles, possibly to lysosomes or to the Golgi complex, before entering into the cytosol [284]. The connection between different parts of the vacuolar system occurs by vesicle-mediated transport which is carried out by budding of shuttle vesicles from a membrane organelle, followed by fusion with the target compartment, This transport process requires a fusion protein for membrane fusion [285].

The fate of the toxin after internalisation may depend on the receptor molecule. Abrin and ricin, which bind to a variety of different surface molecules [267,275], may follow various intracellular routes, one or more of which allows for the toxin transfer into the cytosol. As in the case of liver non-parenchymal cells, macrophages take up ricin via both the galactose- terminated receptor and the mannose-containing receptors. The latter seem to be involved in the most efficient mechanism since the toxicity to macrophages is mostly due to ricin taken up by the mannose receptor pathway, although the amount of ricin taken up by macrophages via galactose-containing cell surface structure is higher than that taken up via the mannose receptor [275]. A possible reason is that ricin internalised via galactosyl residues is recycled to the cell surface more rapidly than the toxin internalised via mannose receptors [270].

A progressively decreasing pH is encountered by molecules that migrate in both the inward- and out- ward-directed pathways. This increasing acidity induces conformational changes which can determine the subsequent sorting of the incoming molecules. Many receptor-ligand complexes dissociate at the mildly acidic pH of the endosome. Afterwards the receptor is recycled back to the cell surface while the ligand is usually delivered to lysosomes. CHO cell mutants with defects in vacuolar acidification were resistant to diphtheria and Pseudomonas toxins and to modeccin, while they were hypersensitive to ricin D [286] and to viscumin

[287]. These different sensitivities could reflect altered trafficking of macromolecules in CHO mutants [286].

Intracellular routing may be influenced by the va- lence of the ligand. In fact, polyvalent conjugates of ricin reached vacuolar and tubulo-vesicular portions of the endosomal system only, whilst native ricin also reached Golgi elements [288].

In receptor-mediated endocytosis of physiological ligands, the fate of these ligands and their receptors may involve distinct intracellular routes, reflecting their specific functions. On the contrary, ricin behaves as a non-specific ligand, gaining access to all sub-compartments of the endosomal-lysosomal and Golgi system [260].

Transfer of the active chain into the cytosol. Toxins with intracellular sites of action may reach their cytosolic target from a number of vesicular and tubular compartments. Monensin, chloroquine and NH4CI, which increase the pH of acidic vesicles (reviewed in Ref. 289), protect cells against modeccin [284] and volkensin [50]. This indicates that the active chains of these toxins, like the A-subunit of diphtheria toxin, require low pH for entry and could be transported across the membrane of lysosomes or of the acid part of endosomes or of Golgi apparatus. On the contrary, the same compounds slightly sensitise cells to abrin, ricin, and viscumin, suggesting that the active chains of these toxins can enter the cytosol of many cell types from a neutral compartment [265,276]. NH4C1 protects macrophages against intoxication when ricin is taken up via the mannose receptor route, whereas it sensi- tises cells when ricin is taken up via the galactose binding sites [275]. Thus, it has been suggested that acidity may be required for translocation of ricin into the cytosol after internalisation via the mannose receptor pathway, whereas ricin bound to galactosyl residues enters the cytosol from a neutral compartment [275,290]. A mouse L-cell mutant with defects in both the late endocytic pathway and the secretory pathway was resistant to modeccin, Pseudomonas exotoxin, and ricin [291]. Resistance to the same toxins was also obtained in CHO cells by altering the structure of the Golgi complex. Thus, these toxins apparently need to be delivered to the TGN before they can be released to cytosol, either from the Golgi or from the lysosomes [292].

The transfer to the cytosol of abrin, ricin, modeccin, and viscumin requires the presence of Ca 2+ in the medium. It is possible that a Ca 2+ flux across the membrane is needed for entry, since inhibitors of Ca 2 + uptake protect the cells against the toxins [265,277,293].

Although it is not clear where toxins can enter the cytosol, the differences observed in requirements for entry suggest that the site of transfer across the membrane may be different for the different toxins. The toxic effect of ricin is possibly coupled to the delivery

257

to the Golgi complex [294]. It has been shown also that inhibitors of glycoprotein synthesis and transport (tunicamycin and swainsonine) sensitise cells to abrin and ricin [295]. This observation suggests that toxins compete with newly formed glycoproteins for transport or for processing [295]. Moreover, a hybridoma cell secreting antibodies against ricin has been found to be resistant to the toxic effect of ricin [296]. Ricin therefore must meet intracellular secretory antibodies before reaching the cytosol. Thus, at least in the case of ricin, a considerable amount of evidence points to the Golgi apparatus as the transfer site [283].

Reduction of the interchain disulphide bond greatly decreases the toxicity to cells of type 2 RIPs, by split- ting the molecule into the two subunits. By contrast, the inhibitory activity of two-chain RIPs on protein synthesis by cell-free systems is very low, unless the two chains are splitted. Hence, the disulphide bond must be reduced before toxins are delivered to ribosomes. However, it is unclear whether this process occurs before, during or after translocation of type 2 RIPs into the cytosol. Recent studies on subcellular distribution of ricin suggests that the reduction of its interchain disulphide bridge occurs to a large extent in the cytosol [290]. CHO mutants with abnormally low levels of reduced glutathione showed a decreased ability to cleave the disulphide bond in an anti-cancer drug-containing conjugate [297]. However, the same cells were able to cleave the disulphide bond of diphtheria toxin and ricin, suggesting an easier reduction catalysed by thiol:protein disulphide oxidoreductases [298].

III-B.2. Internalisation of type 1 ribosome-inactit,ating proteins

The entry mechanism of type 1 RIPs is not well known and only hypotheses can be formulated. Glyco- syl residues are present in most of the type 1 RIPs, which could be internalised after binding to carbohydrate receptors on cell membrane. Peritoneal exudate cells (PEC) internalise gelonin by mannose receptors in a saturable manner. Mannan inhibits the specific binding and changes the pattern of gelonin uptake by PEC to the non-saturable pattern observed in cells lacking mannose receptors. In spite of the differences observed in the uptake, the presence of mannan did not affect the toxicity of gelonin to PEC [299].

The lower cytotoxicity of type 1 RIPs, as compared to that of type 2 RIPs, suggests that receptor-mediated internalisation is not very efficient for type 1 RIPs. This notion is also supported by the lack of correlation between the toxicity to cells and the presence of sugar residues in a given RIP. In fact, saporin-S6 which is devoid of sugars (Table III) is more toxic to many cell types than other type 1 RIPs, which are glycoproteins (Ref. 300 and Table IX below). No specific binding sites for saporin-S6 or momordin I were detected on

258

TABLE IX

Cytotoxieity of some type 1 ribosome-inactivating protein~

Cell type ICs, ~ (/zM)

Caryophyllaceae Cucurbitaceae Euphorbiaceae Phytolaccaceae

dianthin 32 saporin-S6 momorcochin-S momordin I gelonin mapalmin PD-S2 PAP-S

HeLa b EUE b > 3.33 TG b 0.56 Mouse macrophages Trophoblasts 0.03 BeWo m 0.04 Jar m 0.48 Human fibroblasts 1.16 3T3 n L929 ~ Ramos o Pries o NB100 q

0.61 c

0.04

0.05 0.12 0.26 0.63

2.87 d > 3.33 * > 3.33 ~ > 3.33 ~ 1.62 " > 3.33 i > 3.33 ~ > 3.33 k ;~ 3.33 ~

2.04 d 0.40 i 0.88 ~ 1.68 ~ 0.85 I 0.03 ~ 11.115 k 0./12 ~'

0.28 d 0.21 I 0.26 ~ 0.46 ~ I).118 I 0.27 d 0.06 i 0.07 i 0.08 g 11.04 h 0.10 ]

> 3.33 d 1.11 I 0.13 I 1.64 ~ I).76 I 1.09 d 2.86 I 3.08 I 0.61 g 3.09 t

> 3.30 h > 3.33 ~ > 3.33 k > 3.311 ~

1.30 p > 3.33 p

0.006 c 0.32 a 0.1/3 ~ 0.002 I,

:' Concentration inhibiting by 50% cell protein synthesis, b Carcinoma-derived cell lines, c [48], d [85], c [304], f [92], g [47], t~ [94], i [45], i[5], k [1], I [300]. m Chorioncarcinoma cell lines. " Fibroblastic cell line. o Burkitt's lymphoma cell lines, p [305]. q Human neuroblastoma-derived cell line.

B e W o o r H e L a c e i l s s u r f a c e , n o r w e r e d i f f e r e n c e s s e e n

in t h e b i n d i n g o f m o m o r d i n I a n d s a p o r i n - S 6 t o h i g h l y

a n d s c a r c e l y s e n s i t i v e c e l l s [300]. T h i s s u g g e s t s t h a t a

r e c e p t o r - i n d e p e n d e n t m e c h a n i s m o f e n t r y i n t o c e l l s

c o u l d b e p r e s e n t f o r n o n - g l y c o s y l a t e d s i n g l e - c h a i n R I P s

[301].

R I P s c a n b e t a k e n u p by c e l l s t h r o u g h t w o m e c h a -

n i s m s : t h e e n d o c y t o s i s d e p e n d e n t o n t h e b i n d i n g o f

R I P s to e i t h e r t h e g a l a c t o s y l r e s i d u e s ( t y p e 2 R I P s ) o r

t h e m a n n o s e r e c e p t o r s o n t h e ce l l m e m b r a n e , a n d t h e

f l u i d - p h a s e e n d o c y t o s i s w h i c h a l l o w s t h e i n t e r n a l i s a -

t i o n o f m o l e c u l e s w i t h o u t a r e c e p t o r - m e d i a t e d m e c h a -

n i s m [302]. T h e l a t t e r t y p e o f e n t r y t a k e s p l a c e b o t h in

c o a t e d a n d u n c o a t e d p i t s , g i v i n g r i s e t o a n o n s e l e c t i v e

u p t a k e w h i c h is l e s s e f f i c i e n t t h a n t h a t u t i l i s i n g s p e c i f i c

s u r f a c e b i n d i n g s i t e s . G e l o n i n , a n d p o s s i b l y o t h e r t y p e

1 R I P s , c o u l d b e i n t e r n a l i s e d by t h e f l u i d - p h a s e e n d o -

c y t o s i s i n t o c e l l s l a c k i n g t h e r e c e p t o r s f o r t h e g lycosy l

r e s i d u e s p r e s e n t in t h e R I P m o l e c u l e [299].

N o c o r r e l a t i o n w a s o b s e r v e d b e t w e e n B e W o ( h i g h l y

s e n s i t i v e ) o r H e L a ( l e s s s e n s i t i v e ) c e l l s w i t h r e s p e c t t o

u p t a k e o f a n d s e n s i t i v i t y t o s a p o r i n - S 6 o r m o m o r d i n I

[300], s u g g e s t i n g t h a t d i f f e r e n c e s in t h e i n t e r n a l i s a t i o n

a r e n o t n e c e s s a r i l y t h e r e a s o n f o r t h e d i f f e r e n t r a t e s o f

c y t o t o x i c i t y o f t y p e 1 R I P s . N H n C I s l i g h t l y r e d u c e d t h e

c y t o t o x i c i t y o f g e l o n i n [299,303] , w h e r e a s c h l o r o q u i n e

TABLE X

Cytotoxicity of type 2 ribosome-inactivating proteins

The cytotoxic effects of RIPs on various cell types here reported were determined by the inhibition of protein synthesis after a comparable (18 h) treatment. Data may differ from those reported in Table VIII because culture conditions were not strictly comparable.

Cell line iC50 a (M)

Ricin Abrin Modeccin Viscumin Volkensin

t teLa b 1.1" 10-12c 3.9" 10 12 c 2.8" 10-12c 1.7' 10 -9 c 3.5" 10 13 d

EUE b 1.0"10 I~. FM f 4.7'10 llc 8.8"10 12c 5.6"11/ IIIc

Rat macrophages 4.0.10 13 Mouse macrophages 6.0.10 12~ 1.0 10 1i c L929 " 9.0-10 1~ 3T3 h 4.7'10 13c 7.3"10 14c 6.8'10 El¢ L-cells h 4.0" 10 ~4d 501.1h 4.7.10 - t2c 4 . 2 ' 1 0 t 3 c 3.0"10 1to Vero i 3.2.10 -~2c 5.7.10 - t3c 2.8.10 lOc 4.0-10 13'~ NRK i 6.0.10 13c 2.2-10 13c 1.7.10-10c BHK i 1.0"10 sc 2.4" 10 lla Ramos J 2.8.10- 10 k Priess J 9.0' 10-10 k

a Concentration inhibiting by 50% cell protein synthesis, b Carcinoma-derived cell lines, c [265]. a [50]. ° [304]. f Human melanoma cell line. g [275]. n Fibroblastic cell lines, i Kidney-derived cell lines. J Burkitt's lymphoma cell lines, k [305].

259

[299] and monensin [303] potentiated its toxicity. These results suggest that the different cytotoxicity of various type 1 RIPs could be ascribed to differences in their intracellular trafficking.

III-B.3. Effects on mammalian cells RIPs cytotoxicity has been tested by many investiga-

tors on a wide panel of cells, each time with a different purpose, often using non-comparable experimental conditions. In spite of the differences in experimental conditions, it is clear that single-chain type 1 RIPs are much less cytotoxic than type 2 RIPs: IC50 (concentration inhibiting 50% of protein synthesis) in the /zM range for type 1 versus pM range for type 2 RIPs (reviewed in Ref. 5; see also Table VIII).

The cytotoxic effects of RIPs on various cell types, determined by the inhibition of protein synthesis after a comparable (18 h) treatment, are summarised in Tables IX (type 1 RIPs) and X (type 2 RIPs). Consid- erable variations were observed in the toxicity of each RIP to ceils. Saporin-S6, modeccin and volkensin were the type 1 and type 2 RIPs, respectively, generally most toxic to cells. Also, marked differences were observed in the sensitivity of each cell type to different RIPs. Macrophages, trophoblasts, the chorioncarcinoma-derived BeWo cell line, and the human neuroblastoma- derived NB100 cell line seem to be the most sensitive cell types to single-chain RIPs, whereas HeLa cells, EUE cells, human embryonic fibroblasts, L929 mouse fibroblasts and Burkitt's lymphoma-derived cell lines seem to be more resistant. A wider range of cell sensitivity was observed with type 2 RIPs, the highest effect being observed on rat macrophages and fibroblast-derived cell lines.

The lower toxicity of type 1 RIPs is due to the lack of a lectin B chain with consequent poor entry into the cytoplasm. This is confirmed by the high toxicity which can be conferred to single chain RIPs by facilitating their penetration into cells (i) if cells are rendered more permeable to macromolecules, (ii) by including RIPs into carriers capable of delivering them inside cells, or (iii) by linking them to molecules capable of binding to cell membranes.

Permeabilisation of the cells to RIPs can be achieved by physical means, such as short electrical pulses [306], or by biological treatments such as the action of com- plement [307] or viral infection [308]. As a matter of fact, the antiviral activity of type 1 RIPs was attributed to an easier entry of these proteins into the cytoplasm of virus-infected cells, where they damage ribosomes, thus killing the cells and arresting viral reproduction.

Carriers which can transport type 1 RIPs into cells include liposomes [309], erythrocyte ghosts [310] and viral envelopes [311], which all can be fused with cells and deliver their content into the cytoplasm.

The first conjugate of a RIP with a cell-binding molecule was obtained by linking gelonin to concanavalin A to form a complex which was more toxic to cells than the free RIP [92]. The same result was obtained by linking a RIP to the B chain of ricin [312], but the best and more numerous examples of this kind are the immunotoxins constructed by linking a RIP to an antibody reacting with an antigen on the cell surface (see section IV-A).

III-C Effects on laboratory animals and man

III-C.1. Toxicity and lesions The toxicity of plant materials containing type 2

RIPs has long been known. The signs and symptoms induced in about 700 peo-

ple poisoned with ricin have been described [313]. Pathology included bleeding in the serous membranes, haemorrhages in the stomach and intestine, degenera- tive changes in the heart, liver and kidney, and damage in the spleen and lymph nodes.

No report is known of fatal poisoning in humans by type 1 RIPs, although signs of toxicity (fever, rashes) were noticed when trichosanthin was given to induce abortion [314] or to AIDS patients (see section V-B.3 and Refs. 315,316).

All RIPs are strongly immunogenic [317], and many type 1 RIPs and at least ricin among type 2 RIPs are potent allergens (Refs. 318,319; and Barbieri, L., Bolognesi, A. and Stirpe, F., unpublished observations). Ricin induces the formation of IgE not only against ricin itself [318], but also against other antigens administered at the same time [319].

The LDso values of RIPs injected parenterally to mice are reported in Table VIII. The toxicity of type 2 RIPs is much greater than that of type 1 RIPs, due to the presence of the lectin subunit which allows an easy entrance into the cells of various vital organs. The toxicity of type 1 RIPs is increased if their molecule is rendered larger, for instance by linkage to an antibody [320,321] or by polymerisation [322,323].

Much work has been done on the determination of toxic effects of type 2 RIPs in various animals (reviewed in Refs. 2,10). The lethal doses of these proteins may vary greatly among different species: a difference of 20-fold was observed between the LDs0 values of volkensin for the two rodents mouse and rat [50].

The in vivo effects of abrin, modeccin and ricin have been studied in the rat in a strictly comparable way [324-326]. The pathology of animals given lethal doses of these toxins is not identical: only ricin, modeccin and volkensin cause gross liver changes and often asci- tis. A feature of ricin poisoning is the inflammation of intestine and lymphoid organs, in agreement with previous observations [327]. It was from the similarities in

260

histological damages that ricin was suspected to be the poison used in an assassination case [328].

In the liver of rats poisoned with ricin the earliest changes were observed in the cytoplasm and nuclei of non-parenchymal (Kupffer and sinusoidal) cells, which were progressively damaged until they became necrotic [324]. Only after the development of these lesions, degeneration and necrosis of the hepatocytes appeared, probably as a consequence of the destruction of the sinusoidal lining. Necrosis of the macrophage- rich red pulp of the spleen was observed in these animals. That non-parenchymal cells are the first target of ricin was confirmed [329], and the whole pattern is consistent with the preferential uptake of ricin by these cells [330] and with the higher toxicity of ricin to macrophages [275]. More recently, apoptosis was described in lymphoid tissues and intestine of abrin- and ricin-poisoned rats [331]. Apoptotic changes were also observed in tissue cultures of cancer cells treated with ricin [332].

In animals poisoned with modeccin the most severe lesions were observed in the liver [326]. Changes in the rough endoplasmic reticulum and swelling of mito- chondria were well marked in the hepatocytes at 6 h of poisoning, and become more severe with time. Volkensin too brought about severe liver necrosis in the rat (Barbieri, L. and Stirpe, F., unpublished observations).

Abrin did not affect liver, neither hepatocytes nor sinusoidal cells, but brought about severe necrosis of the acinar pancreatic cells [325] and apoptotic changes in the lymphoid tissues and the intestine of the rat [3311.

The effects of these toxins on protein synthesis in vivo paralleled the ultrastructural lesions. Thus, protein synthesis was impaired in the spleen of rats poisoned with ricin [324], in the liver of rats poisoned with modeccin [326], and in the pancreas of rats poisoned with abrin [325]. It was ascertained that in rats poisoned with modeccin, the impairment of protein synthesis was accompanied by damage of the 60S ribosomal subunit. Thus, modeccin (and presumably the other toxins) damages ribosomes in vivo in the same way as it does in vitro. Since the A chains of these toxins all have the same N-glycosidase activity, the different lesions observed in vivo are probably due to different penetration of the toxins into the cells, and thus to diversity of the respective B chains.

The lesions caused by type 2 RIPs are quite different from those observed in animals killed with other inhibitors of protein synthesis. Also, it is noteworthy that no lesions accounting for death could be found in rats killed with abrin [325] or viscumin (Stirpe, F., unpublished results), which suggests that these toxins may bring about some kind of undetected damage, for instance in the nervous system. This notion is indirectly

supported by the observations on the axonal transport of ricin and related toxins (see section III-C.2) and on the extreme toxicity of ricin injected intraventricularly [3331.

Recently, it was observed that ricin [334,335] and viscumin [336,337] induce the release of tumour necrosis factor and interleukins. The contribution of these cytokines to the lesions caused by the toxins is still unknown: they could probably account for the fever observed in ricin-poisoned animals [338] and in patients receiving viscumin-containing preparations [336].

A detailed study on the toxicity of ricin A chain administered i.v. at non lethal doses was performed in rats and monkeys [126]. Necropsies on day 9 after 9 m g / k g of ricin A chain revealed centrilobular necrosis of the liver, some necrosis of the proximal tubules of the kidneys and lesions of the serous acini of salivary glands and pancreas. Apathy, piloerection, decrease in erythrocytes, polymorphonuclear neutrophils and platelet counts were observed, whilst lymphocytosis was present. Albumin and haemoglobin were decreased, cholesterol, triacylglycerols, urea and serum hepatic enzymes were increased. 30 days after treatment most parameters returned to normal ranges.

In a comparative study of the lesions caused in mice by lethal doses of type 1 RIPs [323] it was observed that the organs constantly involved were liver, kidney and spleen. The lesions were essentially cell necrosis, sometimes accompanied by fatty change.

No significant permanent lesions were observed after 14 days in mice treated with various type 1 RIPs at non lethal doses (Barbieri, L., Battelli, M.G. and Stirpe, F., unpublished observations).

I11-C.2. Axonal transport and effects on the nervous system

Ricin and related toxins injected into peripheral nerves can be retrogradly transported along the axon to the neurones, which are killed (suicide transport) [339-346]. Ricin injected intraneurally prevents nerve fibre regeneration [347,348] and formation of neuro- mas after nerve transection [347].

Axonal retrograde transport was observed with all toxins tested [349,350]. However, if the toxins are injected in the central nervous system, only modeccin and volkensin are transported to other areas through the projecting neurones, whereas ricin and abrin are not [341-354]. Thus, modeccin and volkensin are useful tools for the study of neuronal interconnections in the central nervous system.

Moreover, some experiments showed that ricin injected outside nerves into several tissues (anterior eye chamber [355], supramandibular gland [339], dental pulp [346], superior lip [356], lateral rectus muscle [357]) may reach the autonomic ganglia relevant to the injected area. This indicates that ricin injected into

261

tissues is taken up by nerves, presumably at their ends, through a still unknown mechanism.

In other experiments saporin-S6 was linked to the OX7 antibody, which recognises the Thyl.1 antigen, expressed on rat neurones [358]. This conjugate injected intraaxonally damaged neurones, whereas free saporin-S6 did not. The same conjugate was used to localise 5-hydroxytryptamine receptors in the cingulate cortex [359].

These experiments suggest that the transport of toxins is a property associated with their B-chains (or other vector molecules). If this is so, then B-chains might be used to target drugs, growth factors or other molecules to the central nervous system.

The whole subject of neural lesioning induced by RIPs and their antibody-conjugates has been reviewed recently [360,361].

TABLE XI

Abortifacient activity of ribosome-inactivating proteins h~ mice

Activity is indicated as the number of aborted fetuses as compared to total number of implant sites.

RIP Dose Response Refs. (mg/kg) (%)

Gelonin 2 33 378 Luffaculin 4 83 81 Momordin I 2 83 378 Momordin II 2 83 378 PAP-S 2 100 378 Saporin-S6 2 100 378 Saporin-S8 2 100 378 Saporin-S9 2 67 378 Trichosanthin 0.6 100 87 a-Trichosanthin 2.5 100 89

III-C.3. Immunosuppressive activity The high toxicity of type 1 RIPs to macrophages

(Table IX) led to studies of whether they could have immunosuppressive activity. Indeed, momordin I, PAP- S and gelonin suppressed antibody formation in response to T-dependent antigens, delayed rejection of skin allografts, and decreased resistance to allogeneic tumour grafts, provided they were given before, and not together with, or after, the antigen [304,362]. Simi- lar results have been obtained by Leung et al. [363,364] with momordins and trichosanthin. These observations, together with the low toxicity to lymphocytes [1,304,362,365], indicate that RIPs must exert their immunosuppressive activity by interfering with an early step in the immune response, which is consistent with the possibility of an action on macrophages. This is suggested also by the inhibition of MHC class I-re- stricted antigen presentation by gelonin [366].

The property of preventing the mounting of an immune response to a given antigen, without altering the existing immunity against other antigens makes type 1 RIPs unique immunosuppressive agents.

III-C.4. Abortifacient activity Herbal remedies prepared from the root tubers of

Trichosanthes kirilowii, a Cucurbitacea, have been used for centuries in Chinese folk medicine to induce mid- term abortion [367]. A vast literature, mainly from Chinese scientists, exists on the subject [87,368-376]. The active principle responsible for the abortifacient activity has been purified and found to be a protein, trichosanthin, which induces abortion in mice, rabbits and monkeys, but not in rats and hamsters [369], and was reported to have adjuvant therapeutic effect on chorioncarcinoma (review in Ref. 377). Trichosanthin was found to be a RIP [87] and all RIPs tested so far had a similar abortifacient activity in mice [378] (Table XI).

The effects of trichosanthin on pregnancy have been studied in detail [368]: (i) it causes selective necrosis of the syncytiotrophoblasts of placental villi; (ii) the subsequent clot formation in the local circulation induces large areas of infarction; (iii) these changes are accompanied by impairment of functional activities: fall in human chorionic gonadotropin [379] and steroid hormones levels, impairment in the metabolic exchanges, increase in the synthesis of prostaglandins with consequent induction of abortion. These effects are probably due to a high toxicity of RIPs for chorioncarcinoma cells and trophoblasts (Refs. 300,377,380 and Table IX) which is consistent with the high pinocytotic activity of these cells. Presumably other RIPs [84,378,381-385] and plant extracts [374,376,386] induce abortion through the same mechanism. Thus, RIPs could be used to induce abortion in veterinary if not in human medicine.

III-D. Effects on other organisms

The effects of RIPs on plant ceils have been scarcely studied and the results reported in the literature give no clear clues. The growth of carrot cells was inhibited by PAP but was stimulated by ricin D and to a lesser extent by gelonin [232]. Ricin D and gelonin stimulated also the growth of rice cells [232].

The high susceptibility of some protozoan ribosomes to selected RIPs (see Refs. 225-228 and Fig. 8) suggests that RIPs may affect protozoan growth. Unfortu- nately, the only experiments with whole Acanthamoeba castellanii described in the literature concern ricin and PAP which appear to be fairly inactive on the ribosomes of this species [387], although data on PAP are conflicting [228].

Effects on fungi were discussed in detail by Roberts and Selitrennikoff [4]. Fungal ribosomes seem to be a sensitive substrate for RIP (see section Ill-A) and

262

indeed RIPs from barley, rye, wheat and corn blocked the growth of protoplasts of Neurospora crassa [4]. However, of 15 wild type species of fungi screened 12 were not inhibited by barley RIP, two were inhibited weakly, and only one, Trichoderma reesei, was strongly inhibited (IC~ 0 100 nM) [224]. These unespected results could be explained when it was found that the RIP from barley needs the aid of two enzymes ((1-3)- a-glucanase and chitinase) to enter the fungal cell [ 111]. The three proteins together were highly effective in inhibiting the growth of Trichoderma reesei and Fusarium sporotrichoides (barley pathogens), Rhizocto- nia solani (potato pathogen) and Botrytis cinerea (pea pathogen) [111].

Crude preparations of ricin were ineffective on Musca domestica or Locusta migratoria [388-390] as well as non toxic to the codling moth larva, Larpocapsa lasperypsia [391]. More recently, cells from Trichoplusia ni and Spodoptera frugiperda were found to be resistant to ricin, although protein synthesis was inhibited in their cell-free extracts and their ribosomes were depurinated [392]. Both ricin and saporin-S6 were highly toxic to two Coleoptera, Callosobruchus macula- tus and Anthonomus grandis when added to the diet at 0.001-0.0001% of dry weight [393]. In contrast, the same RIPs were largely ineffective against the two Lepidoptera, Spodoptera littoralis and Heliotis trirescens [393] as was ricin to Culex pipiens [394].

III-E. Anti~iral actiuity

III-E. 1. Plant c, iruses It has been known since 1925 that extracts from the

leaves of several plants prevented transmission of the infection to other plants when mixed to suspensions of tobacco mosaic virus [395]. These observations led to the partial isolation of the antiviral principles of P. americana [396-398] and of Dianthus caryophyllus [399,400]. Following the purification of the proteins responsible for the antiviral activity (PAP [78] and dianthins [43], respectively) it was demonstrated that the protein synthesis inhibitory activity present in the plant extracts was due to the same molecules. Subse- quently, it was ascertained that all RIPs tested, including type 2 toxins, prevented infection of Nicotiana leaves by tobacco mosaic virus (Table XII) although at very different concentrations.

Very early investigations showed that plant extracts with antiviral activity did not prevent the infection of autologous plants, but were effective only on heterologous plants [403]. This led to the conclusion that the antiviral principles acted on the host plant rather than on the viruses. Later studies are all consistent with this view [404-406]. Any direct effect of RIPs on the viruses seems excluded by a number of observations: (i) cu- cumber mosaic virus and influenza virus mixed with a

TABLE XII

l~[[2,cts of ribosorne-inactit,ating proteins on t'ira[ m]k, ctions ~)[ Nico- tiana glutinosa plants by tobacco mosaic cirus

RIP Inhibition of viral Rcfs. (nM) multiplication:

No. of lesions (% of controls)

Type 1 RIPs Agrostin 2 1700 42 37 Agrostin 5 1700 1 37 Agrostin 6 1700 3 37 Bryodin-R 1000 18 35 Dianthin 30 34 9 78 Dianthin 32 30 16 78 Gelonin 1800 49 401 MAP ~' 27 50 4()2 Momordin I 1 600 58 401 PAP I 11 44 PAP-II 1 27 44 Saporin-S6 1 700 I) 37 Saporin-S9 1 700 0 37

Type 2 RIPs Abrin c 770 31 401 Modeccin 790 42 401 Ricin D 770 15 4(11

'~ Tests on Nicotiana tabacum cultivar Xanthi nc.

partially purified extract of P. americana regained infectivity after removal of the extract [398]; (ii) similar results were obtained with animal viruses [407,408]; (iii) as far as it is known the structure recognised by the RIPs on the ribosomal RNA is not present in viral RNAs.

With the purification of PAP and the discovery of its effect on ribosomes, the hypothesis was formulated that protein synthesis inhibition was the mechanism through which RIPs exerted their antiviral activity [43]. The antiviral action of RIPs could be due to inactivation of ribosomes of the infected plant cells. It is known that virus infection modifies the permeability selectiveness of cell membrane, thus allowing the access to the cytoplasm of molecules normally excluded [409,410]. RIPs could then enter virus infected cells and, once inside, inactivate the ribosomes and block viral replication [308]. This may account for the reduced number of lesions in virus infected plants treated with RIPs or RIP containing plant extracts. It was proposed that PAP, which is localised extracellularly in pokeweed cells, could enter into cells altered by viral infection, then inactivating ribosomes and arresting viral replication [193]. The same mechanism may oper- ate in the case of ricin, which is also separated from Ricinus ribosomes [189], and presumably of other RIPs active on ribosomes from their own plants [235,411]. This would support an antiviral role of RIPs in plants, although (i) some plant ribosomes seem resistant to their own RIPs (see section III-A), and (ii) some RIP-

containing plant extracts do not inhibit virus infection in autologous plants [403] as mentioned above.

III-E.2. Animal viruses A partially purified preparation of PAP inhibited

viral replication in monkey kidney cells infected with influenza virus [398]; the yield of influenza virus in embryonated hen eggs was also reduced by the same preparation. Other studies confirmed this inhibitory effect by various RIPs both on RNA viruses (polio virus) [407,408], and on DNA viruses (Herpes simplex virus) [45,356,408,412], suggesting that this is a general property of this class of plant proteins. The observed inhibitory effect was accompanied by inhibition of protein synthesis in the treated cells at lower concentrations of RIPs than in non infected control cell cultures. Furthermore, the RIPs used did not show any effect on the infectivity of the virus strains used or on their

I STEP

T

263

capacity to bind to cells [407]. These results indicate that the mechanism of antiviral activity of RIPs in animal systems is probably the same as that suggested for plant systems: (i) increased permeability of, and easier entry of RIPs into, virus infected ceils, (ii) block of protein synthesis, and (iii) reduced viral multiplication.

This mechanism, though, may not be the only one since the effect of trichosanthin on HIV replication has been reported to be separated from protein synthesis inhibition [413]. It has been reported that the com- pound GLQ223, a highly purified formulated preparation of trichosanthin, has a potent inhibitory activity against human immunodeficiency virus in vitro [413,414]. Substantial inhibition of viral replication was obtained at trichosanthin concentrations that did not affect uninfected cultures run in parallel. HIV replication was blocked for at least five days when blood

Primary antibody

Immunotoxin I T containing secondary

antibody

Fig. 10. Treatment with indirect immunotoxins. I step: treatment of cells with the antigen specific antibody of species A. II step: treatment of reacted cells with an immunotoxin prepared with a anti-species A immunoglobulin antibody produced in species B.

264

samples from HIV-infected patients were treated with a single 3 h exposure to trichosanthin. Inhibition of HIV replication by other RIPs was also reported [91,207,415,416].

The mechanism of the action against this specific infection appears still unclear since the mechanism proposed for the antiviral activity in other systems seems not compatible with the results obtained with HIV [413]. Treatment of acutely infected T lym- phoblastoid cells resulted in a decrease in the levels of viral proteins at concentrations of drug not affecting ceil-specific protein or DNA synthesis [91]. Further- more, treated cells showed a selective decrease in the amount of viral RNA, whilst the level for actin RNA (cell-specific) remained unchanged [413].

Phase I and 1 / I I studies of trichosanthin in AIDS patients have been performed [315,316]. Some decrease of p24 antigen and rise of CD4 counts were reported. Side effects included headache, myalgia, fever, fatigue and rash and, in some patients, neurological adverse events. The latter were attributed to factors toxic to brain cells released in vitro by trichosanthin-treated macrophages, more abundantly if macrophages were infected by HIV [417].

IV. Targeting of ribosome-inactivating proteins

In an attempt to selectively kill a given type of harmful or undesired cells, drugs or toxic molecules have been linked to carriers that specifically recognise target cells. A variety of molecules have been used to construct these 'magic bullets'. Antibodies, usually monoclonal, have been the most frequently used carriers, but also lectins, hormones, growth factors, receptors, receptor-binding substances, antigens have been employed. Drugs, enzymes, radioisotopes, or toxins, RIPs among them, have been used as active, 'killer' moieties. The whole subject has been reviewed in two books [418,419], and the use of RIPs for this purpose in several reviews, the most recent one by Ramakrishnan et al. [420].

IV-A. Immunotoxins

Ribosome-inactivating proteins can be linked to antibodies to form 'immunotoxins', specifically toxic to the cells target of the antibodies used. There are several recent reviews on this matter [420-422] and only a few general principles will be discussed here.

lntermolecular bond. Antibodies are usually linked to the toxic moiety by a disulphide bond, less frequently by a thioether bond [423]. Immunotoxins do not inhibit protein synthesis in a cell-free system unless this bond is broken by reduction. This means that the RIP active site is probably hindered in some way by the antibody molecule. It is not known when, and through

which mechanism, the bond is broken in vivo. As a matter of fact, it is not known whether the whole immunotoxin molecule or only the RIP moiety gets access to the cytosol of the susceptible cells.

hldirect immunotoxins. To simplify the in vitro use of immunotoxins an 'indirect' two steps procedure has been developed (Fig. 10). The target cells are first incubated with an anti-cell primary antibody. The cells are then incubated with an immunotoxin containing antibodies against the immunoglobulins of the same species as the primary antibody [424-429]. This approach is very useful because it allows for the use of a variety of different primary antibodies in conjunction with only one immunotoxin. This procedure reduces drastically the biochemical preparative work and the controls for immunotoxin activity and unspecific toxicity.

Chimeric genes. Chemically constructed immunotoxins are often heterogeneous because of random binding of ligands. A way to circumvent this problem is to construct chimeric genes for fusion proteins (reviewed by Ref. 430). A functional recombinant expression plasmid encoding a staphylococcal protein A-ricin A chain fusion protein has been obtained [431]. This new protein retains the binding properties of protein A and the ribosome-inactivating activity of ricin A chain.

IV-A.1. Immunotoxins with type 2 ribosome-inactiL,ating proteins

Immunotoxins have been prepared with both type 1 and type 2 RIPs. The toxin most frequently employed for this purpose is ricin, initially used as whole toxin. However, conjugates prepared in this way have a poor specificity, since the B chain of ricin could bind to, and consequently enter in, virtually all cells unless the experiments were performed in the presence of lactose. Better results are obtained by using the A chain separated from the B chain. In this case the specificity is that of the antibody used, although the entry into cells is a somewhat less efficient process than that observed with the whole toxin. Presumably, this phenomenon is due to lack of the B-chain which, besides binding to cells, seems to facilitate the entry of the active subunit into the cytoplasm [432]. Several experimental designs have been envisaged to overcome this disadvantage: (i) the cytotoxicity of A chain-containing immunotoxins is potentiated by free [433,434] or antibody-conjugated B chain [435]; (ii) immunotoxin are prepared with whole ricin, the B chain binding site of which is modified or 'blocked', in an attempt to prevent the non-specific binding, while retaining the property of facilitating the entry into cells of the conjugate [436]; (iii) the cytotoxicity of A chain conjugates in vitro is enhanced by the use of monensin or lysosomotropic agents [437-443].

A major disadvantage of the A chain of ricin (as of

TABLE XIII

limmunotoxins constructed with ribosome-inactivating proteins type I

RIP Refs.

Barley RIP 448-450 Bryodin 323, 451,452 Dianthin 32 447 Gelonin a 323, 447-471 Luffin 472 Momordin I 323, 447, 451,452, 464, 465, 473 Momorcochin-S 85, 323, 447 PAP(S) h 323, 426, 442, 447, 457, 474-485 PD-S2 94 Saporin-S6 301,320, 321,323, 359, 429, 447, 451,455, 486-506 Trichokirin 91, 323, 477 Trichosanthin 507

a Further references in Ref. 422; b Further references in Ref. 508.

any other two-chain toxin) is the complex and danger- ous preparation, which involves the previous preparation of the toxin. This can be overcome with the use of recombinant A-chains, and, hopefully, in the future the whole immunotoxin molecule will be produced by recombinant technique.

Immunotoxins prepared with the A chains of abrin [444,445] and viscumin [446,447] are few, probably because these toxins are less available and have been less extensively studied than ricin.

IV-A.2. Immunotoxins with type 1 ribosome-inactit~ating proteins

Immunotoxins have been prepared also with several single-chain type 1 RIPs linked to antibodies, usually

265

through a disulphide bond (see Table XIII and reviews in Refs. 420-422). Since none of these RIPs has free and accessible sulphydryl groups, an artificial one must be inserted by the use of appropriate heterobifunc- tional reagents, N-succinimidyl-3-(2-pyridyldithio)pro- pionate (SPDP) [509] being the one most frequently used. This reagent, however, brings about a variable loss of RIPs' enzymatic activity, a loss which in the case of gelonin was rather high [323,447,510], thus limiting the use of this RIP for the preparation of immunotoxins. Better results are often obtained with 2-iminothio- lane or other coupling reagents [91,471,511-514].

In the preparation of immunotoxins, type 1 RIPs seem to offer some advantages over the toxin A-chains, in that they are stable, easy and safe to prepare. The wide variety of type 1 RIPs available may prove useful to overcome the immune reaction in the case of re- peated administration. Moreover, some immunotoxins containing saporin-S6 seem more potent than immunotoxins prepared with the same antibody and the A chain of ricin [491], even when the inhibitory activity on cell-free protein synthesis is comparable [506]. It is possible that, as compared with ricin A chain immunotoxins, saporin immunotoxins are taken up more easily by target cells, or follow a different intracellular route in which they escape inactivation. The latter notion is supported by the different effects of lysosomotropic amines or carboxylic ionophores which enhance the cytotoxic activity of immunotoxins containing ricin A chain [437-439,441,443] or PAP [440], but not that of immunotoxins containing other single-chain RIPs (gelonin [515], saporin-S6 [491], trichokirin [91]). These observations suggest that the entry into cells a n d / o r

RIP

Bifunctional antibody

Fig. 11. Treatment with RIPs and bispecific antibodies.

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the intracellular fate of immunotoxins may be different, and conditioned by the type of RIP moiety they contain. Consistent with this notion an active immunotoxin was obtained with saporin-S6 linked to the non- modulating Campath 1 antibody [492].

Unexpectedly, immunotoxins containing type 1 RIPs have a higher toxicity to animals than free RIPs, although differences were observed in various laboratories [321,323,491]. At least in part, the higher toxicity of immunotoxins is due to the larger size of these molecules which prevents their excretion through the kidneys.

IV-B. Targeting with bifunctional antibodies

A possible means to reduce some difficulties encountered with immunotoxins could be the delivery of RIPs to cells by the use of bifunctional antibodies [516] (Fig. 11). These are hybrid antibodies with one binding site for an antigen on target cells and a second binding

site for a RIP. In this way (i) the toxicity should bc reduced, (ii) if administered separately, the antibody and the RIP, being smaller molecules, could have a better access to the target, and (iii) there is no need of a linker and consequently all RIPs would retain full activity and those which are inactivated by linking reagents could be used. Ricin A chain [517] and saporin-S6 [518] have been targeted with bispecific antibodies. Preliminary clinical trials showed the effec- tiveness of an anti-CD19-saporin-S6 in chronic lymphocytic leukemia and the absence of toxic side effects [504].

IV-G\ Conjugates with non-immune carriers

Ribosome-inactivating proteins can be bound to any molecule capable of acting as a specific carrier which is taken up by, or links to, a cell target. At least in theory, hormones, growth factors, receptors, lectins, antigens, vitamin-binding proteins could be used (Table XIV).

TABLE XIV

Conjugates between RIPs and non antibody carriers

Carrier RIP Refs.

Growth factors EGF ricin 519, 520 ricin A chain 519-524

FGF saporin-S6 525--532 Lectins abrin B chain ricin A chain 533

concanavalin A gelonin 92 ricin A chain 534, 535

diphtheria toxin B chain ricin A chain 536 ricin B chain abrin A chain 533

gelonin 537 modeccin A chain 538 PAP 539

Wisteria floribunda lectin ricin A chain 540 wheat germ lectin ricin A chain 541

Hormones chorionic gonadotropin ricin A chain 542 546 gelonin 547

corticotropin releasing factor gelonin 548-55(t insulin ricin A chain 55 I luteinizing hormone gekmin 552-554

Carbohydrates ~x-Glczg-BSA gelonin 555 monophosphopen tamannose gehmin 556

ricin 557, 558

Antigens CD4 antigen ricin A chain 559, 560 acetylcholine receptor gelonin 456

ricin A chain 561,562 (A, G, C, T)zs-BGG ricin A chain 563 tetanus toxoid ricin 564 thyroglobulin ricin A chain 565 anti-idiotype antibody saporin-S6 487

Miscellaneous asialofetuin ricin A chain 519 avidin ricin A chain 424 az_macroglobuli n ricin A chain 566, 567 protein A ricin 425, 428, 568 tetanus toxoid gelonin 569 transferrin ricin A chain 57[1, 571

saporin-S6 488

As a matter of fact, the first conjugate with a type 1 RIPs was prepared by linking gelonin to concanavalin A[92]. Very recently the A-chain of ricin was linked to the CD4 antigen, with the aim of eliminating HIV-infected cells expressing the CD4-binding virus [481]. Conjugates (mitotoxins) of fibroblast growth factor and saporin-S6 have been prepared (reviewed in Ref. 572).

RIPs linked to antigens responsible for autoimmune diseases are potentially useful in therapy. It should be possible with them to selectively eliminate the immunocompetent cells supporting the diseases. The few experiments performed so far are encouraging (see section V-B.1).

V. Possible uses in experimental and clinical medicine

The most studied application of RIPs is their use as immunotoxins, or conjugates with other suitable carriers. The possible utilisations are numerous since, at least in theory, it should be possible with them to eliminate in a selective manner any type of harmful cells (e.g., malignant, parasitic and harmful immunocompetent cells). An obvious prerequisite for the safe use of immunotoxins is that the antibodies used are specific for unwanted cells, or at least bind only to normal cells which can be safely destroyed. Normal cells that may be sacrificed are those not essential for life (e.g., epithelial cells of the mammary gland) or are easily regenerated from spared precursors (e.g., some lymphocyte subpopulations).

V-A. Anti-tumour therapy

V-A. 1. Ex uiuo purging of bone marrow Autologous or allogeneic bone marrow transplanta-

tion can be life-saving in a number of malignant or haematological diseases. Major obstacles to the allogeneic transplantation are the rejection and the development of the GVHD reaction (see section V-B.2). The contamination by malignant cells must be considered in the case of autologous bone marrow transplantation, practised in tumour patients after supra-lethal chemo- a n d / o r radiotherapy.

Immunotoxins work very well in vitro, and can be used to prevent the difficulties mentioned above. The first suggested clinical applications of immunotoxin were to purge bone marrow of T cells to prevent graft-versus-host disease (GVHD) in the case of allogeneic transplantation [573], or of malignant cells in the case of autologous transplantation [574-576].

In the case of bone marrow purging only the toxicity of the immunotoxin to, and its cross reactivity with, the haemopoietic stem cells are to be avoided, whereas the general toxicity and any cross reactivity with other vital organs can be largely disregarded. In the purging of bone marrow from malignant cells, the main problem is

267

the availability of antibodies (preferably monoclonal) specific for the cells to be eliminated.

Immunotoxins for bone marrow purging were prepared mostly with ricin A chain, but also with whole ricin [577], abrin A chain [578], momordin [473], saporin-S6 [500] and PAP [484]. Highly efficient immunotoxins prepared with ricin or other type 2 RIPs can be used for the ex vivo purging: the unspecific toxicity due to the B chain of the toxins can be avoided by addition of excess lactose which competes with galactosyl-terminated receptors on the cell membranes [579].

The purging of malignant cells from bone marrow with ricin or ricin A chain immunotoxins has been reviewed recently [419,792]. Most immunotoxins were against B-cell [574,575,580], T-cell [581-585] or non lymphocytic [586] leukaemia and lymphomas, and against erythroleukaemic stem cells [587]. Some immunotoxins were also prepared against breast tumour cells using whole ricin [588] or abrin [578] and ricin [589] A chains.

Immunotoxins for bone marrow purging were prepared also with type 1 RIPs. Immunotoxins for this purpose were prepared with PAP and antibodies against leukaemia B- [442] and T-cells [474]. The use of immunotoxins containing PAP [475,479] or ricin [590] in combination with anti-neoplastic drugs was also proposed. An immunotoxin consisting of PAP linked to a B43 (anti-CD19) monoclonal antibody was used to remove CD19 + leukaemia B-cell precursors from autologous bone marrow grafts for ALL patients [484]. The treatment did not affect engraftment and effectively removed target cells, and still most patients relapsed. Relapses occurred also among patients receiving allogeneic grafts, suggesting that the failure of the treatment was due to incomplete elimination of leukemic cells from patients.

Conjugates potentially useful to purge bone marrow were prepared with saporin-S6 linked to antibodies against various B- [474,497] and T-cell antigens, and against the transferrin receptor [501]. These conjugates were highly and specifically toxic to target cells, sparing hematopoietic progenitors. Their effect was not increased by the potentiators used with ricin A chain immunotoxin, but was only enhanced by amantadine [584].

Immunoconjugates with saporin-S6 [493] or momordin [473] and anti-plasma cell antibodies were cytotoxic to myelomatous cells, while sparing the bone marrow stem cells necessary for the success of the graft. In a case of T-cell acute lymphoblastic leukaemia, the bone marrow was purged with an anti-CD5- saporin-S6 immunotoxin [591]. It could be ascertained retrospectively that the reinfused bone marrow was not entirely disease-free: a 0.5% contamination with malignant cells was present, as judged by the recombination

2¢~8

pattern of the T-cell receptor, and the disease relapsed after 3 weeks.

I/A.2. ht i'ico local treatment Immunotoxins containing whole ricin, whose carbo-

hydrate binding site was blocked, has been injected directly into turnouts in mice with established solid tumours. The conjugates were found to be effective in vNo in mice carrying thymoma grafts and in nude mice bearing human tumour xenografts [592]. The use of these conjugates was suggested for the local intratu- mour therapy of selected cancers (i.e., ovarian cancers, brain tumours and [eptomeningeal neoplasia) [593].

The use of RIPs and of their immunotoxins has been proposed also for the control of non neoplastic pathological tissue growths. Ricin has been injected into the terminal proximal segment of transected rat sciatic nerves: the treated nerves showed no evidence of ncuroma formation at dosages of ricin that allowed 1{)0% survival [347]. lmmunotoxins composed of mono- chmal antibodies and recombinant ricin A chain have been proposed to control the anomalous growth of corneal endothelial cells [594], retinal pigment epithelial cells [595,596] fibroblasts [597,598] and lens epithelial cells [599,600] in the eye.

V-A.3. In t'iro systemic treatment The vast majority of the research on immunotoxins

is tocused on their possible use in anti tumour therapy. lmmunotoxins were administered to cancer patients in phase I or I / 1 I clinical trials, reviewed in Refs. 419.42(I,572,601 - 6(13.

So far most immunotoxins used for the treatment of patients were prepared with ricin A chain or blocked ricin. Several trials were conducted on patients with B- and T-cell leukemias and lymphomas [604-609]. Ricin A chain immunotoxins were given also to patients with solid tumours, including malignant melanoma [610- 614], breast carcinoma [615,616] and colon cancer [617,618].

The reports on the administration to human patients of immunotoxins containing type I RIPs published so far are only two: (i) a conjugate made with saporin-S6 and an anti-CD30 monoclonal antibody given to patients with Hodgkin's lymphoma [505] and (ii) a conjugate made with PAP and an anti-CD19 monoclonal antibody [484].

Remissions of various grade, up to complete, were observed in most trials with l eukaemia / lymphoma. Beneficial effects were more modest [617] or absent in the case of solid tumours, possibly because the immunotoxins had no access to malignant cells.

Some toxicity was invariably observed. Fever was the most constant symptom, and other side-effects included dyspnea, fatigue and myalgia, anorexia and nausea, skin rashes, elevation of serum transaminases,

hypoalbuminemia, fluid retention and oedcma, capil- lat-y leak syndrome, the latter duc to release of cytokines [615,616] or to direct endothelial damage [t)lg]. In one trial, neuropathy was observed, and was al- tributed to binding of the immunotoxins to Schwann cells o1 to myelin [615].

Virtually all patients receiving immunotoxins, unless severely immunodepressed, formed antibodies against both the antibody and the toxic moiety. The appearance of these antibodies prevented further administration of the drug, thus severely limiting its potential efficacy.

The overall results of clinical trials with immunotoxins are still modest, in part duc to:

(i) recurrence of the diseases. It should be remem- bered, however, that trials were performed mostly on patients with advanced tumours:

(ii) formation of antibodies preventing prolonged treatment. Hopefully, the immune reaction may be circumvented with the sequential use of conjugates prepared with different RIPs, immunologically distinct from each other, linked to 'humanised ' antibodies (hybrid molecules consisting of the variable region from routine monoclonal antibodies linked to the constant region from human antibodies), and eventually to human monoclonal antibodies:

(iii) toxic side effects. However, effective doses could be administered with generally acceptable side effects. Only in few cases they were severe.

Other foreseeable problems include (i) the shedding from cancer cells of antigens that would bind the immunotoxins, thus diverting them from their target, (ii) thc access of the conjugates to tumour cells could be difficult, especially in the case of poorly vascular- ized solid turnouts of considerable size and (iii) the emergence of variants of the malignant cells, deprived of the antigen target of the immunotoxins. This might be avoided by the use of mixtures of immunotoxins made with antibodies against different cell antigens.

It is generally believed that immunotoxins will be best used to remove small tumour masses, such as the minimal residual deseasc remaining after the removal of primary tumours, but much work is still necessary to achieve this goal.

V-B. Immune disorders

V-B. 1. Au to immune diseases Autoimmune disease could be cured by removing

clones of immunocompetent cells responsible for the autoimmune reaction. This goal might be achieved by targeting toxins, and RIPs among them, to the cells to be eliminated.

An approach to the therapy of autoimmune disease could be the use of anti-lymphocyte immunotoxins. The use of a ricin A chain immunotoxin for the therapy

of rheumatoid arthritis [620] and of an anti-CD5 [621] ricin A chain immunotoxin for the therapy of lupus nephritis has been proposed. Interferon-induced autoimmune diabetes in the mouse was prevented with anti-CD3 and, less efficiently, with anti-Lyl (analogous to human CD5) ricin A chain immunotoxins [622]. With this kind of immunotoxins, however, the whole population of lymphocytes bearing the targeted antigen would be removed, with consequent generalised immunosuppression.

More selective would be the use of immunotoxins constructed with specific anti-idiotype antibodies. This approach has not been explored for autoimmune disease, but anti-idiotype immunotoxins have been prepared with saporin-S6. Using such immunotoxin, immunoglobulin-secreting leukemic cells in guinea pigs were selectively eliminated [487].

A different approach is to use conjugates of toxic moieties with the antigens responsible for the disease. Such conjugates should combine with, and kill, the relevant immunocompetent cells. These 'antigen-toxins' will have two major advantages over antiidiotype immunotoxins: (i) the carrier molecule is a human molecule and, (ii) protein antigens or, even better, their determinant parts, are usually much smaller than antibodies. This may facilitate the construction of a fusion protein between a RIP and the carrier by fusing the coding sequences of the two components.

The feasibility of selectively eliminating antigen- specific B cell responses with antigen-toxin conjugates was demonstrated with a ricin-tetanus toxoid conjugate [564]. With this it was possible to abrogate the anti- tetanus toxin antibody production in vitro, without affecting antibody responses against other non-cross- reacting antigens.

Conjugates for the elimination of auto-reactive B cells in autoimmune diseases were constructed. Ricin A chain conjugated to human thyroglobulin was specifically toxic to auto-reactive lymphocytes from patients with Hashimoto disease (autoimmune thyroiditis) [565]. Similarly, ricin A chain conjugated to nucleosides selectively reduced spontaneous anti-nucleoside antibody production in vitro by lymphocytes from patients with systemic lupus erythematosus [563].

Specific suppression of immune response to acetylcholine receptor was obtained in vitro with a ricin- acetylcholine receptor conjugate. This system was suggested for the therapy of myasthenia gravis [561,562]. That this model could work in vivo was shown with a gelonin-acetylcholine receptor conjugate tested in experimental autoimmune myasthenia gravis in the rat [456]. The loss in functional acetylcholine receptor was significantly smaller in the therapy group than in the untreated experimental autoimmune myastenia graL, is group. The acetylcholine-dependent end plate was almost normal in 75% of the treated animals and the

269

specific antibody titres were lower after treatment with antigen-toxin.

V-B.2. Pret,ention and treatment of graft-L,ersus-host disease

Organ transplantation is increasingly practised in a number of conditions, to substitute for defective organs or, in the case of bone marrow, to replace bone marrow destroyed by treatment. In the case of allogenic bone marrow transplantation, the graft-versus-host reaction exerted by immunocompetent ceils of the graft is still a serious and often life-threatening complica- tion, in spite of the improvements in the d o n o r / recipient immunological matching, and it greatly limits the use of mismatched donors.

The risk of GVHD is reduced and engraftment is facilitated if the organs to be transplanted are rendered free of T lymphocytes, and GVHD can be treated by removing the immunocompetent cells involved. These tasks can be accomplished with immunotoxins, which have been employed to prevent or treat GVHD.

Irradiated C 5 7 B L / 6 mice t ransplanted with B A L B / c splenocytes and bone marrow cells were pro- tected from GVHD if donor cells were pre-treated with an anti-Thyl,2-ricin immunotoxin, in the presence of excess lactose [623,624]. Immunotoxins prepared with ricin and monoclonal antibodies against human T-lymphocyte antigens TA-1, UCHT1 (anti-CD3) and T101 (anti-CD5) were proposed [434,579,625] for GVHD prevention. These authors used whole ricin immunotoxin having observed that they were more efficient than those with the same antibodies linked to ricin A chain [434,624]).

Efficient T-cell removal was obtained with a variety of ricin A chain immunotoxins in the presence of enhancers such as NH4CI [584,626,627], monensin [628] or verapamil [584]. Various other immunotoxins were prepared with ricin A chain linked to antibodies against various lymphocyte antigens (CD2, CD5, CD7, CD8 [629]; CD3, CD4, CD8 [630]; CD3, CD4, CD5, CD7, CD8 [631]; CD2, CD3, CD8 [632]; interleukin-2 receptor B chain [633]). These immunotoxins showed a marked toxicity to target lymphocytes only in the presence of enhancers. In the case of anti-CD4 and anti- CD8 immunotoxins, only a modest cytotoxic effect was obtained even in the presence of NH4C1 [631]. Under- standably, a better and sometimes virtually complete elimination of T ceils without damage to hematopoietic cells was obtained by treating bone marrow cell suspensions with a mixture of ricin A chain immunotoxins made with different antibodies, rather than with a single mono-specific immunotoxin [629-631].

Immunotoxins suitable for T cell removal from bone marrow were prepared also with type 1 RIPs. Potent immunotoxins were obtained with saporin-S6 linked to SOT3 (anti-CD3) and SOTla (anti-CD5) monoclonal

270

antibodies whereas an OKTll -sapor in-S6 (anti-CD2) immunotoxin had less effect [490]. Selective killing ol: CD4 ~ cells was obtained with a ]EC-T4-saporin-S6 monoclonal antibody, and CD4 ~ and CD8 + cells werc eliminated with the indirect method using an F(ab')~ anti-mouse IgG saporin-S6 immunotoxin and anti-CD4 and anti-CD8 monoclonal antibodies [429]. It is noteworthy that the effect of an OKTl-saporin-S6 immunotoxin (anti-CD5) was enhanced by amantadinc but not by NH4CI [491].

Clinical trials were performed with bone marrow allografts from which T-cells were removed with a mixture of TA-I- , UCHT1- and Tl01-ricin immunotoxins [573,634], with a T101-ricin A chain immunotoxin, [635] and with a T101 Fab fragment-ricin A chain immunotoxin [636]. Good engraftment was obtained in all these studies: grade II skin G V H D was observed in 4 out of 17 patients [634] in one trial, and in only 2 out of 38 patients in an other study [636]. In either cases no patients developed severe GVHD, which indicates the feasibility of the approach,

A ricin A chain immunotoxin against the CD5 lymphocytic antigen was given to patients with steroid-resistant G V H D after bone marrow transplantation. A positive responsc was obtained in 16 out of 32 cases, with complete response in nine cases [637]. ]'his was the first and one of the most successful uses of immunotoxins in human therapy.

A B-BI0 (anti-CD25)-saporin-S6 immunotoxin has been proposed for prevention and treatment of G V H D [638]. The use of an anti-CD25 immunotoxin would have the advantage of eliminating only the activated lymphoeytes, leaving intact the remaining lymphocyte population.

lmmunotoxins have been proposed also to remove T cells from organs other than bone marrow to be transplanted. Infiltrating T-lymphocytes, resistant to previous aggressive immunosuppression, were effectively removed from a rejected human renal allograft by perfusion with a saporin-S6-anti-CD5 immunotoxin [497]. Perfusion of the intestine with an anti-CD5-ricin A chain immunotoxin prolonged the survival of a semi-al- Iogenic intestinal graft in the rat [639,640].

V-B.3. AIDS Although the first immunotoxin was prepared with

diphtheria toxin linked to an antibody against a viral agent [641], the use of immunotoxins to combat viral infection has received little consideration. Presumably, the reason is that to suppress infection a complete rcmowd of viruses is required, a difficult goal to achieve in the experiments with cells in culture commonly used to assay the efficiency of immunotoxins. The results in vivo though could be bet ter than those obtained in vitro, because an incomplete, but substantial reduction of the amount of virus in an infected organism could be

sufficient lor the disease to bc controlled by the immune mechanisms. Thc only attempts so far were to eliminate HIV infection with trichosanthin.

Scveral therapeutic stratcgies havc been proposcd to attempt a cure or a remission in the HIV sustained acquired immunodeficiency syndromc with the use of RIPs or of RIP-containing immunotoxins. (i) The efficiency of intravenous administration of trichosanthin is presently under scrutiny in two phase I clinical trials in the USA. The rationalc is that trichosanthin appears to have anti-HIV activity of some still unknown naturc (see section III-E.2). (ii) Surolia and Ramprasad [642] suggested the use of immunotoxins directed towards CD4 + cells. Several immunotoxins with variable spc- cific activity against the C D 4 ' cells have been constructed for this purpose [421,42~L63(t,631,643-645]. The administration of these immunotoxins would greatly diminish the number of virus particles released into the circulation following the dcstruction of the C D 4 ' cells. As a consequence, this reduced number of viral particles released might be neutralised by the antibodies present in the patient blood. (iii) Good targets for immunotoxins are two viral glycoproteins expressed on the surface of HIV-infected cells, namely gpl20, which binds to the CD4 antigen, and gp41, which mediates fusion. Gpl20 has been thc target of rCD4-recombinant ricin A chain [646,647], CD4 peptide-ricin A chain [648] and CD4-PAP conjugates [481] and of ricin A chain [645,649,650] or PAP-S anti-gpl2fl immunotoxins [651], which all inhibited replication of HIV. However, gpl20 is highly wtriablc and heterogeneous, and can shed from infected cells and bind to uninfected CD4 ~ cells. A more stable antigen is gp41, against which effective immunotoxins were prepared with ricin A chain [647,650,652]. In a comparative study with different anti-CDl20, anti-gp41 and anti-gpl60 antibodies, the immunotoxins containing anti-gpl60 polyclonal antibodies from H1V-infected patients had the broadest specificity for different HIV strains and the highest specific activity [650].

Autologous bone marrow transplantation has been proposed as a different approach in the therapy of AIDS, and the various fl)rms of RIP-containing conjugates described above could be employed to eliminate infected cells from the marrow prior to reinfusion. The ex vivo purging would allow the use of chloroquine or other "potentiators" it) enhance the effect of ricin A chain and possibly other immunotoxins [647,652].

VI. Applications in agriculture

Recent progress in the areas of in vitro culture and genetic engineering allows the introduction of specific genes into the desired plant hosts. Bacterial genes have already been successfully engineered in crop plants to confer herbicidal and insect resistance (see for exam-

ples Refs. 653-656). A promising approach to insect pest control is the isolation and transfection of plant genes coding for entomotoxic compounds, as practised with the toxin of Bacillus thuringiensis. Ribosome-inactivating proteins usually show modest inhibitory activity on plant ribosomes (see section III-A) and consequently could be suitable candidates for this experimental approach to parasite control, It could then be possible to identify plant parasites of economic rele- vance whose ribosomes are highly sensitive to a RIP. Transformation of an economically important host plant with the gene for a RIP which is toxic to parasites and is ineffective on the ribosomes of the plant should confer specific resistance, as it seems to occur in the case of transfected tobacco plants [411].

The biological significance of RIPs in nature is not yet known. Amongst the more likely advantages for the plant is then a possible effect on plant pathogenic agents [1]. Indeed, it was recently reported that barley RIP has fungicide activity in synergism with a chitinase and a glucanase [111]. Presumably, these enzymes alter the cell membrane of fungi, allowing the RIP to enter the cytoplasm. The anti fungal activity of barley RIP was further indicated by the increased resistance to fungi of transgenic tobacco plants expressing this RIP [411].

RIPs could also confer resistance to insects, as indicated by the toxicity of RIPs to some larvae (see section I l l -D) and by the effects of RIPs on ribosomes purified from Musca domestica larvae (Fig. 8) which demonstrate that some RIPs may indeed be very potent in damaging insect ribosomes.

VII. Biological role and perspectives

The main question which remains unanswered is the biological role of RIPs. These proteins are frequent among plants and often present at an high concentration. Moreover, the fact that some ribosomes appeared resistant to their action in the test systems used suggests that possibly in some plant materials RIPs were not detected because of the lack of sensitivity of the system used to screen the materials. Thus, RIPs could be more frequent, and possibly ubiquitous in plants, and this strongly suggests a premium for their conser- vation through the evolution.

Several hypotheses have been formulated for the function of RIPs in nature, none of which is fully convincing.

One is a defensive role against predators. This seems plausible only in the case of RIPs type 2 and in the few cases when RIPs type 1 are present at a very high concentration. These cases, however, seem few and can be considered as mutations which were conserved because advantageous, but they cannot account for the widespread presence of RIPs.

271

A metabolic role seems more likely, especially since the enzymatic activity of RIPs was detected, although the purpose of depurination of a specific RNA sequence remains unknown, and actually raises further questions. It seems strange that an enzyme is required at such high concentrations as those sometimes found. This could be due to an accidental mutation, as mentioned above, but could also be due to the natural conditions in which these enzymes work, undoubtedly different from the optimal experimental conditions. One notion assigns to RIPs a regulatory role in protein synthesis [233]. However, a regulation through an apparently irreversible inactivation of ribosomes seems uneconomical and hence unlikely.

The fact that RIPs are usually more concentrated in seeds, may suggest that their function is involved in germination.

A possible role as a defence mechanism against plant pathogens could be of great biological impor- tance, as mentioned in section VI for fungi. A protec- tive effect against viral infections was also proposed, and gained new impetus after the recent findings [235,236] discussed above (sections III-E.1).

Another possibility to be tested is that equivalents of RIPs may exist in organisms other than plants, and that may have escaped detection due to the inadequacy of the test system used.

Acknowledgements

The research performed by the authors was supported by the Consiglio Nazionale delle Ricerche, Rome, within the projects 'Biotecnologie e Biostru- mentazione' , 'Oncologia ' and 'ACRO' , by the Minis- tero dell 'Universita e della Ricerca Scientifica e Tecno- logica, Rome, by the National AIDS Project of the Istituto Superiore della Sanit?a, Rome, by the Regione Emilia-Romagna, Bologna, by the Associazione Ital- iana per la Ricerca sul Cancro, Milan, by the European Economic Community, Brussels, and by the Pallotti's Legacy for Cancer Research.

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