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Preparation and Characterization of Recombinant Proricin Containing an Alternative Protease-Sensitive Linker Sequence Michael Westby, Richard H. Argent, Carol Pitcher, J. Michael Lord, and Lynne M. Roberts' Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. Received February 26,1992
The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990)FEBS Lett. 273,200. Kim, J., and Weaver, R. F. (1988)Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosomeinactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavagesite in recombinant fusions with RTA is discussed.
INTRODUCTION Ricin is a heterodimeric cytotoxin produced in the seeds of the castor oil plant, Ricinus communis. The mature toxin consists of a 32-kDa A chain (RTA) linked by a disulfide bond to a 34-kDagalactose-bindingBchain (RTB) (1).Ricin intoxication of eukaryotic cells is initiated when the holotoxin interacts with cell surface glycoproteins and glycolipidscontaining galactose. This binding is mediated entirely by RTB, which is also believed to play a role in correctly transporting RTA during the ensuing endocytosis (2,3). Membrane translocation of RTA, possibly from a Golgi compartment (4, 5 ) , is then followed by catalytic inactivation of ribosomes in a step mediated entirely by reduced RTA. RTA is an RNA-specific N-glycosidase which acts on 28s or 26s rRNA leading to a specific depurination within a highly conserved region thought to be crucial in the interaction with elongation factors (6). Ricin is synthesized in Ricinus seeds as a preproprotein which is converted to its mature form by a number of coand post-translational modifications (7,8). These begin as the protein is being segregated into the ER lumen and terminate after vesicular transport when the protein is finally deposited in protein body organelles where ricin accumulates. The initial preproricin molecule contains a 35 residue presequence (including a signal peptide), followed by RTA, a 12 amino acid residue linker, and the RTB sequence (9). The final processing steps include proteolytic removal of the linking peptide by enzymes contained within protein bodies (8). A study of the activities of the proricin precursor has shown that although it possesses sugar binding activity, it is unable to depurinate 28s rRNA (IO). Catalytic activity is present only when peptide continuity between RTA and RTB is disrupted. Thus synthesis of RTA as an inactive proenzyme contributes to the safeguards which ensure that RTA does not inactivate endogenous plant ribosomes. Ricin and RTA have been used extensively in a variety of conjugates designed for selective cell destruction (reviewed in ref 11). Conventionally, the toxin is linked
* Author to whom correspondence should be addressed.
to an antibody, lymphokine, or other protein entity by chemical means, which also introduces a reducible disulfide bond. Many such conjugates exert a potent cytotoxic effect upon their target cells. Increasingly, however, recombinant cytotoxic conjugates are being produced directly by expression of the relevant gene fusions. This has been a particularly successful approach with the bacterial toxins diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). By fusing fragments of these toxins with alternative cell binding proteins such as a-melanocyte stimulating hormone (12),soluble CD4 (13), or a single chain Fv antibody fragment (14),cell-specific, single-chain cytotoxins have been generated. Similar single-chain fusions containing RTA are not cytotoxic (15, 16). It is believed that during intoxication, RTA must be released from its cell binding ligand to be competent for membrane translocation. Unlike DT and PE, RTA lacks a specific proteolytic cleavage site recognized by target cell proteases encountered upon cellular uptake. Introduction of a trypsin-sensitive sequence into an RTAprotein A (PA) fusion protein demonstrated for the first time that a noncytoxic fusion protein containing RTA could be converted into a cytotoxic conjugate (16). In the present report we describe the production and characterization of a recombinant variant possessing an alternative protease-sensitive linker separating RTA from RTB. Using proricin as a model recombinant fusion, we reveal that, as in native proricin, the mutant precursor possesses lectin activity but has no RTA activity until treated with the appropriate protease and reduced. Specificcleavage generatesdisulfide-bonded subunits with the biological properties of native holotoxin. This particular arrangement of modified linker and flanking cysteines, which does not significantly perturb the structure of the component polypeptides, may have general applicability in creating disulfide-linked conjugates from single-chain recombinant polypeptides containing RTA. EXPERIMENTAL PROCEDURES Construction of Plasmids. Preproricin cDNA (9)was subjected to site-directed mutagenesis to create a mutant clone with a linker encoding a factor Xa recognition
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Figure 1. Mutagenesis of the proricin linker. (A) DNA and predicted amino acid sequences of the linker regions in wild-type preproricin cDNA (ppRWT) and factor X-site containing preproricin (ppRAXa). Mutated bases are shown in bold type and the amino acids comprising the protease recognition site are underlined. The arrow indicates the predicted site of cleavage. Nucleotide numbers refer to the preproricin cDNA (9). S refers to signal sequence, A, L, and B to RTA, linker, and RTB, respectively. (B)The wild-type and mutant preproricin sequences were ligated into pSP64T as described in the methods and shown here for the wild-type sequence. 5 and 3 refer to the 5’and 3’ untranslated regions of Xenopus 0globin cDNA which, upon transcription, provide stability to the RNA when injected into Xenopus oocytes (18). HinDIII refers to a HindIII site between the SP6 promoter and the BglII expression site. Other sites characteristic of the vector or preproricin sequence are also indicated. sequence (IEGR). The template for mutagenesis was derived from a 552 base pair BglII fragment which spans the preproricin linker region. This fragment was cloned into the BglII site of plC2O-H (17),and an EcoRI and a HindIII fragment were subsequently ligated into M13mp19. Mutagenesis was performed by standard techniques using an oligonucleotide-directed mutagenesis kit (Amersham Corp.) as directed by the manufacturer. Mutations were confirmed by dideoxy sequencing. Oligonucleotides for mutagenesis were synthesized on an Applied Biosystems Model 380 B DNA synthesizer. The eight base pair mismatch to create the factor Xa site was achieved in a single round of mutagenesis using the oligonucleotide 5’-TTTGCTTATAGAAGGCCGGGTACCAAAT-3’. The mismatches are indicated by underlining. The BglII fragment was then substituted into the wild-typepreproricin clone and sequenced. The nucleotide and predicted amino acid sequences corresponding to the
new linker is shown in Figure 1A. Wild-type and mutant proricins were subsequently recloned into the BgZII expression site of pSP64T, a vector which has been successfully used for the production of stable transcripts suitable for expression in Xenopus laevis oocytes (18). Expression of Preproricin Transcripts. Transcripts were synthesized in vitro in the presence of the capping dinucleotide 7-Me(5’)GpppG(5’)OH and SP6 RNA polymerase as described previously (19). Purified RNA was dissolved in water at a concentration of 1mg/mL. Oocytes were injected with approximately 50 ng of RNA, pulse labeled with [35Slmethionine,and homogenized as described previously (19),except that the protease inhibitor phenylmethanesulfonylfluoride was omitted. Proricin was either affinity purified (below) or, standardly, batches of 10 oocytes were homogenized for immunoprecipitation (20).
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Affinity Purification of Proricin Mutants. Recombinant proricins were purified by affinity chromatography using a 1.0 mL column of SeLectin-2 beads (10) (lactose coupled to acrylamide; Pierce, Rockford, IL) equilibratedin oocyte homogenizationbuffer (19). Soluble protein from 10homogenized oocytes was typically passed through the column a total of three times. The column was washed with three 1.0-mL aliquots of oocyte homogenization buffer prior to the elution of bound material in either homogenization buffer or protease digestion buffer (10 mM Tris-HC1,l mM EDTA, 140 mM NaC1, pH 7.6) containing 50 mM galactose. Proteolytic Digestion. Affinity selected proricin was eluted from the SeLectin-2 matrix in protease digestion buffer containing 50 mM galactose. Proricin substrate was then incubated for 60 min at 26 "C with 1/10 volume of human factor Xa (kindly provided by P. Esnouf, The Radcliffe Hospital, Oxford, UK). Factor Xa, at a concentration of 240 pg/mL was stored frozen at -20 "C. Depurination of 285 rRNA. Affinity-selected and protease-treated proricin was incubated for 30 min at 30 "C with 30 pg of salt-washed rabbit ribosomes prepared from a non-nuclease-treated rabbit reticulocyte lysate (Promega) (21) in 1X Endo buffer (25 mM Tris-HC1, 25 mM KC1,5 mM MgC12, pH 7.6) before or after reduction of the interchain disulfide bond using 5% (v/v) @-mercaptoethanol for 30 min at room temperature. RNA was then extracted and analyzed for the RTA-specific modification of ribosomal RNA using the acetic-aniline reagent, exactly as described previously (22). This assay is based on the observation that RTA-modifiedribosomes which carry a 28s rRNA depurinated at a position close to the 3' end, are extremely sensitive to hydrolysis using acetic-aniline. Thus brief aniline treatment of RNA extracted from ribosomes will release a 390 base RNA fragment only if the ribosomes were inactivated with a catalytically active RTA. Non-aniline-treated samples should not release a visible RNA fragment. Purified recombinant RTA on ribosomes was used to give a positive control signal. Other Met hods. Published procedures were followed for translation of in vitro transcripts in wheat germ cellfree lysates (23),sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, immunoprecipitation (20), and enzymic deglycosylation using endo-N-acetyl glucosaminidase H (8). Antibodies for immunoprecipitation were raised in rabbits against glycosylated RTB and crossreact with RTA, RTB, and proricin. RESULTS
Mutagenesis and Cloning. Oligonucleotide sitedirected mutagnesis of the preproricin cDNA was performed to create a factor Xa recognition sequence within the naturally occurring linker which separates the RTA and RTB coding regions. The nucleotide and amino acid sequences of the wild type (pp RWT) and mutant (ppRAXa) are shown in Figure 1A. Preproricin encoding sequences were then cloned into the transcription vector pSP64T as to generate an expression plasmid as illustrated in Figure 1B. Expression and Purification of Proricin. Transcripts were prepared for wild-type preproricin and the variant using SP6 RNA polymerase. Transcripts were microinjected into Xenopus oocytes and the [35S]methionine-labeled products were subsequently analyzed after homogenization and purification by selection on columns of immobilized lactose (Figure 2). In both cases a good proportion of the proricin synthesized possessed
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I 45 Figure 2. Wild-type and mutant proricins synthesized in Xenopus oocytes are glycosylated and bind lactose. [35Slmethionine-labeled homogenates from 10 oocytes were applied to a 1.0-mL SeLectin-2 (Pierce) column as described in the methods. A and B refer to wild-type and mutant proricins, respectively. Lane 1,unbound protein; lane 2, immunoprecipitate of proricin from the unbound fraction; lanes 3 and 4, immunoprecipitated proricin in the first two 0.5-mL column washes with homogenization buffer; lanes 5 and 6, nonimmunoprecipitated material eluted in buffer containing 50 mM galactose; lanes 7 and 8, immunoprecipitates from crude homogenates not treated and treated with endo-N-acetylglucosaminidaseH, respectively. M refers to molecular weight markers. Immunoprecipitations (where appropriate) were performed using rabbit anti-B chain antisera which cross react with both subunits separately and with the proricin precursors. All samples were run by SDSPAGE in reducing buffer. M r 1
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Figure 3. Protease susceptibility of mutant proricin: lane 1, oocyte produced RTB control; lanes 2 and 3, affinity purified proricin eluted in reaction buffer containing 50 mM galactose was incubated with and without factor Xa as described in the methods. Samples are electrophoresed on SDS-PAGE in a reducing sample buffer.
lactose binding ability, as judged by the amount of radioactive proricin released from the SeLectin-2 matrix in buffer containing 50 mM galactose (Figure 2, lanes 2 and 5). As observed previously (IO),the only labeled protein to be retained by the column was theRicinus lectin molecule (Figure 2, lane 5). In this track the sample represents a nonimmunoprecipitated sample. When immunoprecipitated samples of total homogenate (lane 7) were treated with endo-N-acetyl glucosaminidase H (lane 8),the multiple proricin species were converted to single polypeptides of greater electrophoretic mobility (i.e. deglycosylated), confirming that they had become N-glycosylated within Xenopus oocytes. The faster migrating species in the proricin immunoprecipate from total homogenates (lanes 2 and 7) is most likely a nonglycosylated form of proricin which runs with the same mobility as the endo H treated sample (lane 8). The nonglycosylated form is not always seen (e.g. Figure 2B) and appears to be dependent upon the oocyte batch. Protease Susceptibility of the Modified Linker. Affinity purified mutant proricin was treated with protease to confirm specificsusceptibility (Figure 3). Recombinant RTB produced in Xenopus oocytes and affinity purified was used to indicate the electrophoretic mobility of this
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presence or absence of reducing agent. Figure 5B shows that ribosomeswere only modified in the presence of RTA, irrespective of the presence or absence of 5% (v/v) p-mercaptoethanol.
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Figure 4. Cleavage and disulfide bonding of mutant proricin: affinity purified proricin from 10 oocytes were digested for up to 1h with factor Xa asdescribedin the methods and subsequently immunoprecipitated. A and B refer to wild-type and mutant proricins, respectively. All tracks but lane 8 on the SDS-PAG were run in the presence of reducing agent. Lanes 1-5, material eluted from the column incubated in the first galactose-containing wash with factor Xa for 0, 15, 30,45, and 60 min, respectively; lane 6, immunoprecipitateof the unbound material;lane 7, protein eluted in the second galactose wash; lane 8, a duplicate samples of the 60-min protease sample (lane 5) electrophoresed in the absence of reducing agent. M refers to molecular weight markers.
particular subunit (Figure 3, lane 1). Using the mutant proricin and factor Xa, reproducible cleavage products were visualized (Figure 3, lane 2) that were absent in a duplicate incubation minus protease (Figure 3, lane 3). Cleavagewas incompletebut reproducible,using the batch of enzyme provided. The production of three fragments is discussed later. Cleavage and Disulfide-BondFormation. Affinitypurified wild-type or mutant proricins were digested for up to 60 min with factor Xa (Figure 4). Aliquots of each reaction were immunoprecipitated under nonreducing conditions and then subjected to SDS-PAGE in the presence of reducing agent. Wild-type proricin was not cleaved by factor Xa (Figure 4A) whereas the mutant polypeptide was, albeit at low efficiency using the conditions described here (Figure 4B). It would appear that after 15 min there was little further digestion. When an equivalent aliquot of the 60-min mutant digestion was electrophoresed in the absence of reducing agent, the cleavage fragmentshad apparently disappeared,indicating they were covalently coupled by a disulfide bond in the native protein. (Figure 4B, lane 8). Ribosome Inactivation. Figure 5 shows the activities of the various forms of proricin toward mammalian ribosomes. As shown, the RNA fragment diagnostic of RTA activity (seethe methods for explanation)was absent in samples containing cleaved but unreduced proricin (Figure 5A, lanes 1-4), but present if the connecting disulfide bond had been reduced by pretreatment with 5% (v/v) p-mercaptoethanol (Figure 5A, lanes 5-7). The decreasing intensity of fragment in these lanes correlates with the amounts of cleaved substrate added to the ribosomes. Noncleaved proricin was not catalytically active (Figure 5, lane 9, and ref 10). Controls include ribosomes treated with 0.1 ng of recombinant RTA or 10 ng of nonreduced or reduced recombinant RTB (Figure 5A, lanes 10-12, respectively), plus non-aniline-treated samples (Figure 5A, lanes 13-18). To check that the high concentration (5 % (v/v)) of p-mercaptoethanol did not itself interfere with the assay, ribosomes were incubated in the presence of absence of recombinant RTA in the
We show here that it is possible to make proricin containing a novel protease-sensitivecleavage site within the naturally occurring linker region without significantly perturbing the structures and therefore the biological properties of the component A and B chains. The factor X site was recognized by its protease although cleavage was generally poor under the conditions used here. Nevertheless, in the proportion of proricin which was cleavable it is clear that the new linker had not become buried in the fusion protein but remained accessible and presumably surface-exposed. Futhermore, presence of a modified linker did not affect disulfide bond formation between Cys 259 of RTA and Cys 4 of RTB. The natural linker in wild-type proricin (Figure 1A) is cleaved by endoproteases contained within plant cell vacuoles. The cleavage specificity of at least one of these enzymes is known. The unique asparagine-specific endoprotease activity (responsible for cleavage after the terminal Asn 279 in the proricin linker) has been widely implicated in the processing of many plant vacuolar proteins (25). Its activity was recognized some years ago (26), but the enzyme has been purified only recently (27). It is an extremely unstable protease after purification, but nevertheless has been shown to convert several plant proprotein precursors into their mature forms. However a second endoprotease, presumably responsible for generating the mature C-terminus of the RTA subunit in proricin, has not yet been identified. To exert a cytotoxic effect, RTA must be released from RTB intracellularly (16). In native ricin the linker has already been removed during protein maturation, thus the holotoxin requires no further treatment other than intracellular reduction to render it cytotoxic (28). In contrast, the bacterial toxins DT and P E and Shiga toxin are not proteolytically processed during their biosynthesis (29). However, their enzymatic ADP ribosylating or ribosome inactivating fragments are released from the holotoxins as a result of tryspin-like cleavages within a disulfide-bonded loop sequence (29). Such nicking can be revealed experimentally and appears to occur upon endocytosis when the toxins encounter an appropriate serine protease (30). Clearly, this requirement for a releasable toxic fragment has implications for the design of selective cell-killing reagents. In the development of these reagents, immunotoxins (ITS)(antibody-toxin conjugates),have emerged as a major group with potentially enormous clinical value (reviewed in refs 31-34). In the so-called third and subsequent generations of conjugates, tailor-made recombinant proteins with the desired activities are anticipated. Although not yet routine, this approach also extends to the manufacturer of entire single chain antibody-toxin or other recombinant cell-toxin fusion proteins (12-14). We have shown previously that RTA, as part of a recombinant fusion protein containing protein A in place of RTB, was biologically active in vitro but was not cytotoxicunless a cleavable sequence was inserted between the two protein moieties (16). On this previous occasion we used the DT disulfide loop as our protease-sensitive region. However, it was difficult to analyze cleavage and disulfide bonding in vitro due to the presence of multiple tryspin-sensitive sites within the RTA molecule and the
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Figure 5. Cleaved proricin exhibits ribosome inactivating ability only after reduction of the interchain disulfide bond. (A) After incubation with ribosomes total RNA was analyzed as described previously (22). Lanes 1-12 represent extracted RNAs treated with aniline: lanes 1-4, serial dilutions of cleaved but nonreduced proricin; lanes 5-8, serial dilutions of cleaved and reduced proricin; lane 9, reduced but noncleaved proricin; lane 10,O.l ng of recombinant RTA; lanes 11 and 12,lO ng of recombinant RTB, reduced and nonreduced, respectively;lanes 13-18 represent non-aniline-treatedaliquots of RNA samples shown in lanes 1, 2, 5, 6, 10, and 11, respectively. (B) Lanes 1 and 2, aniline-treated RNA after incubation of ribosomes with 1 ng of recombinant RTA, reduced or nonreduced, respectively;lanes 3 and 4, equivalent incubations in which the extracted RNA was not treated with aniline. The arrow indicates the position of the small RNA fragment released after aniline-treatmentof RNA extracted from RTA-modifiedribosomes.
cleavage procedure required judicious use of protease. Futhermore, a different construct containing RTA linked to IL2 by the DT loop did not generate a disulfide-bonded heterodimer upon tryspin cleavage (L.M.R., unpublished). It appears that the DT loop may have limited application depending upon its context in a particular fusion. It is therefore timely to search for other possible joining sequences susceptible to specific cleavage and disulfide bonding. In this report we have used native preproricin as our model to test the potential of an alternative, highly specific cleavage site within the linker. The human factor Xa recognition sequence (IEGR) has been used successfully to create cleavable recombinant proteins on previous occasions (35,36). The minimal change of three amino acid residues to create such a site in the proricin linker was shown here not to prevent accessibility to the active protease nor to detrimentally affect disulfide bonding between cysteines which flank the linker, nor to disrupt the normal biological properties of the precursor substrate. A potentially spurious site (IVGRJ) occurs 16 residues within the RTB sequence. At first it was suspected that the smallest of the three cleavage products (e.g., Figure 3, lane 2, Figure 4B, lanes 2-5) might represent a fragment of RTB anomalously cleaved at just such a site, the larger two fragments representing RTA and RTB released after cleavage at the defined linker site. However, wild-type proricin when treated with factor Xa did not produce such a fragment (Figure 4A). Furthermore, if the smallest fragment were derived from a cleavage site lying outside the cysteines connecting RTA and RTB, it should still be observed when the cleaved proricin is run under non reducing conditions (Figure 4B, lane 8). The fragment was not observedunder these conditions. Its exact nature is therefore unclear but we believe it most likely represents an underglycosylated subunit. RTA and RTB have two potential N-glycosylation sites each (9), but frequently RTA carries just one N-linked oligosaccharide(37). This heterogeneity is visualized as a smear using SDS-PAGE
(Figure 2, lane 2). Endo-N-acetyl-glucosaminidaseH treatment converts the multiple forms of proricin into a single, faster migrating species (Figure 2, lanes 7 and 8). In addition to the partly and fully glycosylated forms of proricin, a nonglycosylated version is also produced in oocytes (compare Figure 2, lanes 2 and 8). This form does not possess lectin activity as judged by its inability to bind to the affinity matrix. Since N-glycosylation, like disulfide bond formation, is an ER-catalyzed event, we assume that the affinity purified material tested in this report is from that proportion contained within the oocyte endomembrane system. Xenopus oocytes were used in this study because we have previously found it to be an excellent system for the expression and analysis of soluble and stable forms of ricin containing the RTB region (10, 19, 24). This contrasts with the RTB made in Escherichia coliwhich was unstable and had a propensity to aggregate (38). Although a good system in which to purify substrate for in vitro analyses, the oocyte clearly has one major drawback. With yields of 1-13 ng of proricin/oocyte, insufficient material can be purified to perform microsequenceanalysis on the cleavage products or to perform a cytotoxicity assessment. We therefore base our assumption that cleavage occurs at the correct site on comparison of the size of the released fragments with control recombinant RTB using SDSPAGE (Figure 3, lanes 1 and 2). Cleavage also generates a disulfide-bonded conjugate possessing the activities previously ascribed to processed proricin (10) (viz., ribosome-inactivating ability associated with correctly processed and reduced holotoxin). This, and previous fiidings with the RTA-protein A fusions (16),leads us to assume the cleaved molecule would possess cytotoxic ability. To take the analysis further, an alternative eukaryotic expression system would clearly be desired for the production of useful amounts of proricin. N-glycosylation appears to be important in conferring solubility and stability to the molecule, or more specifically to the RTB component of the precursor (24). This requirement
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precludes development of a bacterial expression system. However, fusions of RTA-linker sequences with alternative cell binding ligands having less stringent requirements could well be prepared on large scale using a bacterial host. Recombinant mutant RTB molecules deficient in galactose (cell) binding (24),or in some other property, may be produced in association with RTA carrying a factor Xa site at its C-terminus as described in this report. This strategy would ensure correct folding of the mutant RTB moiety and production of stoichometric amounts of both subunits. The cleaved proricins made this way could then be coupled by more conventional means to a cell-targeting antibody to produce versions of the highly potent holotoxin-containingITS (11). However, the usefulness of the factor Xa cleavage site in conjuctionwith RTAgoes further. Since the presence of eight additional residues at the C-terminus of RTA does not negate its ribosome inactivating-or RTB-reassociation abilities, one might conceive the design of recombinant fusions containing RTA, the modified linker, and the first few residues of RTB (to provide a connecting cysteine) with a whole range of alternative cell-binding ligands. Other cleavage sites for use in recombinant fusions containing RTA or other plant ribosome inactivating proteins may also be considered. Mutation of proricin to generate an a-thrombin-specific site (IVPRGS) (Figure 1A) has also been accomplished (data not shown). Upon expression in Xenopus oocytes the precursor is soluble and sensitive to thrombin and the disulfide bond is correctly formed. Thus lack of an asparagine-specific protease within the endocytic compartments of target eukaryotic cells need no longer preclude construction of cytotoxic recombinant fusions containing RTA. ACKNOWLEDGMENT
We thank Dr. P. Esnouf of The Radcliffe Hospital, Oxford, for the gift of factor Xa and IC1plc for recombinant RTA. This work was supported by the UK Science and Engineering Research Council via Grant GR/G 00877. LITERATURE CITED (1) Olsnes, S., and Phil, A. (1982) In Molecular Action of Toxins and Viruses (P. Cohen, and S. Van Heyningen, Eds.) pp 51105, Elsevier, Amsterdam. (2) Youle, R. J., Murray, G. J., and Neville, D. M. (1979) Ricin linked to monophosphopentamannose binds to fibroblast lysosomal hydrolase receptors, resulting in a cell-type specific toxin. Proc. Natl. Acad. Sci. U.S.A. 76, 5559-5562. (3) McIntosh, D. P., Edwards, D. C., Cumber, A. J., Parnell, G. D., Dean, C. J., Ross, W. C. J.,and Forrester, J. A. (1983) Ricin B chain converts noncytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent. FEES Lett. 164, 17-20. (4) van Deurs, B., Sandvig, K., Petersen, 0. W., Olsnes, S., Simons, K., and Griffiths, G. (1988) Estimation of the amount of internalized ricin that reaches the trans-Golgi network. J. Cell. Biol. 106, 253-267. (5) Sandvig, K., Prydz, K., Hansen, S. H., and van Deurs, B. (1991)Ricin transport in Brefeldin A-treated cells: Correlation between Golgi structure and toxic effect. J. Cell. Biol. 115, 971-981. (6) Endo, Y., Mitaui, K., Motizuki, M., and Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. J. Biol. Chem. 262, 551-559. (7) Lord, J. M. (1985) Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean. Eur. J . Biochem. 146,403-409.
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