The expression of functional ricin B-chain in Saccharomyces cerevisiae

Biochimica et Biophysica Acta, 950 (1988) 385-394

385

Elsevier BBA 91846

T h e e x p r e s s i o n of functional ricin B - c h a i n in S a c c h a r o m y c e s

cerevisiae

P.T. Richardson, L.M. Roberts, J.H. Gould and J.M. Lord Department of Biological Sciences, University of Warwick, Coventry (U.K.)

(Received6 May 1988)

Key words: Ricin; B chain; Heterologousgene expression; (R. communis seed); (S. cerevisiae)

Yeast cells transformed with plasmids containing ricin B-chain coding sequences expressed this heterologous protein. When ricin B-chain was expressed in a form which resulted in its deposition in the yeast cytosol it formed insoluble aggregates which were devoid of galactose-binding activity. In contrast, when DNA fusions were constructed, in which the B-chain coding sequence was preceded by either the preproalpha-factor leader sequence or the native preproricin signal sequence, the recombinant B-chain products were soluble and biologically active. Both the homologous yeast signal peptide and the heterologous plant signal peptide directed the expressed product into the lumen of the yeast endoplasmic reticulum. As a result, the recombinant B-chain products were processed at the N-terminus, glycosylated and folded into an active conformation, presumably stabilized by correct intrachain disulphide bond formation.

Introduction Ricin,

a

heterodimeric protein

present

in

Ricinus communis seeds [1], is one of the most

potently cytotoxic compounds known. It consists of a toxic subunit (the A-chain) which enzymatically inactivates 60 S subunits of eucaryotic ribosomes, joined by a single disulphide bond to a galactose-specific lectin subunit (the B-chain) [2]. Ricin A-chain is an N-glycosidase which acts by cleaving a specific adenine residue from 28 S r R N A (adenine 4324 from rat liver 28 S rRNA) [3,4]. The B-chain is believed to have two important functions in the intoxication process. Firstly, it binds ricin to cell-surface galactose residues through its sugar-binding sites [5], and secondly, it facilitates translocation of toxic A-chain

Abbreviation: PBS, phosphate-bufferedsaline. Correspondence: J.M. Lord, Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, U.K.

across intracellular membranes into the target cell cytoplasm during the ensuing endocytosis [6]. These two B-chain functions apparently reside on separate domains of the polypeptide [7]. Ricin A- and B-chains are initially synthesized together within a single precursor polypeptide. The preproricin gene encodes a precursor consisting of a 35-residue N-terminal leader sequence, which includes, but which does not entirely consist of, an N-terminal signal sequence, followed by the mature A-chain sequence (267 residues), which is joined to the mature B-chain sequence (262 residues) by a 12-residue linker peptide [8]. During ricin biosynthesis in Ricinus seeds, the N-terminal signal sequence mediates the cotranslational translocation of the nascent precursor into the lumen of the endoplasmic reticulum. The signal sequence is cleaved during this step, and proricin is coreglycosylated and disulphide bonds are formed, including the disulphide bond which ultimately joins the mature A- and B-chains of ricin. From the endoplasmic reticulum, proricin is transported via the Golgi apparatus and Golgi-derived vesicles

0167-4781/88/$03.50 © 1988 ElsevierScience Publishers B.V. (Biomedical Division)

386

to the protein bodies. Within the protein bodies, acid proteinase(s) processes proricin to the mature heterodimeric form [8]. There is at present considerable interest in the therapeutic potential of ricin-based immunotoxins, hybrid molecules in which either ricin A-chain alone or whole ricin is chemically conjugated to a cell-reactive monoclonal antibody [9]. In general, A-chain immunotoxins have the advantage of high specificity, but they frequently exhibit low toxicity, presumably because, in the absence of B-chain, the A-chain is not readily translocated into the target cell cytoplasm. In contrast, whole ricin-containing immunotoxins are extremely toxic, but exhibit low specificity, since the B-chain galactosebinding capacity overrides the targeting specificity conferred by the antibody. It is now appropriate to resolve this problem by protein engineering. The gene encoding preproricin has been cloned [10] and the X-ray structure of the native toxin has been determined [11]. The ricin B-chain residues critical for binding to galactose are therefore known and can be altered by site-directed mutagenesis. Modified B-chains will ultimately be expressed from mutant cDNAs in a heterologous system. The ability to express biologically active ricin B-chain is a prerequisite for this approach, and in the present paper, we demonstrate that yeast is a suitable system for this purpose. In order to increase the chances of obtaining wild-type recombinant polypeptides folded into a biologically active (galactose-binding) conformation, the B-chain coding sequence must be preceded by DNA encoding an N-terminal signal sequence. A homologous yeast signal sequence (from the alpha-factor precursor) and the native signal sequence from preproricin both effectively translocate the nascent product into the lumen of the yeast rough endoplasmic reticulum. Materials and M e t h o d s

Reagents. Isotopes, linkers and enzymes for molecular biology were purchased from Amersham International (Amersham, U.K.). Endo-Nacetylglucosaminidase H was from Miles Laboratories (Elkhart, IN, U.S.A.). Yeast expression vector pMA91 [12] and host strain MT302/lc were kindly provided by Drs. A.J. and S.M. Kingsman

(Oxford, U.K.) and the alpha-factor gene plasmid, pDS6a3 [13], was kindly provided by Dr. J. Rothblatt (EMBL, F.R.G.). General methods. Plasmid DNAs were prepared by the alkaline lysis method [14] and purified by centrifugation in caesium c h l o r i d e / e t h i d i u m bromide. Restriction endonuclease digests and ligations were carried out as recommended by the suppliers. Endo-N-acetylglucosaminidase treatment of immunoprecipitates were performed as described previously [15]. Construction of the expression plasmids. The yeast expression vector pMA91 [12] was modified by converting the unique BgllI restriction site into an XhoI site by adding XhoI linkers. The vector generated is designated pMA91X. A DNA fragment containing ricin B-chain coding sequence, preceded by an XhoI site and an initiation codon, was derived from preproricin by oligonucleotide site-directed mutagenesis. A 1954 bp PstI fragment containing preproricin cDNA [9] was cloned into M13mpl9. An XhoI site and ATG were created at positions 759 and 763, respectively, at the 3'-end of the ricin A-chain sequence using a synthetic oligonucleotide (22-mer) carrying three mismatches. This was annealed to single-stranded template and used as a primer for second-strand synthesis using the Klenow fragment of E. coli DNA polymerase I and T4 DNA ligase [16]. Bacteriophages containing the desired mutation were identified by colony hybridization using 32p-labelled mutagenic oligonucleotide as probe. The mutation was confirmed by DNA sequencing [17]. The ricin B-chain-encoding fragment was excised from M13mp19 RF DNA using XhoI and SalI and ligated into XhoI-restricted pMA91X to generate pRIC7. This expression plasmid directs the expression of a fusion protein in which the ricin B-chain sequence is preceded by 24 extra N-terminal amino acids (see Results and Discussion for details). To construct an alpha-factor leader-ricin Bchain fusion, a HindlII site was firstly created at the 5'-end of the B-chain coding region at position 827. This was accomplished using a synthetic oligonucleotide (18-mer) carrying three mismatches by the methods described above. Proricin-encoding DNA was excised from mutant RF D N A by PstI digestion and subcloned into PstI

387 digested pUC8', a form of pUC8 which had been previously modified to remove the polylinker HindlII site. This generated an intermediate from which a B-chain-encoding HindlII-SalI fragment was isolated and religated into pUC8 digested with HindlII and Sail to generate pUC8RB. Plasmid pDS6a3, containing the complete a-factor gene, was restricted with EcoRI and end-filled by incubation with T4 DNA polymerase. XhoI linkers were added, this ligation reconstituting an EcoRI site. On removing excess XhoI linkers, the alpha-factor gene promoter is lost. This plasmid, pdS6a3X, was digested with HindlII and the large fragment was isolated and ligated with HindlIIlinearized pUC8RB. After digestion with XhoI and Sail, the smaller fragment containing alphafactor leader-ricin B chain was isolated and ligated into XhoI-digested pMA91X to generate pRIC12. PreB-chain, comprising the ricin B-chain coding sequence from pUC8RB, to which the native ricin signal sequence was attached, was prepared as described previously [18]. The XhoI-SalI fragment of pUC8preRB was ligated into pMA91X to generate pRICK Expression of ricin B-chain in S. cerevisiae. Yeast (strain M T 3 0 2 / l c ) spheroplasts were transformed by the method of Hinnen et al. [19] as modified by Beggs [20] using pMA91X, pRIC7, pRIC12, pRIC8 or plasmids containing the inserts in the wrong orientation with respect to the phosphoglycerate kinase promoter. Plasmid rescue from yeast transformants confirmed that the D N A was unaltered during transformation (data not shown). Cultures were grown in complete synthetic medium lacking leucine [21]. Harvested cells were resuspended in 10 mM N a H 2 P O 4 (pH 7.2), 150 mM NaCI, 100 mM lactose, 40 /~g/ml phenylmethylsulphonyl fluoride, supplemented where appropriate with 0.2% ( w / v ) sodium dodecyl sulphate a n d / o r 10 mM dithiothreitol. Cells were lysed by vortexing with glass beads. When appropriate, yeast proteins were radiolabelled by growing 50 ml cultures to early log phase, harvesting and resuspending in 50 ml of medium lacking methionine. After a further 1 h at 30 o C to deplete the cells of free methionine, they were reharvested and resuspended in 2-3 ml of m e d i u m s u p p l e m e n t e d with 100 # C i [35S]methionine (approx. 1000 C i / m m o l ) as the sole methionine source. After incubation for 15-30

min at 30 o C, unlabelled methionine was added to a final concentration of 1% (w/v), and the incubation was continued for a further 30 min prior to cell lysis. Proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by fluorography [22]. Immunoprecipitation using antibodies raised in rabbits against either ricin or purified ricin B-chain was performed as described previously [22]. Unlabelled proteins were transferred from gels to nitrocellulose filters [23]. The filters were probed with anti-B-chain or anti-ricin antibodies, and immunoreactive species were identified using the b i o t i n / s t r e p t a v i d i n method (Amersham) or with 125I-labelled protein A followed by fluorography. Purification of recombinant ricin B-chain aggregates. Yeast cells expressing B-chain cytoplasmically from pRIC7 were lysed in the absence of detergent and subjected to low-speed centrifugation in order to remove cell debris. Aggregates of recombinant B-chain were then sedimented from this first supematant by centrifugation at 12000 × g for 10 min. The pellet was resuspended in 5 M urea and 1% (v/v) Triton X-100 and the sample was recentrifuged [24]. Refolding of denatured B-chain. Purified recombinant ricin B-chain aggregates were resuspended in 6 M guanidine hydrochloride/100 mM dithiothreitol by rotating overnight at room temperature. Portions (0.5 ml containing 200 /.tg) of denatured B-chain were then refolded by slow dialysis at 4 ° C firstly into PBS containing 7 M urea, 100 mM dithiothreitol and gradually into a series of PBS solutions of decreasing urea and reducing agent concentrations, culminating in a final dialysis against PBS. In all, seven dialysis steps were used giving sequential, equivalent dilutions (6 M urea/85 mM dithiothreitol; 5 M u r e a / 7 0 mM dithiothreitol, etc.). Assay for B-chain galactose-binding activity. Recombinant ricin B-chain was assayed for lectin activity by its ability to bind to immobilised asialofetuin as described previously [18]. Briefly, asialofetuin was used to coat the wells of a microtitre plate. After blocking and washing, samples containing recombinant B-chain were added, and, after washing, biologically active B-chain, which bound to the asialofetuin, was measured by adding rabbit anti-ricin B-chain antibodies followed

388

by t25I-labelled protein A. By preparing a calibration curve using native ricin B-chain, this radioimmunoassay was used to quantitate active recombinant ricin B-chain production. Results and Discussion

Construction of expression plasmids A ricin B-chain-encoding insert was derived from preproricin c D N A by site-directed mutagenesis in M13, as described in Materials and Methods, and as illustrated in Fig. la. The XhoI-SalI fragment, containing the B-chain coding region, was inserted into pMA91X immediately down-

(a) Preproricin signal 5'i

A chain

(pRCL617)

linker r//A

Bchain ~3'

.ATC ATA GCT CTC ATG GTG TA

AGA

A.

TAT CGA GAG CTC TAC ATA TCT A mutagsnic oligo

.ATC ATA GCT CTC GAG ATG TAT AGA TCG GCA. Xhol

Met~

(b) Xhot 5' [ f / / J

B chain

Sall 3'

stream from the constitutive yeast phosphoglycerate kinase promoter and upstream from the phosphoglycerate kinase transcription terminator (Fig. lb). The product expressed from this construction will be a fusion protein in which the last 12 residues from the carboxy-terminus of ricin A-chain and the 12 residues of the linker region precede the entire B-chain (262 residues). The region mutagenized at the 3'-end of the A-chain was chosen largely for convenience. However, depending on the expression strategy, the presence of this A-chain terminal segment may be advantageous, since this region contains the last A-chain cysteine codon which may ensure correct disulphide bond formation during translation or refolding. This terminal A-chain cysteine residue normally forms a disulphide bond with the first B-chain cysteine, leaving the remaining eight Bchain cysteine residues to form intrachain disulphide bonds in pairwise combinations in the Nto the C-direction [2]. This construct was designated imperfect B. In the second construction, a near-perfect fusion between the complete yeast alpha-factor leader (89 codons [25]) and ricin B-chain was made (Fig. 2). There are only two additional codons, which encode the last two residues of the proricin linker region [9], between the alpha-factor leader and the B-chain. This construct was designated alpha-B. In a final construct, the ricin B-chain D N A was preceded in frame by D N A (35 codons) encoding the native ricin signal sequence, exactly as described elsewhere (not shown) [18]. This construct was designated pre-B and, in common with imperfect B and alpha-B, was inserted into the XhoI expression site of pMA91X.

Expression of imperfect B

2u or" i

AMP R

Fig. I . Construction of a yeast expression p|asmid. (a) I n t r o duction o f a X h o | site and initiation codon at the 3'-end of the

A-chain sequence in preproricin c D N A by site-directed mutagenesis. (b) B-chain containing XhoI-SatI fragment was excised from mutated preproricin and cloned into the Xhol expression site of pMA91X. In the vector, the open box represents yeast D N A sequences and the solid line pBR322 sequences.

The expression vectors pMA91X, pRIC7 and pRIC7r (where the imperfect B-chain insert is in the wrong orientation for expression) were used to transform yeast to leucine prototrophy. A Northern blot of total R N A from untransformed yeast and from the various transformants was probed with a nick-translated ricin B-chain c D N A fragment. Cells transformed with pRIC or pRIC7r produced a transcript of the expected size (data not shown).

389

H

x

P

ppapPo-alpha

/ / / ~ ~

I~ Pstt. ltgate pPortctn fragment

/

EcoRt. ena fill. /

R B

addXhol linkers s Bs

xR

P

@'

Sall. Htndltl

~

B

H

Ad111 RB

Htndttt

H

sp~B xA

Xho1-Sall 11gate into pHA91X.Xhot

i~v,5,R

H B

Fig. 2. Construction of a yeast alpha-factor leader - ricin B-chain expression plasmid. A HindlII site was created in the linker region of preproricin by site-directed mutagenesis in M13mplg. Preproricin was excised and subcloned into PstI-digested modified pUC8', and a ricin B-chain-encoding fragment was isolated and religated into pUC8 to generate pUC8RB. Piasmid pDS6a3 was modified by deleting the yeast alpha-factor promoter (pro), generating pDS6a3X. This plasmid was digested with HindIII, and the large fragment released was ligated with HindIII-linearized pUC8RB. This produced an in-frame fusion between the yeast alpha-factor leader and ricin B-chain, which was then excised as an XhoI-SalI fragment and ligated into the XhoI expression site of pMA91X, to generate pRIC12.

390

1

2

3

4

5

6

7

TABLE I

kD

~.~ 92 68

45

......

U 3 0

Fig. 3. Expression of ricin B-chain in yeast. Yeast transform a n t s were radiolabelled with [3SS]methionine and the cells were lysed as described. Total labelled proteins (lanes 1-3) or immunoprecipitates obtained using rabbit anti-ricin B-chain serum (lanes 4 - 6 ) was separated by SDS-PAGE and visualized by fluorography. Lanes 1 and 4, pMA91X; lanes 2 and 5, pRIC7; lanes 3 and 6, pRIC7r; lane 7, molecular-weight markers.

Expression of imperfect B-chain was confirmed by labelling transformants with [35S]methionine and separating total proteins by SDS-polyacrylamide gel electrophoresis. Yeast transformed with pRIC7 produced a prominent 35S-labelled polypeptide (Fig. 3, lane 2, arrow) which was not present in lysates from ceils transformed with pMA91X or pRIC7r (Fig. 3, lanes 1 and 3, respectively). The molecular weight of this polypeptide was 31 500, the expected size of recombinant imperfect B-chain, and it was immunoprecipitated by rabbit antibodies raised against ricin B-chain (Fig. 3, lane 5). Further, when total proteins from pRIC7 transformants were separated by SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose, this band reacted strongly and exclusively with rabbit antibodies to ricin B-chain, as visualized by the biotin/streptavidin assay (not shown). The stability of the expressed B-chain in yeast cells was examined by pulse-labelling transformants with [35S]methionine for 30 min followed by further incubation for periods of up to 4 h with

LECTIN ACTIVITY O F R E C O M B I N A N T RICIN B - C H A I N Microtitre plate wells were coated with asialofetuin (20 /~g in 200 #1 of 100 m M N a H C O 3 (pH 9.2)) for 3 h at room temperature. The coating solution was removed and the wells were washed three times with PBS containing 0.1% ( v / v ) Tween 20. The wells were blocked by adding 200 /~1 of PBS/Tween-containing 0.2% ( w / v ) bovine serum albumin. After washing, purified ricin B chain or test material was added, and the volume was adjusted to 200 /*1 with blocking solution. After 3 h at room temperature, the wells were evacuated and washed before adding a purified rabbit IgG fraction (20 # g in 200 /zl blocking solution) containing polyclonal antibodies raised against ricin B chain. After an overnight incubation at 4 ° C, the wells were evacuated and washed before adding 125I-labelled protein A (5.105 cpm per well of a 5 #Ci per mg stock in 200 #1 of blocking solution) for 3 h at room temperature. The wells were washed three times in PBS/Tween, three times in PBS and once in water, excised from the plate and counted in an LKB g a m m a counter. Purified ricin B-chain

Recombinant ricin B-chain

A m o u n t assayed (ng)

cpm

transformant

cpm

0 1 2 5 10 15 20

2600 3712 4164 8899 15896 20199 25578

pRIC12 pRIC12r pRIC8 pRIC8r pRIC7 pRIC7r pRIC7 denatured pRIC7r denatured pRIC7 refolded pRIC7r refolded

8892 2819 11 182 2432 2036 2819 2008 2132 21389 2554

10 + 10 m M lactose 10 + 100 m M lactose

4029 2132

excess unlabelled methionine. No evidence for selective degradation of B-chain was obtained (data not shown). Yeast proteins were routinely extracted in the presence of SDS to facilitate electrophoretic analysis. When proteins extracted in the absence of SDS were applied to a Sepharose 4B column, there was no apparent retention of B-chain, presumably because it was unable to bind galactose. Failure to bind galactose was confirmed by the asialofetuin binding radioimmunoassay (Table I). The bulk of the B-chain isolated from yeast in the absence of SDS formed insoluble aggregates, which could be recovered by low-speed centrifugation (Fig. 4, lane 4). Contaminating proteins were readily removed from the B-chain aggregates by washing with u r e a / T r i t o n X-100 solution (Fig. 4, lane

391

1

2

3

4

5

6

i ij!ill !~i!~ !i!ill~!~

•

~ ~ ~i!!i~ i ~ ~ ~ ii~

~

Fig. 4. Purification of aggregated ricin B-chain. Yeast transformants were incubated with [35S]methionine and lysed. After a low-speed spin to remove large debris, the lysates were centrifuged at 12000 x g for 10 rain. The pellets were resuspended in 5 m M urea and 1% Triton X-100, and the samples were recentrifuged. Proteins were separated by SDS-PAGE and visualized by fluorography. Lanes 1 and 2, total proteins from pRIC7r and pRIC7, respectively; lanes 3 and 4, 12000× g pellets from pRIC7r and pRIC7; lanes 5 and 6, washed pellets from pRIC7r and pRIC7.

6). The remaining single polypeptide reacted with ricin B-chain antibodies after Western blotting (not shown). The absence of functional lectin activity of cytoplasmically expressed aggregated Bchain is not surprising. During biosynthesis in R. communis seeds, preproricin is cotranslationally translocated into the lumen of the endoplasmic reticulum, where correct folding and disulphide bond formation are accomplished [15]. The expression construct used here did not include an appropriate signal sequence to ensure that such a translocation step would occur. As expected, the recombinant product was not glycosylated: no decrease in molecular weight was observed after digestion with endo-N-acetyl-glucosaminidase (not shown). Attempts to refold denatured imperfect B-chain were partially successful in that some biological activity was restored (Table I). The yield of suc-

cessfully folded product was, however, less than 5% of the total. Imperfect B-chain is a fusion protein, and the additional 24 amino-acid residues at the N-terminus may have hindered correct refolding under the conditions used here.

Expression of alpha B and pre-B To direct the recombinant polypeptide into the yeast endoplasmic reticulum, constructs were prepared in which the B-chain sequence was preceded by t h e homologous yeast alpha-factor leader (pRIC12) or the native ricin signal sequence (pRIC8). Total yeast proteins obtained from lysed transformants were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters. The filters were probed with mixed polyclonal rabbit antibodies raised against ricin A- and B-chains, and bound antibodies were visualized using biotin/streptavidin or 12sIlabelled protein A. Immunoreactive species were present in extracts of cells transformed with pRIC12 (alpha-B) (Fig. 5, lanes 2 and 3) and pRIC8 (pre-B) (Fig. 6, lane 1), but were not present in extracts of control cells which had been transformed with either vector alone or with vector containing the appropriate insert in the reverse orientation. The size of the recombinant B-chain products was in the expected molecular-weight range, assuming that segregation into the yeast

; ~(

: 1 1 ~ (!~27~ 3 i i ~ i ~

¸ 4¸¸¸¸

5

Fig. 5. Expression of alpha-factor leader-ricin B-chain in yeast. Yeast transformants were lysed and total proteins were separated by SDS-PAGE and transferred to nitrocellulose. The blot was incubated with rabbit anti-ricin serum and bound antibody was visualized using biotinylated protein A and peroxidase-streptavidin. Lane 1, ricin; lanes 2 and 3, pRIC12 transformants; lanes 4 and 5, pRIC12r transformants. The arrows indicate native ricin A- and B-chains.

392 endoplasmic reticulum occurred and was accompanied by core-glycosylation. The contention that the recombinant products were glycosylated was confirmed by endo-N-acetyl-glucosaminidase digestion. Enzymic deglycosylation reduced the apparent molecular weight of both the alpha-B and pre-B products to that expected for non-glycosylated B-chain (Fig. 7, lanes 2 and 6). This result also shows that both the homologous yeast signal sequence (the alpha-factor leader) and a heterologous plant signal sequence (the native preproricin sequence) were capable of directing the segregation of expressed ricin B-chain into the lumen of the yeast endoplasmic reticulum, the site of N-glycosylation [26]. We assume that the presence of minor immunoreactive products, observed particularly in the case of alpha-B (Fig. 5, lanes 2 and 3), may reflect heterogeneity in glycosylation. The deglycosylated pre-B product is the same apparent size as the deglycosylated alpha-B product, indicating that the N-terminal signal sequences were endoproteolytically cleaved during segregation of the nascent polypeptides. The alpha-factor leader contains 89 amino-acid residues and is believed to be composed of a cleavable

I

2

3

4B

Fig. 6. Expressionof prericin B-chain in yeast. Yeast transformants were lysed, and total proteins were separated by SDSP A G E and transferred to nitrocellulose. The blot was incubated with rabbit anti-ricin serum and bound antibody was visualized using 125I-labeiledprotein A and fluorography.Lane 1, pRIC8 transformant; lane 2, ricin; lane 3, pRIC8r transformant. The arrows indicate native ricin A- and B-chains.

N-terminal endoplasmic reticulum translocation signal followed by a signal for secretion [25]. It is therefore significantly larger than the native ricin leader sequence, which contains 35 residues. The removal of the alpha-factor leader sequence further indicates that the endoplasmic reticulumsegregated alpha-B product progressed through the yeast secretory pathway, at least as far as the Golgi apparatus. In the Golgi apparatus, the product of the yeast KEX2 gene acts by cleaving after the Lys-Arg residues at the carboxyl end of the alpha-factor leader [27]. Previous studies have reported that the preproalpha-factor leader directs the secretion of heterologous proteins into the yeast culture medium [28-31]. In the present study, we found that around 10% of the total alpha-B product was secreted, while the remainder was retained within the cells or within the periplasmic space (data not shown). This low level of secretion may reflect the molecular size or some other property of the ricin B-chain. Since preproalpha-factor leader has been found to efficiently direct the secretion of the heterologously expressed 400 kDa envelope glycoprotein derived from Epstein-Barr virus [32], the failure to secrete ricin B-chain efficiently seems unlikely to be due to size per se. The limiting factor apparently resides in the post-endoplasmic reticulum yeast secretory pathway, because we found no evidence for unsegregated, non-glycosylated alpha-B (Fig. 7, lane 6). In the case of the pre-B product, no evidence for secretion was obtained. Both the alpha-B and the pre-B products were biologically active, as d e t e r m i n e d by the galactose-binding radioimmunoassay (Table I). This may in part reflect the generation of correct disulphide bonds within the recombinant B-chain products as a consequence of their cotranslational segregation into the lumen of the yeast endoplasmic reticulum. In keeping with this, centrifugation analysis showed that the segregated alpha-B product was soluble (not shown). A major aim of our research is to delineate the ricin B-chain domain which is responsible for its ability to (a) bind the sugar galactose and (b) facilitate ricin A-chain translocation across a membrane and into the cytoplasm of a target cell. If these two properties are conferred by physically and functionally distinct domains within the B-

393

Q, --

+

--

+

--

+

-

-I-

F i g . 7. Oligosaccharide removal from recombinant ricin B-chain. Recombinant ricin B-chain was recovered from yeast lysates by immunoprecipitation and was separated by SDS-PAGE without ( - ) or after ( + ) incubation with endo-N-acetyl-glucosaminidase. Proteins were visualized by using [125I-labelled protein A and fluorography. Lanes 1 and 2, pRIC8 transformants; lanes 3 and 4, pRIC8r transformants; lanes 5 and 6, pRIC12 transforrnants; lanes 7 and 8, pRIC12r transformants.

c h a i n as r e p o r t e d [7], it s h o u l d b e p o s s i b l e to e l i m i n a t e o n e o f t h e m selectively. U s i n g site-dir e c t e d m u t a g e n e s i s o f ricin B - c h a i n c D N A to alter c o d o n s for a m i n o - a c i d r e s i d u e s r e s p o n s i b l e for galactose binding, mutant B-chains lacking lectin a c t i v i t y b u t still a b l e to p o t e n t i a t e A - c h a i n t r a n s l o c a t i o n will b e p r o d u c e d . S u c h a m u t a n t B - c h a i n w o u l d h a v e c o n s i d e r a b l e p o t e n t i a l for i m m u n o t o x i n c o n s t r u c t i o n , p r o m i s i n g to c o u p l e h i g h p o t e n c y w i t h h i g h specificity [33]. T h i s a p p r o a c h r e q u i r e s a heterologous expression system capable of produci n g b i o l o g i c a l l y active r i c i n B - c h a i n . I n the p r e s e n t r e p o r t , we s h o w t h a t y e a s t is s u i t a b l e for this p u r p o s e , p r o v i d e d t h a t the r e c o m b i n a n t r i c i n Bc h a i n p r o d u c t is d i r e c t e d i n t o the yeast e n d o p l a s m i c r e t i c u l u m , w h e r e we a s s u m e c o r r e c t foldi n g a n d d i s u l p h i d e b o n d f o r m a t i o n take place. References

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