Cytotoxic ribosome-inactivating lectins from plants.

A class of heterodimeric plant proteins consisting of a carbohydrate-binding B-chain and an enzymatic A-chain which act on ribosomes to inhibit protein synthesis are amongst the most toxic substances known. The best known example of such a toxic lectin is ricin, produced by the seeds of the castor oil plant, Ricinnus communis. For ricin to reach its substrate in the cytosol, it must be endocytosed, transported through the endomembrane system to reach the compartment from which it is translocated into the cytosol, and there avoid degradation making it possible for a few molecules to inactivate a large proportion of the ribosomes and hence kill the cell. Cell entry by ricin involves the following steps: (i) binding to cell-surface glycolipids and glycoproteins bearing beta-1,4-linked galactose residues through the lectin activity of the B-chain (RTB); (ii) uptake by endocytosis and entry into early endosomes; (iii) transfer by vesicular transport to the trans-Golgi network; (iv) retrograde vesicular transport through the Golgi complex and into the endoplasmic reticulum (ER); (v) reduction of the disulfide bond connecting the A- and B-chains; (vi) a partial unfolding of the A-chain (RTA) to enable it to translocate across the ER membrane via the Sec61p translocon using the pathway normally followed by misfolded ER proteins for targeting to the ER-associated degradation (ERAD) machinery; (vi) refolding in the cytosol into a protease-resistant, enzymatically active structure; (vii) interaction with the sarcin-ricin domain (SRD) of the large ribosome subunit RNA followed by cleavage of a single N-glycosidic bond in the RNA to generate a depurinated, inactive ribosome. In addition to the highly specific action on ribosomes, ricin and related ribosome-inactivating proteins (RIPs) have a less specific action in vitro on DNA and RNA substrates releasing multiple adenine, and in some instances, guanine residues. This polynucleotide:adenosine glycosidase activity has been implicated in the general antiviral, and specifically, the anti HIV-1 activity of several single-chain RIPs which are homologous to the A-chains of the heterodimeric lectins. However, in the absence of clear cause and effect evidence in vivo, such claims should be regarded with caution.

Using cardiac phase to order reconstruction (CAPTOR): a method to improve diastolic images.

A method is proposed to reconstruct multiphase images that accurately depicts the entire cardiac cycle. A segmented, gradient-recalled-echo sequence (FASTCARD) was modified to acquire data continuously. Images were reconstructed retrospectively by selecting views from each heartbeat based on cardiac phase rather than the time elapsed from the QRS complex. Cardiac phase was calculated using a model that compensates for beat-to-beat heart rate changes. Images collected using cardiac phase to order reconstruction (CAPTOR) depict the entire cardiac cycle and lack the temporal gap that is characteristic of prospectively reconstructed sequences. Time-volume curves of the left ventricle capture the contribution of atrial contraction to end-diastolic volume (EDV). Transmitral phase-contrast flow measurements show a second peak inflow (alpha wave) that is absent in the standard sequence. Because atrial contraction contributes to ventricular EDV, images using CAPTOR potentially may provide a more reliable measure of EDV, stroke volume, and ejection fraction than standard techniques.

Resistance to geminivirus infection by virus-induced expression of dianthin in transgenic plants.

Ribosome-inactivating proteins (RIPs) are naturally occurring plant toxins that exhibit antiviral activity against a diverse range of plant and animal viruses. Here, the action of dianthin, a potent RIP isolated from Dianthus caryophyllus, has been exploited to engineer resistance to a plant DNA virus, African cassava mosaic virus (ACMV), in transgenic Nicotiana benthamiana. To achieve this, dianthin has been expressed from the ACMV virion-sense promoter that is transactivated by the product of viral gene AC2. This avoids the need for constitutive expression of the RIP, facilitating the regeneration of phenotypically normal plants, and ensures transgene expression is localized to virus-infected cells. When challenged with ACMV, transgenic plants produce atypical necrotic lesions on inoculated leaves, indicative of dianthin expression, viral DNA accumulation is significantly reduced in these tissues, and plants exhibit attenuated systemic symptoms from which they recover. This phenotype holds for isolates of ACMV but not for other geminiviruses, suggesting that AC2 homologues from the latter are unable to efficiently transactivate the ACMV promoter.

The action of pokeweed antiviral protein and ricin A-chain on mutants in the alpha-sarcin loop of Escherichia coli 23S ribosomal RNA.

The alpha-sarcin loop of Escherichia coli 23S rRNA is a universally-conserved structure involved in the binding of elongation factors Tu and G and is the site of action of the ribosome-inactivating proteins (RIPs). One such group, the N-glycosidase RIPs, act by the removal of a single adenine residue (A2660) believed to exist in a GAGA-containing tetraloop structure. The action of two RIPs, the catalytic A-chain from the heterodimeric toxic lectin ricin (RTA) and the single-chain RIP pokeweed antiviral protein (PAP), which are known to be highly homologous in tertiary structure, was determined on native ribosomes or naked 23S rRNA containing mutations designed to affect the structure of the GAGA tetraloop. One such mutant rRNA containing G2663C, which abolishes the potential tetraloop by disrupting the Watson-Crick base-pair involved in closing it, resulted in a loss of depurination by RTA, but not by PAP. A similar result was observed for mutant G2661A. The double mutant C2658G + G2663C, which restores the tetraloop-closing base-pair but in the reverse orientation, resulted in sensitivity to both PAP and RTA, as in the wild-type. Thus, the tetraloop structure is required for the action of RTA, but not of PAP, and unlike RTA, PAP does not require G at position 2661. RNA containing the G2664C mutation, which lies outside the tetraloop, served as a substrate for both PAP and RTA. The comparison of the recognition elements for PAP and RTA was made with naked (deproteinised) rRNA, because RTA does not act on E. coli ribosomes. However, PAP is active on E. coli ribosomes, and it was found that the action of PAP on ribosomes containing the above mutations paralleled exactly that on the corresponding naked rRNAs. It is concluded that the recognition elements for PAP and RTA differ and may account, at least in part, for the fact that PAP but not RTA catalyses the depurination of E. coli ribosomes.

Wheat ribosome-inactivating proteins: seed and leaf forms with different specificities and cofactor requirements.

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.

Mutational studies on the alpha-sarcin loop of Escherichia coli 23S ribosomal RNA.

The alpha-sarcin loop, located in domain VI of Escherichia coli 23S rRNA, is a universally conserved sequence involved in the binding of elongation factors to the ribosome and is the site of action of ribosome-inactivating proteins. Six mutations were created in this loop with the aim of establishing whether the mutant 23S rRNA could be assembled into functional ribosomes. In order to distinguish between plasmid-derived (mutant) and chromosome-derived (wild-type) 23S rRNAs, an oligonucleotide tag sequence was introduced into the plasmid-borne 23S rRNA gene. The tag sequence had no apparent effect on ribosome assembly or function. Two of the bases mutated (at positions A2660 and G2661) have been implicated in the binding of both elongation factor Tu and elongation factor G to the ribosome [Moazed, D., Robertson, J. M. & Noller, H. F. (1988) Nature 334, 362-364]. A further two bases (at positions C2658 and G2663) have been proposed to form a Watson-Crick base pair involved in the formation of a tetraloop structure required for ribosome function [Szewczak, A. A., Moore, P. B., Chan, Y. L. & Wool, I. G. (1993) Proc. Natl Acad. Sci. USA 90, 9581-9585]. It is inferred that the identity of the bases at positions 2658 and 2663 are of critical importance for ribosome structure and function, and that this function cannot be restored by a second mutation which potentially restores a Watson-Crick base pair, but with reversed position. Of five single mutants (each mutant containing one of the mutations C2658G, A2660G, G2661A, G2663C and G2664C) and one double mutant (containing both mutations C2658G and G2663C) only the two mutants with the single mutations G2661A and G2664C were incorporated into ribosomes at a level comparable to that of 23S rRNA expressed from a wild-type plasmid. However, the G2664C mutation resulted in a decrease in growth rate and a gradual loss of viability. rRNAs containing the G2663C single mutation and the C2658G and G2663C double mutation showed reduced incorporation into 50S subunits and these did not enter into ribosome couples.

Pokeweed antiviral protein (PAP) mutations which permit E.coli growth do not eliminate catalytic activity towards prokaryotic ribosomes.

Pokeweed antiviral protein (PAP) has N-glycosidase activity towards both eukaryotic and prokaryotic ribosomes. This is in marked contrast with the A chains of type 2 ribosome inactivating proteins (RIPs) such as ricin and abrin, which inactivate only eukaryotic ribosomes. A recent report described spontaneous mutations in PAP that implicated specific amino acids to be involved in determining the activity of PAP towards prokaryotic ribosomes. As part of an ongoing study into RIP--ribosome interactions these mutations were specifically recreated in a PAP clone encoding the mature 262 amino acid PAP sequence. Mutants were tested for their N-glycosidase activity by analysing the integrity of eukaryotic and prokaryotic ribosomes after mutant protein expression. Mutations of F196Y and K211R, either individually or within the same clone, were active toward both classes of ribosome, indicating that these amino acid positions are not involved in differentiating ribosomal substrates. Mutation R68G led to a protein that appeared to be inactive towards prokaryotic ribosomes, but also very poorly active towards eukaryotic ribosomes. This mutation is currently under further investigation.

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes.

Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.

Characterisation of saporin genes: in vitro expression and ribosome inactivation.

A. Saponaria (soapwort) genomic library was screened with a PCR-derived saporin-specific gene probe. The nucleotide sequences of three saporin genomic clones were determined. One of the clones contained a full-length saporin coding sequence whilst the other two were truncated. A hybrid full-length saporin coding sequence was constructed using the two truncated clones. An SP6 promoter sequence and in-frame initiation codon was added to each of the coding sequences using PCR. In vitro translation of saporin coding sequence transcripts in rabbit reticulocyte lysates resulted in the specific depurination of 28S RNA. This indicated that the saporin sequences encoded functional polypeptides with RNA N-glycosidase activity.

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3' end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A2660, which lies in a sequence that is highly conserved in all species.

Ribosome inactivating proteins of plants.

Many plant tissues produce single chain proteins which can enzymatically remove a specific adenine residue from ribosomal RNA. Although these proteins are potently toxic to isolated ribosomes, they are non-toxic to intact cells, being unable to gain access to their ribosomal substrate. In certain plants however, the gene for the ribosome inactivating protein has fused with a gene encoding a galactose-specific lectin. This generates heterodimeric proteins which can bind to and enter target cells, and which are among the most potent cytotoxins known.

Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation.

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.

Ribosome inactivation by ricin A chain: a sensitive method to assess the activity of wild-type and mutant polypeptides.

When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves.

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.

Binding Sites of E. coli DNA-dependent RNA polymerase on spinach chloroplast DNA.

Under stringent conditions E. coli DNA-dependent RNA polymerase holoenzyme binds selectively to some spinach chloroplast DNA fragments generated by restriction endonucleases. The strongest of these binding sites, as judged by the initial rate of complex formation, are located in the large single-copy DNA region (Crouse et al. 1978) of this molecule and correspond in map location with known protein coding sequences. Some of these binding sites have characteristics of complex formation comparable with those of the PR and PL promoters of phage lambda DNA.Binding sites located close to the rRNA operons on the chloroplast DNA bind polymerase less strongly than those described above. Since the rRNAs are the most abundant transcription products in vivo and in isolated chloroplasts (Hartley and Head 1979; Bohnert et al. 1977) this suggests that the E. coli and chloroplast enzymes do not recognize all of the major promoters in chloroplast DNA with the same efficiency of binding.We have investigated in detail one region of the chloroplast DNA from spinach which contains three strong binding sites. This region has been shown to contain at least the gene for a 32,000 dalton protein (Driesel et al. 1980) which is most probably the so-called photogene (Bedbrook et al. 1978).One of these three E. coli RNA polymerase binding sites is not more than approximately 150 by apart from what, by hybridization studies using isolated mRNA, we know to be the coding sequence for this protein.The results suggest that for some genes on the chloroplast DNA the bacterial RNA polymerase may be used to search for transcription initiation sites.

The synthesis of chloroplast high-molecular-weight ribosomal ribonucleic acid in spinach.

Illuminated suspensions of chloroplasts isolated from young spinach leaves show incorporation of [3H]uridine into several species of RNA. One such RNA species of Mr 2.7 x 10(6) shows sequence homology with both the chloroplast 23-S rRNA (Mr = 1.05 x 10(6)) and 16-S rRNA (Mr = 0.56 x 10(6)), as judged by DNA/RNA competition hybridization. Leaves labelled in vivo with [32P]orthophosphate in the presence of chloramphenicol accumulate labelled RNAs of Mr 1.28 x 10(6), 0.71/0.75 x 10(6) and 0.47 x 10(6). The 1.28 x 10(6)-Mr RNA shows 80.5% sequence homology with the 1.05 x 10(6)-Mr rRNA and the 0.71/0.75 x 10(6)-Mr RNA mixture shows 76% sequence homology with the 0.56 x 10(6)-Mr rRNA. We conclude that the pathway of rRNA maturation in spinach chloroplasts is similar to that of Escherichia coli.

The synthesis and origin of chloroplast low-molecular-weight ribosomal ribonucleic acid in spinach.

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA species which co-electrophorese with 5-S rRNA and tRNA, but show very little incorporation into 4.5-S rRNA. Chloroplast 4.5-S rRNA is labelled in vivo after a distinct lag period relative to 5-S rRNA and tRNA. The kinetics of labelling in vivo of chloroplast 5-S rRNA are similar to those of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, whereas the kinetics of labelling of the 4.5-S rRNAare similar to those of mature 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs. Chloramphenicol inhibits the labelling of chloroplast 4.5-S rRNA in vivo, and concomitantly inhibits the processing of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, but has little effect on the appearance of label in chloroplast 5-S rRNA. DNA/RNA hybridization using 125I-labelled RNAs suggests that chloroplast DNA contains a 2--3-fold excess of 4.5-S and 5-S rRNA genes relative to the high-molecular-weight rRNA genes. Competition hybridization experiments show that the immediate precursor to the 1.05 x 10(6)-Mr rRNA effectively competes with 125I-labelled 4.5-S rRNA for hybridization with chloroplast DNA, and is therefore a likely candidate for a common precursor to both the 1.05 x 10(6)-Mr and 4.5-S rRNAs.