2.1. Purification and Expression of Dianthins
Dianthin-30 and dianthin-32 were first purified to homogeneity from the leaves of
D. caryophyllus L. by chromatography on carboxymethyl (CM-)cellulose, pH 6.5 [
1]. The apparent molecular masses determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are 29,500 and 31,700 Da respectively, and origin for the naming [
1]. Isoelectric focusing provided a single band with a basic isoelectric point of 8.65 for dianthin-30 and 8.55 for dianthin-32, consistent with their chromatographic behavior on CM-cellulose [
23]. As determined by rocket immunoelectrophoresis and by the ability to inhibit protein synthesis, dianthin-30 is present throughout the entire plant while dianthin-32 is located only in leaves and growing shoots [
24]. In the older parts of the plant, dianthin contributes to 1% to 3% of the total extractable protein, whereas much less is present in the younger parts [
24].
The inhibitory activity of dianthin-30 and dianthin-32 was unchanged on pre-incubation at 37 °C for 1 h in the presence of 1% 2-mercaptoethanol, or after freezing and thawing ten consecutive times, or after keeping at 37 °C for 18 h, but was completely abolished by boiling for 20 min [
1]. Freeze-dried dianthin-32 retained full activity after solubilization while freeze-dried dianthin-30 was poorly soluble and could not be tested [
1].
Dianthin-29 was isolated and purified from frozen leaf material by affinity chromatography on Blue 2 S-Sepharose and subsequent cation exchange chromatography on Mono S [
2]. The apparent molecular mass in SDS-PAGE is 29,000 Da [
2].
Antibodies are helpful tools for the purification and detection of proteins. Strocchi et al. produced a polyclonal rabbit antiserum against dianthin-32, which showed no cross-reactivity to lychnin from
Lychnis chalcedonica L. seeds, pokeweed antiviral protein from
Phytolacca americana L. roots (PAP-R), trichokirin from
Trichosanthes kirilowii Maxim. seeds, and colocin-1 from
Citrullus colocynthis (L.) Schrad. seeds, week cross-reactivity to saporin-S6 from
S. officinalis L. seeds, momordin from
Momordica charantia L. seeds and momorcochin-S from
Momordica cochinchinensis (Lour.) Spreng. seeds, and high cross-reactivity to bryodin-R from
Bryonia dioica Jacq. roots [
25]. This antiserum also showed week or medium cross-reactivity with two RIPs isolated from leaves of
Phytolacca dioica L. [
26] and RIPs isolated from the seeds of
Saponaria ocymoides L.,
Vaccaria hispanica (Mill.) Rauschert [
27] and
Basella rubra L. [
28], and from the skinned fruit of
Cucurbita moschata Duchesne [
29]. A rabbit immune antiserum raised against saporin-S6 did not cross-react with dianthin-32 [
30]. Porro and colleagues produced five different highly specific anti-dianthin-32 monoclonal antibodies that revealed no cross-reactivity to momochin from
M. cochinchinensis (Lour.) Spreng. and momordin, and two of them also exhibited no cross-reactivity to saporin-S6, and gelonin from
Gelonium multiflorum A. Juss. However, all of them bound to dianthin-30, three of them even better than to dianthin-32 [
31]. The affinity constants of these anti-dianthin monoclonal antibodies ranged between 1.7 × 10
10 M
–1 and 3.3 × 10
8 M
–1 [
31]. This shows that antibodies against dianthin often exhibit cross-reactivity to RIPs from related plants and sometimes also to RIPs from unrelated plants, as observed for bryodin. Interestingly, four serum samples collected from 17 persons who were employees in a research laboratory and were in daily or in rare contact with RIPs contained specific immunoglobulin E (IgE) against dianthin-30, gelonin, momordin, pokeweed antiviral protein from seeds (PAP-S), saporin, ricin from
Ricinus communis L. seeds and volkensin from
Adenia volkensii Harms roots. In contrast, asparin from
Asparagus officinalis L. seeds and lychnin did not show any IgE binding. Strikingly, among the other plant proteins, RIPs were exclusively recognized by IgE in immunoblots [
32].
An important alternative to purification from natural sources is heterologous expression, however, as RIPs inhibit eukaryotic ribosomes, expression in eukaryotic cells is excluded and even expression in prokaryotes is sometimes not possible [
11]. Nevertheless, dianthin-30 can be expressed in bacterial systems. This was first described by Legname and colleagues who cloned the cDNA encoding for dianthin-30 into the expression plasmid pKK 233.2 for protein production in
E. coli strain JM109 [
33]. The low amount of 20 µg/L recombinant protein was attributed to the inhibitory effect of dianthin-30 on bacterial protein synthesis as it was shown before that the 23S rRNA of
E. coli is also a target for dianthin [
34]. The yield was substantially increased to 10 mg/L by use of the pET11d expression vector [
14]. The authors expressed dianthin-30 and a deleted form (Δ255–270), lacking the putative 16 amino acids long pro-signal sequence, and determined the IC
50 after purification via cation exchange chromatography on Mono S to 0.29 nM, 0.37 nM, and 0.61 nM for dianthin-30 Δ255–270, recombinant dianthin-30 and natural dianthin-30 respectively, measuring the inhibition of (
14C)-leucine incorporation into protein using a rabbit reticulocyte system [
14]. Investigations by circular dichroism spectroscopy indicated that the natural and the recombinant forms of dianthin-30 possess the same secondary structure composition, accounting for an α + β type architecture [
14]. A hexahistidine-tagged dianthin-30 was expressed by Gilabert-Oriol and co-workers in pET11d using
E. coli Rosetta 2(DE3) pLysS and purified by Ni-nitrilotriacetic acid affinity chromatography eluting at 125 mM imidazole [
13]. Mass spectrometry showed a single peak of 29,531 Da, indicating that the
N-terminal methionine is cleaved off in a bacterial post-translational modification [
13]. Circular dichroism measurements conducted after different storage times at −20 °C, 4 °C and 25 °C indicated that the protein stability was not affected by the storage and the protein conformation remained the same. The protein was stable up to 60 °C, where alterations of the structure commenced and achieved 50% denaturation at 66 °C and complete denaturation above 72 °C [
13].
2.2. Primary and Spatial Structure of Dianthin-30 and Its Catalytic Center
Rabbit antibodies raised against dianthin-30 were used to identify a full-length dianthin precursor cDNA clone from a lambda gt11 expression library [
35]. The cDNA was 1153 base pairs in length and encoded a precursor protein of 293 amino acid residues, of which the first 23
N-terminal amino acids represented the signal sequence [
35]. A comparison of the amino acid sequence of dianthin-30 revealed 83% homology with gypsophilin-S from the seeds of
Gypsophila elegans M. Bieb. [
36], 80% homology with saporin-S6 [
21] but only 17% with trichosanthin from
T. kirilowii Maxim. and 20% with ricin A-chain [
35] (
Figure 1). The
C-terminal region contains an
N-glycosylation site and shows homology to a
C-terminal propeptide present in several plant vacuolar proteins, such as wheat germ agglutinin and barley lectin [
35], which is necessary for proper sorting of the lectin to vacuoles [
37]. The major sugars observed at the glycosylation site are mannose, glucose and glucosamine for dianthin-32, and xylose and glucose for dianthin-30 [
23].
The three-dimensional structure of dianthin-30 was first predicted by a computer model applying homology modeling on the basis of two known RIP structures from PAP and ricin A-chain [
40]. The results demonstrate that, despite the similarity of the topology of the binding site, differences in the electrostatic potential can account for experimentally observed differences in substrate recognition and binding for the investigated RIPs [
40]. Experimentally, the structure was independently solved by Kurinov and colleagues for recombinantly expressed dianthin-30 (Δ255–270) (Protein Data Bank (PDB) IDs: 1LP8, 1LPC, 1LPD including complexes with adenyl-guanosine and cyclic AMP) at 1.7 Å resolution [
41], and by Fermani et al. for dianthin-30 purified from leaves of
D. caryophyllus L. (PDB ID: 1RL0) at 1.4 Å resolution [
38,
42]. Despite some varieties in the loop regions, the typical folding for RIPs is conserved. The structures of dianthin-30 and saporin-S6 fit quite well and both show a protein segment containing strands β7, β8 and β9 that is shorter than in other RIPs, however, the surface electrostatic potential in the active site region distinguishes dianthin-30 from saporin-S6 [
38] (
Figure 2). While the active site of saporin-S6 is characterized by a negative potential, dianthin-30 contains both a negative potential and an extended positive region in the catalytic cavity [
38] corroborating the conclusions from the computer model on differences in the electrostatic potential for PAP and ricin A-chain [
40]. The four key residues in the catalytic center, Tyr-73, Tyr-121, Glu-177 and Arg-180, are fully conserved in dianthin-30, saporin-S6, PAP, momordin, trichosanthin, lychnin, bouganin, gelonin, bryodin and ricin A-chain [
38,
43,
44]. Molecular modeling studies of the interactions of dianthin-30 with a single-stranded RNA heptamer predicted a potent anti-human immunodeficiency virus (HIV-) RNA activity due to the unique surface topology and charge distribution in its 20 Å RNA binding cleft. The estimated release was 352 ± 27 pmol adenine per microgram of RNA per hour [
41]. Anti-HIV effects were observed for both dianthin-30 and dianthin-32 [
45,
46].
2.3. Enzymatic Activity and Biological Function
All RIPs mediate the release of adenine-4324 (number referred to rat) from the 28S rRNA of eukaryotic ribosomes [
9,
10]. For dianthin-30 and dianthin-32, this was indirectly confirmed by Reisbig and Bruland who demonstrated that ribosomes remain active when dianthin-treated 40S subunits are combined with untreated 60S subunits but become inactive when treated 60S subunits are combined with untreated 40S subunits [
24]. The release of adenine-4324 results in an aldehyde radical at C1 of the ribose [
9] and this aldehyde group inhibits the activities of the eukaryotic elongation factor (eEF1A)-dependent aminoacyl-tRNA binding to the inactivated ribosome and eEF1A-dependent guanosine-5’-triphosphatase (GTPase), but increases eEF2-dependent activity [
49]. The catalytic activity of RIPs is mainly determined indirectly by in vitro translation assays using rabbit reticulocyte lysate. Similar IC
50 values between 0.133 nM and 0.61 nM were determined for dianthin-30 in different studies (
Table 2). Dianthin-30 and dianthin-32 also inhibited protein synthesis in wheat germ extracts at 0.35–0.70 nM [
24], while for ricin A-chain, a similar effect requires more than 800 nM [
50]. Moreover, ribosomes from several species are sensitive to their own RIPs, including dianthin-32 [
2], saporin [
51], and PAP [
52]. Notably, the enzymatic activity must not be confused with cytotoxicity as complete ricin possesses a cell binding domain (B-chain) while dianthin can enter cells only by chance. The enzymatic activity of dianthin-30 (Δ255–270), which lacks the glycosylation site, is comparable to that of full-length dianthin-30, indicating that the C-terminus and the sugar residues are not involved in the
N-glycosidase activity [
14]. For saporin-S3 that shares (as saporin-S6) 80% homology with dianthin-30 [
21], a complete loss of in vivo activity is observed in the double mutant E176K, R179Q (KQ-mutant) [
53], two residues of the catalytic center (residues 177 and 180 in dianthin-30) [
38]. Therefore, it can be expected that in dianthin-30, the analogous KQ-mutant will also result in enzymatic activity loss.
As mentioned before, in several cases, RIPs can also inhibit prokaryotic ribosomes. Ferreras and colleagues investigated the effects of 29 different type 1 and type 2 RIPs on polyuridylic acid-directed polyphenylalanine synthesis carried out by purified ribosomes from
Streptomyces lividans. Only five of them exhibited an IC
50 below 1 µM including dianthin-32 (331 nM) and as the most effective RIP, crotin-3 (19 nM) from
Croton tiglium L. seeds while dianthin-30 had an IC
50 of 6.5 µM [
12], indicating that dianthin-30 is more suitable for heterologous expression in bacteria than dianthin-32.
Cenini and colleagues examined the effect of dianthin-32 on ribosomes of the ciliate
Tetrahymena pyriformis and the amoeba
Acanthamoeba castellanii [
58], and of both dianthin-30 and dianthin-32 on ribosomes of the parasites
Trypanosoma brucei rhodesiense and
Leishmania infantum [
56]. None of the type 2 RIPs ricin, abrin from
Abrus precatorius L., modeccin from
Adenia digitata (Harv.) Engl., viscumin from
Viscum album L., and volkensin, and the type 1 RIPs gelonin and bryodin had any effect on phenylalanine polymerization by
T. pyriformis ribosomes while PAP-S (IC
50 = 1570 nM) and saporin-S6 (IC
50 = 2630 nM) had a weak effect, momordin a moderate effect (IC
50 = 300 nM) and dianthin-32 a strong effect (IC
50 = 30 nM) [
58]. The sensitivity of
A. castellanii ribosomes to these RIPs was higher than that of
T. pyriformis RIPs but a strong effect was only observed for abrin (IC
50 = 100 nM), saporin-S6 (IC
50 = 17 nM) and again, dianthin-32 (IC
50 = 7 nM), indicating that the efficacy of RIPs on eukaryotic ribosomes is dependent on the RIP and species [
58] (
Table 2). This was further corroborated by investigating the effect on
T. brucei and
L. infantum ribosomes where ricin, modeccin, viscumin, volkensin, gelonin, momordin, mochin from
M. cochinchinensis (Lour.) Spreng., bryodin, trichokirin and barley (
Hordeum vulgare L.) RIP had no or only very week effects (IC
50 > 1.2 µM) while abrin, PAP-S, PAP-R, saporin-S6, saporin-S9 as well as dianthin-30 and dianthin-32 had strong effects, the IC
50 ranging from 153 nM down to 5 nM [
56]. It is notable that different publications describe RIP isolates from
M. cochinchinensis (Lour.) Spreng. that are called mochin, momochin, momorchin and momorcochin. As sequence data are missing in most of the publications, it remains unclear whether these names describe identical RIPs or isoenzymes. Indeed, in many cases, it is even unclear whether the isolate is a uniform protein. Taylor et al. investigated the rRNA depurination activities of five RIPs using yeast and tobacco leaf ribosomes. PAP-L, dianthin-32, tritin from wheat (
Triticum aestivum L.) germ,
H. vulgare L. RIP and ricin A-chain were all active on yeast ribosomes with dianthin-32 being the most active (IC
50 = 0.019 nM) but only dianthin-32 (IC
50 = 0.63 nM), PAP (IC
50 = 0.13 nM) and ricin A chain (IC
50 = 20.8 nM) were active on tobacco ribosomes [
52] (
Table 2).
The enzymatic activity of RIPs is not highly specific for 28S rRNA. Other nucleic acids including plasmids, herring sperm DNA (hsDNA), poly(A) and bacterial rRNA might also be recognized as substrate by a number of RIPs [
62]. Hartley and co-workers showed that bacterial 23S rRNA can be deadenylated by the type 1 RIPs dianthin-30, dianthin-32, PAP-L and PAP-S but not by the A-chains of the type 2 RIPs ricin and abrin [
34]. This was proven by the release of a fragment of 243 nucleotides from the 3′ end of 23S rRNA following aniline treatment of the RNA [
34]. It is known that deadenylation renders the surrounding phosphodiester bonds highly susceptible to hydrolysis after treatment with aniline [
9,
10]. The position of deadenylation by dianthin-32 was found to be A-2660, which lies in a sequence that is highly conserved in all species [
34]. Roncuzzi and Gasperi-Campani described a DNA-nuclease activity for dianthin-30, saporin-S6 and gelonin in addition to the
N-glycosidase activity [
63]. In double-stranded, supercoiled pBR322 plasmid DNA, they identified four cleavage sites for dianthin-30 and saporin-S6, and two cleavage sites for gelonin, while ricin did not show any nuclease effect [
63]. It is questionable whether the observed DNase activity is indeed present. Instead, the phenomenon might be rather a consequence of an
N-glycosidase activity, which alters the torsional stress of the supercoiled DNA with subsequent break of the DNA strand [
64]. Topologically active dianthin-30 and dianthin-32 was also observed for other plasmids including pGEM4Z and pBlueScript SK
+ [
45,
65,
66].
The recognition of other substrates can be used to determine the catalytic activity of dianthin directly instead of using indirect effects in translation assays. The principle of all these assays is to determine the amount of adenine released in the presence of the enzyme. Heisler and colleagues developed a colorimetric assay that is conducted in a single multi-reaction incubation step and allows enzyme kinetic measurements [
67]. The key step is the conversion of released adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally result in three inorganic phosphate ions per adenine molecule that are quantitated by a color-generating phosphorolysis reaction [
67]. The activity for dianthin-30 was determined for the 60S ribosomal subunit, 28S-rRNA, mitochondrial DNA (mtDNA), hsDNA and poly(A). Compared to ricin A-chain and saporin-S3 (which is almost identical to saporin-S6 [
21]), the adenine release was highest for dianthin-30 when using mtDNA and hsDNA as substrate, the maximum value reached for hsDNA with 775 picomoles adenine release per picomole of RIP per hour of incubation while ricin A-chain was the most active RIP on 28S-rRNA, exhibiting a release of 185 pmol/pmol/h [
67] (
Table 3). In this article, the enzymatic activity of a RIP is always expressed as picomoles adenine release per picomole of RIP per hour of incubation (pmol/pmol/h). If other units were used in the literature sources, magnitude and unit were converted accordingly. For more clarity of the unit, we did not reduce the unit fraction to h
–1, which would be correct but more indistinct. For all three RIPs, the release was below the detection limit of 10 pmol/pmol/h when using the 60S subunit or poly(A) as substrate. Fermani and co-workers quantified released adenine by HPLC/MS ESI [
43]. Using this sensitive method, they measured a release of 0.39 to 0.72 pmol/pmol/h for dianthin-30, lychnin, momordin I, ricin A-chain, bouganin, and PAP from rat ribosomes while saporin-S6 was most active exhibiting a release of 1.91 pmol/pmol/h. Adenine release from hsDNA was an order of magnitude higher for dianthin-30, saporin-S6, bouganin and PAP compared to lychnin, momordin-I and ricin A-chain [
43], corroborating the results from Heisler and colleagues. Adenine release from poly(A) was only detectable for dianthin-30 (0.54 pmol/pmol/h) and saporin-S6 (>30 pmol/pmol/h) [
43]. Weng successfully applied a poly(dA) 30mer and detected released adenine by thin-layer chromatography (TLC) and TLC-densitometry measuring UV absorbance at 260 nm. For the optimization of the assay, deoxy-adenine oligonucleotides with different lengths were used [
68]. The release from the poly(dA) 30mer was substantially better than from an RNA that mimics the ribosomal sarcin loop and determined to 110 pmol/pmol/h for dianthin-30 [
68]. It is unclear why the strikingly low activity of dianthin-30 observed by Fermani et al. and Heisler et al. for poly(A) [
43,
67] compared to Weng is attributed to the defined length of 30 residues or to the deoxynucleotides used by Weng. In another study where hsDNA was used as substrate, adenine release by saporin-S3 was 10-fold higher than observed by Fermani et al. for saporin-S6, and 5-fold higher for ricin A-chain [
69].
Lubelli et al. described an immuno-polymerase chain reaction assay to detect dianthin and ricin, a method suitable to quantify low amounts of these RIPs (down to 0.01 pg/mL) independent of their enzymatic activity [
70]. In this assay, dianthin was detected with a primary and secondary biotin-labelled antibody, and a biotinylated reporter DNA was bound to the secondary antibody using streptavidin as a bridge. Quantitation occurred by polymerase chain reaction of the reporter [
70].
As early as 1925, Duggar described the anti-viral effect of pokeweed juice on tobacco plants affected with the tobacco mosaic virus [
75]. The active protein was isolated in 1969 [
76] and the effect on the larger ribosomal subunit was shown in 1973 [
77]. The abbreviation PAP originally stood for
P. americana L. peptide but was later used as pokeweed antiviral protein. Stirpe et al. first described the antiviral effect of dianthins [
1]. The authors infected tobacco leaves with the tobacco-mosaic virus in the presence and absence of dianthin-30 and dianthin-32 and observed almost complete inhibition of virus-mediated lesions at 300 nM [
1] (
Table 4). This was confirmed by Taylor and colleagues who observed that PAP and dianthin-32 fully inhibit the formation of local lesions at 300 and 3000 nM and more than 80% at 30 nM and 60 nM, whereas tritin,
H. vulgare L. RIP and ricin A-chain were essentially ineffective [
52]. Foà-Tomasi et al. infected HEp-2 cells with herpes simplex virus-1 (HSV-1) or with poliovirus-I in the presence of dianthin-32, PAP-S, gelonin, and a RIP from the seeds of
M. charantia. All proteins investigated reduced the viral yield, decreased HSV-1 plaque-forming efficiency (
Table 4), and inhibited protein synthesis more in infected than in uninfected cells, presumably caused by entering infected cells more easily [
78]. As expected, the potency of RIPs on protein synthesis in cells is not the same as in cell-free systems. In the latter, PAP-S,
M. charantia inhibitor, dianthin-32 and gelonin act in a decreasing order of efficacy whereas in both uninfected and virus-infected HEp-2 cells, dianthin-32 and PAP-S are more potent than the
M. charantia inhibitor and gelonin, demonstrating that other factors are involved in cells, such as the rate of RIP penetration and degradation [
78]. This is undergirded by the observation that binding and uptake of saporin-S6 and momordin by choriocarcinoma BeWo and cervical carcinoma HeLa cells are not correlated to cell toxicity [
79]. Batelli and co-workers determined the IC
50 for inhibition of cell protein synthesis by dianthin-32, saporin-S6, bryodin-R, momordin, gelonin and PAP-S on different cell lines and showed that human trophoblasts and BeWo cells are most sensitive while human embryonal fibroblasts, choriocarcinoma JAR cells and ovarian carcinoma TG cells were less affected. In particular, on fibroblasts, the efficacy of dianthin-32 and saporin-S6 was better than that of the other tested RIPs [
79].
A strategy how plants can defend themselves against viruses was experimentally demonstrated by Hong et al. by applying the dianthin-30 coding sequence including the
N-terminal 23 amino acid signal peptide to engineer resistance to the African cassava mosaic virus in the transgenic tobacco species
Nicotiana benthamiana Domin by using a promoter that is transactivated by a viral gene product [
80]. When challenged with the virus, transgenic plants produced atypical necrotic lesions on inoculated leaves, indicating dianthin-30 expression, moreover, viral DNA accumulation was significantly reduced, and plants exhibit attenuated systemic symptoms from which they recover [
80]. By using a potato virus X vector to express the transactivator protein from the African cassava mosaic virus directly in plants, the authors confirmed that amplification of dianthin-30 activity in transgenic plants is indeed mediated by the viral gene product [
81]. When dianthin-30 is constitutively expressed in
Nicotiana tabacum L. cv. Wisconsin 38, the plants are not able to survive, however, dianthin-30 does not hamper the development of rice (
Oryza sativa L. subsp.
indica cv. Pusa Basmati1) although all transgenic rice plants harbored and expressed the complete dianthin-30 gene [
82]. Notably, the transgenic lines showed reduction of sheath blight symptoms in the range of 29 to 42% [
82].