Ribosome-inactivating proteins (RIPs) and their important health promoting property

Abstract

Ribosome-inactivating proteins (RIPs), widely present in plants, certain fungi and bacteria, can inhibit protein synthesis by removing one or more specific adenine residues from the large subunit of ribosomal RNAs (rRNAs). In addition to the well-known RNA N-glycosidase activity, RIPs also possess other enzymatic activities such as polynucleotide adenosine glycosidases (PAG), DNase-like activity, superoxide dismutase and phospholipase. The anti-tumor activity of RIPs is one of the most important biological properties. In this review, we have summarized the distribution, sub-cellular location, characteristic expression of genes, enzymatic activity and cellular entry mechanisms of RIPs and further discussed the phylogenic and molecular evolution of RIP genes. We hope to draw attention to the pharmacological properties, effective construction of immunotoxins for clinic usage and potential future applications in cancer therapy of RIPs.

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1. Introduction

Ribosome-inactivating proteins (RIPs), members of N-glycosidases (EC3.2.2.22) and produced by various organisms including bacteria, fungi, and plants, are a diverse group of toxins sharing ribosome inactivating activity.^1,2 Ricin was the first RIP found in Ricinus communis L.³ In addition, Shiga and Shiga-like toxins were found in Shigella spp. and Escherichia coli.⁴ RIPs can inactivate prokaryotic and eukaryotic ribosomes by removing a specific adenine residue from the highly conserved and surface-exposed sarcin/ricin loop (SRL: GAGA) of 26S or 28S rRNAs.³ As the depurinated 50S/60S subunits are incapable of binding to corresponding elongation factors (EF-2/GTP complex), the protein syntheses are arrested at the translocation step, leading to autonomous cell death.⁵

Plant RIPs are considered to be “natural antibiotics” due to their multiple properties such as anti-viral, anti-bacterial, anti-fungal, and insecticidal activities.⁶ RIPs can enhance resistance of host plants to pathogenic bacteria in wild and transgenic plants.⁶ Moreover, RIPs also have many health beneficial activities, including anti-tumor, anti-cancer and anti-mutagenic properties. Plant extracts containing RIPs have been traditionally used to prevent virus and pest and to induce abortion particularly in China.⁷

Having multi-biological properties, RIPs have broad applications in the fields of toxicology, pharmacology, structure biology, biotechnology, biology and agronomy.⁸ Several RIPs, such as ricin, trichosanthin (TCS), pokeweed antiviral protein (PAP), Shiga-like toxins and momorcharin, have been well characterized and studied for their biochemical and molecular properties.

1.1 Types of ribosome-inactivating proteins

Based on subunits, molecular weight, isoelectric point (pI) value, structure of the genes and mature proteins, RIPs are classified into three types, type I, II and III, detailed in Table 1 and Fig. 1.^9,10 Type I RIPs are significantly more common than type II RIPs, whereas type II RIPs are more cytotoxic than type I RIP relatives.⁴

Table 1 Distribution and activities of most RIPs

Source	RIP	Activity	Ref
Type I RIPs
Amaranthus viridis	Amaranthin	N-Glycosidase, in vitro translational inhibition	129
Momordica balsamina	Balsamin	N-Glycosidase, in vitro translational inhibition	130
Bougainvillea spectabilis	Bouganin	Adenine polynucleotide glycosylase, protein synthesis inhibition	8
Saponaria officinalis	Saporin-R1	N-Glycosidase, ribosome-inactivating activity, protein synthesis inhibition, antitumor, cytotoxic activity, in vitro translational inhibition	26
	Saporin-R2	N-Glycosidase, in vitro translational inhibition	26
	Saporin-R3	N-Glycosidase, in vitro translational inhibition	26
	Saporin-S5	N-Glycosidase, antivirus, protein synthesis inhibition	131
	Saporin-S6	N-Glycosidase, protein synthesis inhibition, apoptosis induction, cytotoxic activity genomic DNA fragmentation activity, antiproliferative effect	132
Phytolacca americana	PAP-I	N-Glycosidase, protein synthesis inhibition, antitumor, antivirus	28
	PAP-II	N-Glycosidase, protein synthesis inhibition, antivirus, in vitro translational inhibition	28
	PAP-III	N-Glycosidase, protein synthesis inhibition	28
	PAP-S1	N-Glycosidase, protein synthesis inhibition	133
	PAP-S2	N-Glycosidase, protein synthesis inhibition	133
Lychnis chalcedonica	Lychnin	Adenine polynucleotide glycosylase, protein synthesis inhibition	8
Momordica charantia	MAP30	N-Glycosidase, anti-HIV, antivirus, antitumor, cell-free translational inhibition protein synthesis inhibition	65
	α-Momorcharin	N-Glycosidase, abortifacient, antitumor and anti-HIV activity, antifungal activity, immunosuppressive, antibacterial activity, DNase-like activity	134
	β-Momorcharin	N-Glycosidase, abortifacient, antitumor and anti-HIV activity, immunosuppressive, protein synthesis inhibition	134
	γ-Momorcharin	N-Glycosidase, protein synthesis inhibition, in vitro translational inhibition	135
	δ-Momorcharin	In vitro translational inhibition	136
	ε-Momorcharin	In vitro translational inhibition	136
Momordica cochinchinensis	Cochinin	N-Glycosidase, antitumor activity, protein synthesis inhibition	137
Gelonium multiflorum	Gelonin	N-Glycosidase, antitumor activity, protein synthesis inhibition, DNase activity	27
Lyophyllum shimeji	Lyophyllin	Antimitogenic, HIV-1 reverse transcriptase inhibitory activity, antifungal activity	138
Trichosanthes kirilowii	Trichosanthrin	N-Glycosidase, cell-free translation inhibition, immunomodulatory, antitumor, anti-HIV	139
	S-Trichokirin	N-Glycosidase, protein synthesis inhibition	139
Phytolacca heterotepala	Heterotepalins	Polynucleotide adenosine glycosidase activity, N-glycosidase activity	140
Mirabilis expansa	ME1	N-Glycosidase, protein synthesis inhibition, antibacterial activity	141
Flammulina velutipes	Velin	N-Glycosidase, cell-free translational inhibition	142
	Flammin	N-Glycosidase, cell-free translational inhibition	142
Type II RIPs
Ricinus communis	Ricin	N-Glycosidase, protein synthesis inhibition, antitumor, antivirus, apoptosis induction ytotoxic activity	13
Abrus precatorius	Abrin	N-Glycosidase activity, protein synthesis inhibition, apoptosis induction	143
Adenia lanceolata	Lanceolin	Polynucleotide glycosylase activity, cell-free translational inhibition, hemagglutinating	51
Adenia stenodactyla	Stenodactylin	Polynucleotide glycosylase activity, cell-free translational inhibition, hemagglutinating	51
Cucurbita foetissima	Foetidissimin II	N-Glycosidase, anticancer activity, cell-free protein synthesis inhibition	144
Sambucus ebulus	Ebulin I	N-Glycosidase, protein synthesis inhibition	145
Viscum album	Mistletoe	Antitumor activity, immunomodulatory activity, N-glycosidase	146
Viscum articulatum	Articulatin D	N-Glycosidase, hemagglutinating activity, cell-free translational inhibition	147
Cinnamomum camphora	Cinnamomin	Antitumor activity, N-glycosidase	148
Type III RIPs
Hordeum vulgare	JIP60	N-Glycosidase, cell-free translational inhibition, defense activity	16
Zea mays	Maize b32	N-Glycosidase, in vitro protein synthesis	17

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Fig. 1 Schematic representation of the mature forms of three types of ribosome-inactivating proteins.

Type I RIPs have single basic polypeptide chains with molecular weights about 30 kDa and pI values around 9.0, sharing several highly conserved active cleft residues as well as secondary structure within the active site region. They vary distinctly in overall sequence homology and post-translational modifications. TCS, saporin, PAP, and alpha-momorcharin, found both in apoplast and symplast, were all typical type I RIPs.^9,11,12 Most newly synthesized type I RIPs are named as proRIPs, containing N- and C-terminal extensions with respect to mature peptide chains.¹³ N-terminal regions often serve as signal peptides, leading the entry of proRIPs into endoplasmic reticulum (ER) and Golgi apparatus for post-translational modifications. For example, PAP's open reading frame is 939 nt in length, which codes for the mature PAP protein (262 amino acids), a 22 amino acids N-terminal signal peptide, and a 29 amino acids C-terminal peptide.¹⁴

Consisting of a N-glycosidase A-chain (30 kDa) and a lectin-like B-chain (30 kDa), type II RIPs (56–69 kDa), such as abrin and ricin, are heterodimeric proteins linked by a molecular disulfide bond. The B-chain can facilitate the entry of A-chain into cytoplasm through binding to galactosyl terminated receptors (β-1,4-linked galactose residues) at the target cell surface.¹³ In addition, relatively non-toxic type II RIPs (three to four logs less toxicity than toxic RIPs) have also been found, such as Ricinus communis agglutinin (about 120 kDa), cinnamomin, ebulin l1, ebulin r1, and nigrin b.⁴ The newly synthesized type II RIPs, such as ricin, are inactive pre-proproteins which need the post-translational modifications and proteolytic cleavage to become active.¹⁵

Type III RIPs contain an amino-terminal domain resembling type I RIPs and a carboxyl-terminal domain with unknown function, including barley JIP60 (ref. 16) and maize ribosome-inactivating protein b32.¹⁷ This type of RIPs is much less prevalent than the other two types. Activation of type III RIPs requires the removal of carboxyl-terminal domain. Active type III RIPs are similar in charge and enzymatic activity to type I RIPs, thereby type III RIPs are sometimes considered to be specific type I RIPs. Type III RIPs are first synthesized as inactive precursors of pro-RIPs, and the active proteins are produced through subsequent proteolytic modification event.^18–20

1.2 Distribution and expression of RIPs

RIPs have been isolated from 50 plant species which cover 17 families.²¹ Plant families, including Cucurbitaceae, Caryophyllaceae, Phytolaccaceae, Poaceae, Euphorbiaceae and Nyctaginaceae, are the rich sources of RIPs.^4,22 Major RIPs are produced by various higher plants. However, there are no RIPs found in Arabidopsis thaliana. To avoid inactivating self ribosomes, newly synthesized RIPs were targeted to cell walls, vacuoles and protein bodies as a sequestering mechanism, and may be released or induced in response to pathogen infection or injury.²³ Once pathogen enters target cells, RIPs regain access into cytoplasm and promote their activity by impairing host ribosomes.²⁴ For example, the 29 kDa constitutive PAP is localized in cell wall matrix of leaf mesophyll cells.¹⁴

RIPs are present in different plant tissues, and the levels of RIPs vary among different organs ranging from a few micrograms to hundreds of milligrams per 100 g fresh tissue.²⁵ They are particularly rich in seeds and fruits yet poor in leaves and stems. Isoforms of RIPs may exist in different organs, or even co-exist in one organ. Taken Saponaria officinalis L. as an example, saporin-L1 and saporin-L2 are found in leaves; saporin-R1, saporin-R2 and saporin-R3 are identified in root; while saporin-S5, saporin-S6, saporin-S8, and saporin-S9 are rich in seeds.²⁶ Tritin-S and tritin-L, with different sizes, charges, ribosome substrate specificities, and cofactor requirements, have been purified from Triticum aestivum L. germ and leaves, respectively.²⁷ PAP (29 kDa) is constitutively expressed in all pokeweed leaves, PAP II (30 kDa) is induced in summer leaves, while PAP-S 29.8 kDa is expressed only in seeds.²⁸ In vitro, all these PAP isoforms exhibit high bioactivity.^14,29,30

Expression of RIPs is affected by developmental stage, environmental condition and hormones.³¹ Moreover, the elevation of RIP expression level has also been observed under environmental stress, virus infection, and senescence. Expression of PIP2, an antiviral protein, was hindered in leaf and root of Phytolacca insularis L., but would be activated when seedling grew up to six week old.³² Additionally, mechanical wounding, jasmonic acid, and abscisic acid all could cause the systemically inducible expression of PIP2, but salicylic acid could not.³² JIP60 is a jasmonate-induced protein, and its expression was found to be induced by desiccation, damage and senescence.¹⁹

1.3 Phylogenic and molecular evolution of RIPs

Multigene families have been reported for RIPs.³³ Previous model on origin and evolution of RIP gene family indicated that the ancestral RIP domain originated in flowering plants, then was acquired by some bacteria through horizontal gene transfer (HGT) accidentally.³⁴ Additionally, RIP genes could be lost from host during evolution. For example, RIP genes cannot be found in Glycine max, but its closely related species Abrus pulchellus is a RIP-expressing plant, indicating that the loss of RIP gene might take place during the evolution of G. max. RIP gene loss also occurred in the order of Brassicales, as no RIP genes exist in Arabidopsis thaliana and Brassica rapa.

At least two different A-chain paralogous genes might be fused to B and C chains independently, leading to the current type II and type III RIP genes, respectively. The fusion of enzymatic A chain and lectin-binding B chain might take place once in flowering plant lineage, as most of type II RIPs have been found only in plants.²² The deletion of B-chain might also occur. For type III RIPs, fusion between the enzymatic A domain and the unknown C domain took place in Poaceae.²² A novel type III RIP gene has been found in the dicot Cannabis sativa, and several type III genes have been found in fungi, suggesting that the fusion of A domain and C domain occurred before plants and fungi diverged.³⁴

However, there are several drawbacks in this model.^34,35 Novel RIP evolution model indicates that the RIP domain was present in the common ancestor of bacteria, archaea and eukaryotes initially. Then, several paralogous RIP genes had evolved before the three domains of life diverged. Moreover, multiple gene duplication and gene loss events of paralogous genes took place, leading to a high heterogeneity in the number of RIP genes among different organisms. More than one paralogous genes suffered multiple duplications, and produced various RIPs after the plant lineage diverged. Specifically, type II RIPs took place through the fusion of one plant paralogous RIP domain to a lectin domain, but some type II RIP genes suffered the deletion of lectin domain and gave out “secondary” type I RIPs. Similarly, a paralogous RIP gene was fused to C-chain domain before the divergence of fungi and plants, resulting in type III RIPs.³⁴ However, RIP genes have not been found in Archaea, the third domain of life.

1.4 Cellular entry mechanism of RIPs

As toxic proteins, RIPs need to enter cells to inactivate ribosome by the virtue of RNA N-glycosidase and other enzymatic activities. RIPs bind to cell surface receptors, cross cell membrane through endocytosis, then enter cytosol via retrograde transport from Golgi apparatus to ER.⁹ Different RIP molecules entry cells through different pathways. Only a few toxin molecules are taken up by target cells, and the inhibition of protein synthesis can be detected 30 min after the internalization of RIP. A single RIP molecule, like ricin, was found to be sufficient to induce target cell death.²¹ We will take TCS (type I RIP) and ricin (type II RIP) as examples to discuss the cellular entry mechanisms of different RIPs.

1.4.1 Mechanisms of cellular entry and apoptosis induction for type I RIPs. Lacking receptor-binding chain, type I RIPs appear difficult to enter target cells. Several specialized animal cells can import type 1 RIPs through endocytosis. TCS, a type I RIP isolated from root tuber of Trichosanthes kirilowii Maxim (Cucurbitaceae family), is specific for the negatively charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG). The C-terminus of TCS interacts with the negatively charged phospholipid-containing cell membrane through electrostatic force and hydrophobic interaction, which can be affected by both pH and ionic strength.^36,37 Acidic microenvironment of cell membrane redistributes charges on some residues, leading to salt bridge breakage and charge–charge repulsion that partially denatures TCS molecules to “molten globular state” and allows their membrane insertion.³⁸ Sometimes, receptors are necessary for the cellular entry of TCS. For instance, TCS binds to lipoprotein receptor-related protein 1 (LRP1) of cell membrane when entering trophoblasts, JAR cells, and choriocarcinoma BeWo cells.³⁹ It enters proximal tubule epithelial cells by binding to megalin and inserts into HIV infected cells via chemokine receptors.⁴⁰ However, the cell surface binding of saporin is mediated by members of the low density lipid (LDL)-related family receptors, and the intracellular movement of saporin relys on the Golgi-mediated retrograde transport.

As summarized in Fig. 2, TCS inhibits cell proliferation through different pathways and mechanisms for different tumor cells. In HeLa cells, the inhibition of cell proliferation by TCS is through suppressing the PKC/MAPK signaling pathway, reducing the activities of adenylyl cyclase and protein kinase C (PKC), and activating caspases 8, 9, 3.^41,42 In hepatoma HepG2 cells, TCS reduced the expression of nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2), leading to cell apoptosis.⁴³ In chronic myelogenous leukemia K562 cells, TCS down-regulated the expression of genes associated with cell proliferation, such as p210 (Bcr-Abl), protein tyrosine kinase (PTK), and heat shock protein 90 (Hsp90).⁴⁴ In OVA-specific T cell, TCS elevated NO content through inducing the expression of nitric oxide synthase (iNOS) and inhibited antigen-specific T cell activation via the NO-mediated apoptosis pathway.⁴⁵ TCS elicited an increase in cytosolic calcium and induced apoptosis in HeLa cells.⁴¹ In JAR cell line, TCS stimulated the production of ROS.⁴⁶ In murine prostatic cancer RM-1 cells treated with TCS, the protein expression of Bax is up-regulated.⁴⁷ Furthermore, mitochondria and ER stress pathway are involved in TCS-induced apoptosis.⁴⁸

Fig. 2 Mechanisms of cell apopotis induced by TCS.

1.4.2 Mechanisms of cellular entry and apoptosis induction of type II RIPs. Type II RIPs can cross plasma membrane and enter cytosol via endocytic pathway (Fig. 3). Mannose-containing RIPs, such as ricin, bind to mannose receptors of cell membrane through β-1,4 linked galactose residues and are internalized by virtue of clathrin-dependent endocytosis, as evidenced in Vero cells.^49,50 However, some other cytotoxic type II RIPs, such as lanceolin and stenodactylin, are internalized by mechanisms independent of clathrin-coated pits.⁵¹

Fig. 3 Putative mechanism of ricin entering target cytoplasm.

When ricin molecules enter cells, they are firstly delivered to early endosomes, from where most are recycled back to cell surface or delivered to lysosomes for proteolytical degradation.⁴⁹ Only a few ricin molecules, no more than five percent, reached trans-Golgi network (TGN), from where ricin toxins undergo retrograde transport from Golgi to ER lumen. The KDEL region of ricin A chain interacts with KDEL receptors. Moreover, the retrograde transport of ricin from Golgi to ER is through the coatomer protein I (COP-I)-coated vesicles.⁵² In ER lumen, dissociation occurs between ricin RTA and RTB subunits. The unfolded RTA acts as an ERAD (ER-associated protein degradation) candidate, leading to transportation from ER lumen into cytosol via translocation machineries.⁵³ In cytosol, RTA may be proteolytically degraded in the ERAD pathway.⁵² The remaining RTA may access translational machinery and inhibit protein synthesis of target cells. Other structurally and functionally equivalent type II RIPs may have the similar mechanism of entering target cells.

2. Enzymatic activities of RIPs

Lack of lectin-binding B chain, type I RIPs are reported to be less cytotoxic than most of the type II RIPs.⁵⁴ Once being introduced into cells, type I RIPs are highly toxic. Besides the well-known RNA N-glycosidase activity, polynucleotide: adenosine glycosidases (PAG) activity, and DNase-like activities, other activities including superoxide dismutase, phospholipase, antioxidant, antiviral and insecticidal properties have also been observed for RIPs.^55,56 RIPs purified from Trichosanthes kirilowii cell cultures possess chitinase activity.⁵⁷ Ricin and saporin have insecticidal properties against Coleopteran species.⁵⁸

2.1 RNA N-glycosidase activity and inhibition of protein synthesis

The site-specific RNA N-glycosidase activity is a common characteristic to all RIPs. Therefore, proteins with glycosidase activity can be categorized as RIPs.⁹ Shiga toxin (Stx), produced by Shigella dysenteriae and cytotoxins (Stx1, Stx2, as well as variants) produced by E. coli serotypes, share the RNA-N-glycosidase activity with plant RIPs.⁵⁹ Ribosomes of bacterial, fungal, plant and mammalian cells are all subjects of RIPs. It needs to be noted that all RIPs could inactivate eukaryotic ribosomes, but several RIPs, including PAP, Shiga toxin, and Mirabilis antiviral protein, could also inactivate prokaryotic ribosomes.⁶⁰

Naked rRNAs are 10⁴ to 10⁵-fold less sensitive to RIPs than intact ribosomes, indicating the important role of ribosomal proteins in RIP-rRNA interactions.³ Kingdom specificity of ribosome substrate for certain RIPs might also be due to the interaction of RIP with ribosomal proteins. Ricin A-chain was found to interact with ribosomal proteins L9 and L10e of mammalian ribosome.⁶¹ PAP was cross-linked to yeast ribosomal protein L3.⁶² TCS interacts with human acidic ribosomal proteins P0, P1 and P2, the lateral stalk of eukaryotic ribosome.⁶⁰ In particular, K173, R174 and K177 in the C-terminal domain of TCS interact with conserved DDD motif at the C-terminal of P2 through forming charge–charge interactions.⁶⁰ Interaction between TCS with flexible C-terminal tails of stalk proteins (P0/P1/P2), could benefit TCS to its SRL substrate more efficiently, and induce conformational changes on TCS, which will increase its enzymatic activity.⁶⁰ Moreover, the binding of TCS to ribosome also changes the conformation of ribosome, making SRL site more susceptible to be depurinated.⁶⁰ However, more work are still needed to clarify these interaction.

RIPs can recognize and cleave N-glycosidic bond at the specific adenine in the highly conserved single-stranded sarcin–ricin loop (SRL, “GAGA”, Fig. 4). The SRL locates in domain VII of rat liver 28S rRNA, which is vital for interaction with elongation factors. The deadenylated site is unstable and can be cleaved through β-elimination reaction, whereby the released 3′ end (about 400 nucleotides) of 28S rRNA can be detected easily by electrophoresis (Fig. 4). In addition to 28S rRNA of mammalian animals, the Saccharomyces cerevisiae 26S rRNA, Oryza sativa 25S rRNA, E. coli 23S rNRA and 16S rRNA are all substrates for RIPs.¹⁰

Fig. 4 Active sites of RIPs in 28S rRNA.

Alpha-momorcharin could release Endo's band (about 400 bp) from naked rice rRNAs with the ratio of 15.4 ng of alpha-momorcharin to 1 ng of rRNA.²⁵ Tritin, a RIP purified from wheat, can depurinate A³⁰²⁴ from 26S rRNA of native yeast (Saccharomyces cerevisiae) ribosomes, the same as ricin A-chain.²⁷ PAP-H can release a 360-nucleotide diagnostic fragment from 26S rRNA through ribosome-inactivating activity.⁶³ Ricin, saporin-S6, and Mirabilis expansa RIPs all can depurinate fungal ribosomes isolated from Rhizoctonia solani, Trichoderma reesei, Alternaria solani, and Candida albicans with different enzymatic activities. Beetin isolated from Beta vulgaris can depurinate both rabbit and Vicia sativa rRNAs.⁶⁴ MAP30, recombinant MAP30, α-momorcharin, recombinant α-momorcharin, β-momorcharin, γ-momorcharin, δ-momorcharin, ε-momorcharin and charantin are all potent inhibitors of protein synthesis in rabbit reticulocyte cell-free lysates.³⁸ Interestingly, MAP30 cannot enter and inactivate ribosomes of uninfected (normal) cells, therefore, there is no inhibition of protein synthesis.⁶⁵

Activities of momorcharins on 28S rRNA are in K⁺ and NH₄⁺ ions-dependent manners.⁹ The γ-momorcharin exhibits RNA N-glycosidase activity toward rat liver ribosomes in a dose-dependent manner. Requirements for macromolecular cofactors have been verified for translational inhibitory activity of RIPs.⁶⁶ Gelonin, barley RIP, PAP, and tritin-S all require ATP for maximal activity.²⁷ The tRNA^Trp, lacking one or two nucleotides at the 3′-CCA end, is a cofactor for gelonin.⁶⁶

2.2 Polynucleotide: adenosine glycosidases (PAG) activity

Enzymatic activity of RIPs is not limited to rRNAs, but also to DNA, mRNAs, poly(A), natural and synthetic polynucleotides in a sequence context-independent manner, releasing multiple adenines and sometimes guanines, well-known as polynucleotide: adenosine glycosidases (PAG) activity.^67,68 PAP possesses depurination activity towards influenza virus, poliovirus, herpes simplex virus, TMV RNA, genomic HIV-1 RNA, brome mosaic virus (BMV), lymphocytic choriomeningitis virus (LCMV) and tobacco etch virus (TEV) RNA both in vitro and in vivo in a concentration-dependent manner.^10,69–73 Saporin-L1 is active toward mammalian nuclear and mitochondrial DNA.⁷⁴ All 52 well known RIPs can depurinate DNA, while some can release adenine from adenine-containing polynucleotides.⁶⁷

2.3 DNase-like activity

DNase-like activity against supercoiled plasmid has been reported for RIPs, converting supercoiled double-stranded DNA into nicked circular molecules at low concentrations, and into linear conformation at high concentrations.^68,75 In this regard, activities of RIPs are similar to DNA glycosylases, which function to remove only inappropriate or damaged bases in cells. Subsequently, the damaged DNAs are often cleaved at the apurinic/apyrimidinic (AP) sites by DNA glycosylases.⁵⁹ Divalent Mg²⁺ ions can enhance the DNA cleavage activity of RIPs, as cation-binding site(s) may exist in the DNA cleavage domain of RIPs. Moreover, the interaction between DNA molecules and RIPs is not sequence-specific, but conformation-specific.⁷⁶ In particular, the negative supercoiled DNA is the preferential substrate of RIPs.

Some RIPs, such as ricin, dianthin 30, saporin, gelonin, cinamomin, α-momorcharin, camphorin, α-sarcin and luffin can cleave and even linearize single-stranded M13 phage DNA and double-stranded supercoiled DNA.^68,75,77 DNA can also be damaged by Shiga toxin in vitro both at acidic and physiological pH.⁷⁸ However, single-stranded DNA breaks induced by plant RIPs are attributed to both the release of adenine from multiple sites and the weakening of DNA sugar-phosphate backbone.⁷⁸ In our research, α-momorcharin exhibits strong DNase-like activity over broad pH (3.0–8.0) and temperature (25–55 °C), and the Tyr⁹³ amino acid is shown to be a critical residue for the DNase-like activity.⁶⁸

3. Multiple applications of RIPs

3.1 Significant roles in plants and breeding of pesticide-free agricultural crops

RIPs may regulate cell proliferation and development through inhibition of protein synthesis.⁷⁹ Moreover, RIPs might also regulate metabolic processes of some cell organelle, such as mitochondria, endoplasmic reticulum, and Golgi apparatus among others. The elevated expression of RIPs might account for the metabolic regulation under seed maturation, tissue senescence and environmental pressures. Contents of seed-derived RIPs, such as ricin and alpha-momorcharin, decrease gradually during seed germination, indicating that amino acids of RIP molecules might serve as C and N sources for the newly synthesized proteins important for seedling growth.

3.1.1 Development of natural bio-pesticides. RIPs are a type of anti-pathogenic protein toxins, which are important in plant defence against foreign pathogenic invaders, including bacteria, fungi, and even plant-eating animals.¹⁰ Type I RIPs have direct effect towards yeast and plant pathogenic fungi.⁸⁰ As typical antifungal protein/pathogenesis-related proteins, RIPs can inhibit synthesis of the fungal cell wall, disrupt cell wall structure and function, interact with plasma membrane and potential fungal intracellular targets, cause changes in the membrane potential, and impede cell division or macromolecular synthesis.^81,82 Synergistic inhibition of fungal growth by RIP, chitinase, together with glucanase purified from barley seeds has been observed.⁸³

PAP-H, secreted from pokeweed hairy roots into rhizosphere, can protect host from pathogen infection.⁶³ Cucurbita moschata RIP can impede the growth of Phytophthora infestans, Erwinia amylovora, and Pseudomonas solanacearum.⁸⁴ Hairy melon RIPs and luffacylin purified from Luffa cylindrica seeds also bear antifungal activity.⁸³ Alpha-momorcharin exhibits significant antifungal activity towards Fusarium solani and Fusarium oxysporum.²⁵ Mirabilis expansa root RIPs are active at microgram levels against several soil-borne bacterial species, as well as pathogenic and nonpathogenic fungi.⁵⁵ BE27, a RIP isolated from Beta vulgaris L. leaves induced by hydrogen peroxide and salicylic acid, displayed strong antifungal activity against green mold Penicillium digitatum through rRNA N-glycosidase activity.⁸⁵

The well-known antifungal activity and antivirus activity of RIPs indicate that plant extracts containing RIPs have great potential in the preparation of bio-pesticide, and such natural products would be a sustainable alternative to the use of synthetic pesticides. The usage of non-phytotoxic and biodegradable plant extracts to resist pathogenic fungi and viruses have attracted lots of attention since 1990s, as they exert minimal environmental impact and pose little danger to consumers.⁸⁶ Exogenous application of PAP protects heterologous plants from viral infection.⁸⁷ In our study, M. charantia seed extract containing α-momorcharin effectively inhibited mycelial growth of F. solani with an IC50 of 108.934 μg mL⁻¹.

3.1.2 Breeding of agricultural crops with reduced or free of pesticides. RIPs can increase host resistance against pathogenic organisms and reduce the usage of agricultural chemicals, resulting in the minimal pesticide containing or pesticide-free agricultural crops. Therefore, many genetically engineered plants with RIPs have been produced. For example, PAP-transgenic plants have enhanced resistance against fungal and viral infection.^87,88 Compared with wild tobacco, transgenic tobacco with a type III RIP is more resistant to maize-eating insects, such as Lasioderma serricorne and Manduca sexta.⁸⁹ Alpha-momorcharin enhances defence response in transgenic tobacco and Oryza sativa plants against diverse viruses and pathogenic microbials.^90,91 Vigna mungo (L.) Hepper transformed with a RIP gene, greatly arrests the growth of C. cassiicola (25–40%) over the wild-type plants, leading to the increase of black gram production.⁹² Transgenic tobacco transformed with curcin 2 gene shows an increased tolerance to tobacco mosaic virus (TMV) by delaying the development of systemic symptoms of TMV, as well as fungal pathogen Rhizoctonia solani through reducing the damage caused by fungal disease.⁹³ When transgenic Oryza sativa seedlings expressing TCS gene were inoculated with spores of rice blast fungus pathogen Pyricularia oryzae, the lesions on leaves were much less severe, and the seedling survival rate and whole plant weight were higher than those of non-transgenic control plants.⁹⁴

3.2 Significant roles of RIPs in insects

Many insects have widespread associations with symbiotic bacteria, and the insect symbionts often perform important roles for host survival, such as supplementing nutrition, and even providing protection against natural enemies.⁹⁵ RIPs have show great potential for the insect defensive symbiont. In relation to the ecologically important defensive symbiosis, bacterial endosymbiont Spiroplasma could effectively protect its host woodland fly Drosophila neotestacea from the parasitism of nematode Howardula aoronymphium by encoding a RIP related to Shiga-like toxins, SpRIP, which would specifically depurinate the 28S rRNA of H. aoronymphium.⁹⁵

3.3 Multiple biomedical applications of RIPs

3.3.1 Use in traditional medicine. RIPs have been traditionally used as anti-tumor, anticancer, abortifacient and anti-HIV agents in China, India, and other developing countries. For instance, TCS has been used as abortifacient drug in early and mid-gestation for centuries in China.⁵⁴ Currently, TCS is still a clinical medicine for abortion. Unfortunately, TCS can cause follicular atresia and degeneration of ovulated oocytes, elicits death of syncytiotrophoblasts of placental villi, and inhibition of embryo development.⁹⁶ Saporins, PAP, and gelonin have abortifacient activity on pregnant mice.⁹⁷ Riproximin, a new type II ribosome-inactivating protein isolated from Ximenia americana, has distinct potential for cancer treatment in African traditional medicine.⁹⁸ Therefore, RIPs could be used in modern pharmaceutical industry as novel antibiotics against pathogenic microorganisms.

3.3.2 Anti-tumor activity and the construction of RIP-based immunotoxins. RIPs are highly toxic to malignant cells than to normal cells, which might be due to changes in receptor concentration on malignant cell surfaces or altered intracellular transport of toxins.⁹⁹ Plant RIPs have shown strong antitumor activity both in vitro and in vivo, as shown in Table 2. Anti-tumor potency of RIPs varies dramatically. Ricin can induce 50% cell apoptosis at concentrations lower than 1 ng mL⁻¹, whereas some type II RIPs isolated from elderberry display no significant effects even at 1 mg mL⁻¹.¹⁰⁰ The involvement of caspases, caspase-like proteases, serine proteases, as well as poly(ADP-ribose) polymerase have also been observed in cell apoptosis induced by RIPs.¹⁰¹

Table 2 Overview of RIPs (type I and type II) with different antitumor activities

RIP	Source	Types of tumor	Study model
Type I RIPs
α-Luffin	Luffa cylindrica	Placental choriocarcinoma, hepatoma, breast cancer	In vitro
Gelonin	Gelonium multiflorum	Bladder cancer	In vitro
MAP30	Momordica charantia	Brain glioblastoma, breast carcinoma, epidermoid carcinoma, liver hepayoma, myeloma neuroblastoma, prosterate carcinoma	In vitro
MCP30	M. charantia	Prostrate cancer	In vitro
α-Momorcharin	M. charantia	Lung, colon, liver, breast and epidermal cancer	In vitro
PAP I	Phytolacca americana	T-Cell leukemia virus	In vitro (mouse)
Saporin	Saponarja officinalis	Ovarian teratocarcinoma, prostrate cancer	In vitro
Trichosanthrin	Trichosanthes kirilowii	Hepatocellular carcinoma	In vitro
Type II RIPs
Ebulin I	Sambucus ebulus	Cervix epithelioid carcinoma	In vitro
Foetidissimin II	Cucurbita foetodissima	Adenocarcinoma and erythroleukemia	In vitro
Mistletoe	Viscum album	Ovarian, colorectal, renal and breast cancer	In vitro, in vivo (human)
Nigrin b	Sambucus americana	Cervix epithelioid carcinoma	In vitro
Abrin	Abrus precatorius	Sarcoma 180 cells and Ehrlich ascites tumor cells	In vitro
Ricin	Ricinus communis	Breast cancer, lymphoma, leukemia	In vitro

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TCS can inhibit cell growth of cultured ovulated oocytes, human macrophages, HIV-infected macrophages, as well as cancer cells such as cervix, lymphoma, and choriocarcinoma among others.^54,96,102 TCS manifests significant inhibitory effects on HeLa cervical cancer cells through heightening cytosolic calcium and suppressing intracellular cAMP/protein kinase C (PKC) levels via PKC inhibition, as well as causing apoptotic cell death.^41,103,104 Furthermore, the participation of endoplasmic reticulum stress pathway in TCS-induced HeLa cell apoptosis has also been evidenced by up-regulation of ER chaperone immunoglobulin heavy chain-binding protein (BiP) and C/EBP homologous protein (CHOP) and activation of caspase 4.⁴⁸ TCS can inhibit growth of chronic myelogenous leukemia K562 cells and acute T cell leukemia Jurkat cells through the down-regulation of p210 Bcr-Abl and its downstream signals, as well as PKC inhibition and caspase 3 activation.^44,48 In cervical cancer CaSki cells, TCS inhibits DNA (cytosine-5)-methyltransferase 1 (DNMT1) enzyme activity and down-regulates levels of DNM1 mRNA and protein.^32,105 MAP30 and rec-MAP30 all exhibit antitumor activity against central nervous system, breast, melanoma and myeloma tumors, prostate, and epidermoid carcinomas.⁹ In addition, recombinant luffin can effectively inhibit proliferation of human placental choriocarcinoma JEG-3, hepatoma HepG2, and breast cancer MCF-7 cell lines in dose- and time-dependent manners.¹⁰⁶ Recombinant Elaeis guineensis Jacq type 2 RIP, EgT2RIP, showed growth inhibitory activity against breast cancer cell lines MCF-7 and non-tumorigenic breast epithelial cell line MCF-10A.¹⁰⁷

However, major adverse effects, short plasma half-life, neurotoxicity and strong anti-genicity of RIPs limit their wide application as therapeutic agents. For example, plasma half-life of TCS is 8.4–12.7 min in vivo, so frequent administration is needed to maintain an effective therapeutic concentration in blood.¹⁰⁸ Repeated use of RIPs may elicit antigenic responses which might diminish their biological activity. When being administered to patients with AIDS, TCS can induce mild to severe anaphylactic responses and neurological disorders, such as myalgia, nausea, diarrhea and flu-like symptoms.¹⁰⁸ Therefore, chemical modifications and genetic engineering of RIPs have been performed, targeting at increased specificity, reduced antigenicity, prolonged half-life and tempted mechanism elucidation of their roles in cell apoptosis. Ricin, TCS, and momorcharin have been combined with targeting moieties (specific antibodies, growth factors, hormones, lectins, etc.) to yield tumoricidal immunotoxins (ITs), also known as “magic bullets”. Furthermore, the half-life of immunotoxins can be improved further if the disulfide bond between monoclonal antibodies (MAb) and the toxin is formed in a hindered fashion using derivatizing agent 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio) toluene (SMPT).¹⁰⁹

RIP-based immunotoxins have been tested clinically on solid tumor cells and especially on refractory haematological malignancies, thus offering great potential as therapeutic agents.^110,111 For example, TCS conjugated with colloidal gold can specifically enter cultured trophoblast and choriocarcinoma JAR cells via LDL receptor-related protein-1 (LRP1) receptor-mediated endocytosis, in which calcium ion signaling might be involved.^39,112 Immunotoxin EGF-TCS is toxic to hepatoma cells both in vitro and in vivo.¹¹³ Dexamethasone can enhance the effects of TCS on HepG2 cell apoptosis through inhibiting NF-kB signaling pathway.⁴³ Ricin A-based immunotoxins can be taken up nonspecifically by macrophages and hepatic nonparenchymal Kupffer cells through the binding of carbohydrate residues present on ricin A-chain with mannose receptors on cell membrane.¹¹⁴ Ricin B-chain has been shown to be a good adjuvant-carrier.¹¹⁵ However, ricin-immunotoxins are more efficient than RTA-immunotoxins, indicating that RTB may be important for the intracellular routing and play vital roles in the translocation of RTA across cellular membrane to cytosol.¹¹⁶ Moreover, RTB in immunotoxin can protect RTA from proteolytic processing by cathepsins.¹¹⁷ Blocked ricin (bR), in which the two galactose-binding sites of native ricin are blocked by affinity ligands, has at least a 3500-fold lower binding affinity and more than 1000-fold less cytotoxic than native ricin for Namalwa cells.¹¹⁸ Transferrin-CRM107, a conjugate of human transferrin and a genetic mutant of diphtheria toxin (CRM107) lacking native binding activity, had a 50% reduction in recurrent malignant brain tumors in the regional therapy experiment.¹¹⁰ L6-Ricin is useful for in vitro purging of autologus bone marrow from solid tumor-patients and for in vivo regional therapy of L6-positive carcinomas, as monoclonal antibody L6 recognizes the specific determinant expressed on carcinomas of lung, colon, breast, and ovarian.¹¹⁹

3.3.3 Anti-viral activity and mechanism of RIPs. Type I RIPs possess broad-spectrum antiviral activities toward plant, animal, fungal viruses, and human immunodeficiency virus (HIV) in the virus-infected cells.^69,120 It has been found that the anti-viral activities of RIPs are not always due to the inhibition of cellular DNA and protein synthesis. The mechanisms through which RIPs exert their antiviral capability need further investigation.

PAP can inhibit the production of human T-cell leukemia virus I (HTLV-1), a delta retrovirus and a causative agent of adult T-cell leukemia.¹²¹ PAP can depurinate nucleotides from gag open reading frame and decrease the translational efficiency of HTLV-1 gag/pol mRNA, suppressing the synthesis of viral proteins.¹²¹ MAP30 can inhibit hepatitis B virus (HBV) DNA replication and the expression of HBV antigen, down-regulate replicative intermediates, and reduce HBsAg secretion in dose- and time-dependent manners.¹²² Two maize RIP variants can inhibit HIV viral replication in human T-lymphocytes.¹²³ Luffin P1, the smallest RIP purified from Luffa cylindrica seeds, has anti-HIV-1 activity in HIV-1 infected C8166 T-cell lines, and can bind with HIV Rev response element through charge complementation.¹²⁴ TCS interacts with HIV-1 viral envelope, exploits sorting strategy to prevent virus dissemination and shows cytotoxicity to HIV-infected macrophages and lymphocytes.^108,125

3.4 Application of RIPs in gene therapy

Toxic gene therapy, also known as suicidal gene therapy relying on the potency of gene products to kill transfected tumor cells and the transfection ability of transfection vehicles, has gained enormous interest in these years, particularly for the treatment of cancer. Toxins, including RIPs, have shown great potential in suicide gene therapy. Two mammalian transfection plasmids, pSAP containing gelonin gene and pGEL containing saporin gene, could induce significantly augmented cytotoxic effects towards several cancer cell lines (HeLa, U87, 9L, as well as MDA-MB-435) at only 2 μg mL⁻¹ gene concentration.¹²⁶

4. Modification of RIPs

To prolong plasma half-life and reduce immunogenicity, RIPs can also be made to conjugat with water-soluble, non-immunogenic, non-antigenic, nontoxic, and US Food and Drug Administration (FDA)-approved polymers, such as dextran and polyethylene glycol (PEG). PEG can also mask the antigenic sites to prevent antibody-binding and reduce antigenicity.¹²⁷ As an example, bitter melon α-momorcharin, chemically modified with the 20 kDa (mPEG)₂-Lys-NHS, has reduced immunogenicity and prolonged plasma half-life.¹²⁸

Protein engineering is also used for RIP modification to reduce antigenicity of RIPs. To reduce the adverse effects of TCS as a drug, the C terminal domain without any active sites have been systematically deleted, resulting in a less antigenic and biological activities-retained variant. Compared with wild type TCS, the antigenicity of C7 variant (the last seven amino acid residues 241–247 at the C-terminus being deleted) was decreased by 2.7 times in vitro ribosome-inactivating activity and 10 times in vivo cytotoxicity.³⁸ Moreover, site-directed mutagenesis has also been used to reduce antigenicity of TCS, yet retaining the ribosome inactivation activity.

5. Conclusions

As a special type of proteins with multiple properties, RIPs hold great promise as candidates for anti-viral, anti-bacterial, anti-fungal, anti-tumor, anti-cancer and immunosuppressive. Moreover, RIPs exert other functions including seed storage, defence against predators or parasites, and they even have a role in aging and stress. This review provides in depth insights into various properties of RIPs and their potential wide application in agriculture and medicine. In-depth understanding of these multifunctional proteins and their physiological functions in plants, animals and human beings will not only shed light on the mechanisms of RIPs' occurrence, pharmacological properties, but also facilitate their application in agriculture as biopesticides and in medicine as anticancer agents.

Acknowledgements

This work was supported by research grants from Huanggang Normal Univerisity (2014022203), and Fund of Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains (2015TD07).

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