Plant Derived Ribosome Inactivating Proteins: An Overview

 

Nitin Kumar, Satyendra Singh, Manvi  and Rajiv Gupta*

Department of Pharmacognosy, Faculty of Pharmacy, Babu Banarasi Das National Institute of Technology and Management, Dr. Akhilesh Das Nagar, Faizabad Road, Lucknow 227 105 U.P (India)

*Corresponding Author E-mail: rajiv961@rediffmail.com

ABSTRACT:

Many plants accumulate proteins that are commonly referred to as ribosome-inactivating proteins (RIPs). Biological effects described to these proteins go back to ancient times because of the high toxicity of seeds of castor bean (Ricinus communis) and jequirity bean (Abrus precatorius), as well as anti-HIV, the abortifacient activity of some plants like Trichosanthes kirilowii and Momordica charantia, rely on the presence of RIPs. Pokeweed antiviral protein, and ricin, previously described as ribosome-inactivating proteins were shown to damage single-stranded DNA by removal of a protein-specific set of adenines. RIPs are enzymes and some have multiple enzymatic activities. These enzymes are expected to damage DNA rather than participate in repair processes. All RIPs depurinated DNA extensively and some released adenine from all adenine-containing polynucleotides. The entire class of plant proteins, called ribosome-inactivating proteins, may be classified as polynucleotide: adenosine glycosidases. The significance of this DNAase activity to the biological function of these plant proteins along with their toxicity effect to animal cells remains to be fully understood.

 

 

INTRODUCTION:

‘Ribosome-inactivating protein’ inactivates animal ribosomes before the structure and enzymatic activity. RIPS are grouped into two types: Type-1 RIPs are single chained such as trichosanthin and type 2 RIPs are double chained such as ricin. The A chains of type-2 RIPs possess the RIP activity and B chain bind the proteins to eukaryotic cell surface and help a chains to enter the cytosol. Both trichosanthin and ricin are RNA-N glycosidases and both inactivate ribosomes by cleaving the n- glycosidic bond of A⁴³²⁴ of 28S rRNA in a hydrolytic fashion¹. The identification of ricin was an important milestone in biochemistry because for the first time a well-defined biological activity was described to a plant protein. Moreover, abrin, a similar toxic protein from the seeds of A. precatorius, also played an important role in the early development of immunology. Since the isolation and characterization of ricin, many structurally and functionally related proteins have been identified in various plants². The interest in RIPs focused on possible medical and therapeutical applications because several of these proteins were found to be more toxic to tumor cells than to normal cells, and hence offered a theoretical opportunity to develop antitumor drugs that selectively target tumor cells ³.

 

Along with the efforts to develop some RIPs into anticancer compounds, attempts were made to find out what RIPs do and how they act. This led to the finding that RIPs are RNA N-glycosidases that inactivate ribosomes through a site-specific deadenylation of the large ribosomal RNA^4,5. RIPs are also capable of inactivating many nonribosomal nucleic acid substrates^6,7,8. The enzymatic activity enhances exploitation of the unique properties and activities of RIPs for diverse applications like immunotoxins⁹, abortifacients¹⁰, and anti-human immunodeficiency virus agents^11,12.

 

CLASSIFICATION:

After the mode of action of RIPs on ribosomes was elucidated, the term RIP was used exclusively for these N-glycosidases. This is important because other types of proteins that inactivate or damage ribosomes by other mechanisms (e.g., RNases or proteases) are not considered RIPs. Two major classes are distinguished: holo-RIPs and chimero-RIPs. Holo-RIPs consist exclusively of a single RNA N-glycosidase domain. Most holo-RIPs consist of a single, intact polypeptide of 30 kDa and are usually referred to as type 1 RIPs. Besides these classical type 1 RIPs, there are also a few examples of type 1 RIPs in which the original 30 kDa polypeptide is proteolytically processed so that the protein consists of two shorter polypeptides held together by noncovalent interactions. These proteins have been named type 3 RIPs. Chimero-RIPs are constructed of one or more protomers consisting of an N-glycosidase domain linked to a structurally different and functionally unrelated domain. Most chimero-RIPs are so-called type 2 RIPs. Type 2 RIPs may be toxic or non toxic. Besides the classical type 2 RIPs, a 60 kDa RIP (called JIP60) has been identified in barley (Hordeum vulgare) that consists of an amino-terminal domain resembling type 1 RIPs linked to an unrelated carboxyl-terminal domain with unknown function¹³.

 

DISTRIBUTION OF RIPs IN PLANTS:

RIPs were initially detected in plants, mostly in family Angiospermae, in both mono- and dicotyledons, in mushrooms¹⁴ and in an alga, Laminaria japonica¹⁵. Some examples of plants¹⁶ having Ribosome-inactivating proteins are given in table 1, 2 and 3.

 

Table 1: Type 1 ribosome-inactivating proteins

Name and part of  Plant	Name of RIP
Agrostemma githago seeds	Agrostins
Amaranthus viridis leaves	Amaranthin
Asparagus officinalis seeds	Asparins,
Basella rubra seeds	Hispin,
Beta vulgaris leaves	Beetin27
Bryonia dioica leaves, roots	Bryodins
Citrullus colocynthis seeds	Colocins
Momordica balsamina seeds	Momordin II
Phytolacca americana leaves, seeds	Pokeweed antiviral proteins (PAP)
Trichosanthes kirilowii roots, seeds	Trichosanthins, Trichokirin
Dianthus barbatus leaves	Dianthin 29
Gelonium multiflorum seeds	Gelonin
Saponaria officinalis leaves, roots, seeds	Saporins
Laminaria japonica leaves	Lamjapin
Lyophyllum shimeij fruiting bodies	Lyophyllin

 

Table 2: Type 2 toxic ribosome-inactivating proteins

Name of Plant  and part	Name of RIP
Abrus precatorius seeds	Abrins
Abrus pulchellus seeds	Pulchellin
Ricinus communis seeds	Ricins
Viscum album leaves	Viscumin

 

Table 3: Type 2 non-toxic ribosome-inactivating proteins

Name of Plant and part	Name of RIP
Cinnamomum camphora seeds	Cinnamomin
Cinnamomum porrectum seeds	Porrectin
Cucurbita foetidissima root	Foetidissimin
Sambucus nigra bark, fruits and seeds	Nigrin b, Nigrin f

 

BIOLOGICAL ACTIVITY:

Both type 1 and type 2 RIPs have been identified on the basis of a well-defined biological activity. Type 2 RIPs were discovered more than a century ago when Stillmark isolated the toxic principle from castor bean seeds. The high toxicity of ricin was attributed to its agglutinating activity, which means that the carbohydrate binding activity of type 2 RIPs was recognized long before their enzymatic activities and their inhibitory activity on protein synthesis¹⁷. Type 2 RIPs owe their carbohydrate binding activity to the B-chain, which contains two or possibly three binding sites¹⁸. Various biological activities of RIPs has been reported and some of them has been mention below.

 

Cytotoxicity:

Cytotoxicity of type 2 RIPs is determined by the binding of the B-chain to a sugar-containing receptor on the cell surface. Ricin, causes 50% cell death at concentrations below 1 mg/ml whereas some elderberry type 2 RIPs show no effect at 1 mg/ml¹⁹.

 

Type 1 RIPs were discovered in 1925 when Duggar and Armstrong observed that Phytolacca americana antiviral protein (PAP) inhibits the transmission of tobacco mosaic virus (TMV) in plants²⁰. However, only in 1978 PAP was recognized as an inhibitor of protein synthesis²¹. Many, but certainly not all, type 1 RIPs are antiviral proteins. Unlike some type 2 RIPs, type 1 RIPs are not cytotoxic and do not behave as toxins because they are not able to cross the cell membrane on their own. Some specialized animal cells, however, can import type 1 RIPs by endocytosis and subsequently become sensitive to the RIP activity. The five tested RIPs show very similar characteristics².

 

Table 4: The five tested RIPs showing inhibition of protein synthesis  Full-size table

Ribosome-inactivating protein	Level in tissue (seeds) (mg/ 100 g)	Inhibition of protein synthesis		Toxicity to mice LD50 (mg/kg)
		Cell-free IC50 (nM)	HeLa cells                        IC50 (nM)
Gelonin	250–300	0.40	>3300	40
Lychnin	160	0.17	>3300	9.3
Momordin	150–180	0.06	>3300	7.4
PAP-S	100–180	0.04	3400	2.6
Saporin S-6	270–400	0.037	610–2340	4.0

 

Abortifacient activity:

Trichosanthin, a ribosome-inhibiting protein obtained from Trichosanthes kirilowii roots and seeds is currently used in China to induce early and mid-term abortion with over 95% success rate and minimal side effects. The abortifacient effect of trichosanthin is described to the active uptake of the RIPs by trophoblasts. Trichosanthin (0·3 mg/25 g) and momorcharin (0·2 mg/25 g) given intraperitoneally to mice on Days 4 and 6 of pregnancy led to an inhibition of implantation²². Ricin, abrin, and PAP inhibit cell-free protein synthesis by irreversibly inactivating the ribosomes in such a way that the function of elongation factors EF-1 and EF-2 is blocked²³.

 

Antiviral activity:

Dodecandrin, a newly discovered ribosome-inhibiting protein, has been isolated and purified from the leaves of the plant, Phytolacca dodecandra. Dodecandrin has a molecular weight of approx. 29,000. It cross-reacts with antiserum prepared against pokeweed antiviral protein from Phytolacca americana and exhibits similar requirements for antiribosomal activity. It is more basic than pokeweed antiviral protein, and comparison of the first 30 amino-terminal residues of the two proteins reveals 83% homology. This level of homology is greater than that between pokeweed antiviral protein and pokeweed antiviral protein S, another antiviral protein found in P. americana. Such conservatism in sequence, coupled with the high efficiency of the proteins in deactivating ribosomes and with their abundance in plant tissue, suggests that they serve an important function in the life of the plant, as a defense against infection ²⁴.

 

Anti-HIVactivity:

GAP31 (gelonin anti-HIV protein of 31kDa) is an anti-HIV protein which we have identified and purified from a medicinal plant, Gelonium multiflorum. It is capable of inhibiting HIV-1 infection and replication. GAP31 also exhibits DNA topoisomerase inhibitor activity and RNA N-glycosidase activity²⁵.

 

Recent advances in trichosanthin; a ribosome-inactivating protein with multiple pharmacological properties has been also reported. Trichosanthin (TCS), a ribosome-inactivating protein extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii has multiple pharmacological properties including abortifacient, anti-tumor and anti-HIV. It is traditionally used to induce abortion but its antigenicity and short plasma half-life have limited the repeated clinical administration.  Studies on structure–function relationship and mechanism of cell entry are also covered. Recently, TCS has been found to induce apoptosis, enhance the action of chemokines and inhibit HIV-1 integrase. These findings give new insights on the pharmacological properties of TCS and other members of ribosome-inactivating proteins²⁶.

 

Immunological effects (immunogenicity, allergenicity and immunosuppression):

The knowledge of the immunogenicity of RIPs goes back to Paul Ehrlich, who obtained the very first antibodies against ricin. Ricin²⁷ and all RIPs are strongly immunogenic, and their administration to animals gives rise to formation of antibodies, with cross-reaction only among RIPs from plants belonging to the same family²⁸. Ricin administration induced an IgE response, and also enhanced the response against other antigens given at the same time. Formation of IgE has been observed in mice after administration of several type 1 RIPs²⁹.

 

RIPs have immunosuppressive effects, both on humoral and cell-mediated response, in that their administration prevents formation of antibodies and retards graft rejection³⁰. This only if RIPs are given before the antigen, suggesting that they interfere with an early step in the immune response.

ENZYMATIC ACTIVITIES:

It is now generally accepted that all RIPs are enzymes and that some have multiple enzymatic activities.

 

Classical enzymatic activities Site-specific RNA N-glycosidase activity toward ribosomes:

Although all RIPs exhibit RNA N-glycosidase activity toward ribosomes, there are marked differences in substrate specificity. Both RIPs and ribosomes contribute to the apparent substrate specificity. Since the rRNA target structure is universally conserved, differences in sensitivity between ribosomes most likely reside within the ribosomal proteins, which may either allow or prevent access of the RIPs to the sarcin/ricin loop. Vater et al. identified rat liver ribosomal proteins L9 and L10e as the binding target of the ricin A-chain ³¹, whereas yeast ribosomal protein L3 was identified as the binding factor of PAP ³². The specific interaction between PAP and L3 probably explains the broad-spectrum activity of PAP toward ribosomes from species of different taxonomic groups because L3 is highly conserved in ribosomes. Differences in activity and ribosome substrate specificity are also due to differences in the structure of different RIPs. PAP-ricin A-chain protein hybrids were created and examined for activity on rabbit reticulocyte and E. coli ribosomes. According to the results of these experiments, the amino-terminal half of the hybrid proteins determine the substrate specificity.  RIPs do not affect ribosomes of the plant in which they are expressed; recent studies have unambiguously demonstrated that RIPs are fully capable of deadenylating and inactivating nonspecific ribosomes³³.

 

Site-specific RNA N-glycosidase activity toward ribosomal RNA:

Although the rRNA in native ribosomes is the preferred substrate for RIPs, naked (deproteinized) rRNA and a synthetic 35-residue oligoribonucleotide that mimics the sarcin/ricin loop can also serve as the substrate for RIP activity³⁴.

 

Polynucleotide: adenosine glycosidase activity:

Saporin-L1, a type 1 RIP from the leaves of Saponaria officinalis, is capable of removing multiple adenine residues from various nucleic acid substrates ³⁵. RIPs present in the starting material in some plant species and tissues are assayed translation inhibitory and polynucleotide adenine glycosylase activities of edible plant extracts. Plants having translation inhibitory activity and adenine glycosylase activity are mention in table 5.

 

Polynucleotide: guanosine glycosidase activity:

Highly purified gelonin, momordin, PAP-S, and saporin-S6, these RIPs are able to remove bases other than adenine³⁷.

 

RNase activity:

Mock et al. demonstrated that preparations of α and especially β momorcharin (corresponding to momordin I and momordin II, respectively) possess RNase activity³⁸.

 

 

Table 5: Plants having translation inhibitory activity and adenine glycosylase activity³⁶

Family	Genus Species Subspecies or variety	Common         name	Translation inhibitory activity		Adenine glycosylase activity
			Specific activity (U g−1  of basic protein) × 10−6	Tissue activity (U g−1 of starting tissue)	Specific activity (U g−1 of basic protein) × 10−3	Tissue activity (U g−1 of starting tissue)
Asteraceae	Cichorium intybus	Chicory	<0.01	<1.00	610	0.7
	Cichorium endivia	Cultivatedendive	0.05	0.13	367	1.1
	Cynara scolymus	Artichoke	0.02	<1.00	31,300	26.2
Solanaceae	Capsicum annuum	Red pepper	0.02	1.30	833	49.6
	Lycopersicon esculentum	Cherry tomato	<0.01	<1.00	500	39.4
	Solanum melongena	Egg plant	<0.01	<1.00	633	13.1
Chenopodiaceae	Beta vulgaris	Common beet	1.25	161.00	216,000	27,800
Apiaceae	Apium graveolens dulce	Celery	<0.01	<1.00	110	10.0
	Apium graveolens rapaceum	Celeriac	0.30	10.20	2160	73.5
	Daucus carota	Carrot	0.26	0.30	920	11.8
	Foenuculum vulgare	Sweet fennel	0.01	0.3	140	3.7
	Pastinaca sativa	Parsnip	0.08	3.11	7520	280.3
	Petroselinum crispum	Parsley	<0.01	<1.00	270	29.5
Fabaceae	Glycine max	Wild soybean	<0.01	<1.00	80	8.1
	Phaseolus vulgaris	Kidney bean	<0.01	<1.40	319	45.0

 

 

DNase activity:

Dianthin 30, saporin-S6, and gelonin indicated that these type 1 RIPs exhibit nuclease activity toward single-stranded DNA³⁹. MAP30, one of the type 1 RIPs from seeds of the bitter melon (Momordica charantia), have been used to support the idea that RIPs are DNA glycosylase/AP lyases⁴⁰. In addition, it was suggested that the DNA glycosylase/AP lyase activity of MAP30 makes the HIV long terminal repeat unsuitable as a substrate for the HIV integrase as well as the DNA gyrase and that the DNA glycosylase/AP lyase may contribute to the antiviral and antitumor activities of MAP30.

The anti-HIV-1 activity has been desscribed to the inhibition by MAP30 and gelonin of the three specific reactions catalyzed by the viral integrase⁴¹.

 

RIPs AS PLANT DEFENSE PROTEINS:

RIPs are studied primarily because of their unique biological activities toward human and animal cells and the perspectives they offer for antiviral and antitumor activities in therapeutical applications. Highly toxic type 2 RIPs like ricin and abrin are believed to protect the seeds they inhabit against plant-eating organisms. Though the oral toxicity of most other type 2 RIPs is much lower than that of ricin, the accumulation of large quantities of these low-toxicity proteins (e.g., in vegetative storage tissues) probably offers some protection against phytophagous invertebrates and/or herbivorous animals. It has been suggested that abundant type 2 RIPs are storage proteins that can be used as a specific defense proteins if the plant is challenged by a predating organism⁴².

 

The active protein component was isolated and named as pokeweed antiviral protein. Subsequently, purified RIPs from many sources have been shown to potently inhibit the infection of test plants with diverse plant viruses. All RIPs with proven antiviral activity toward plant viruses are type 1 RIPs except the type 2 RIP from Eranthis hyemalis⁴³.

 

CONCLUSION:

The properties of RIPs raises some hope for their utilization for various purpose along with possible applications in medicine and also in plant sciences. In former, they have been studied as immunotoxins or as antiviral agents, mainly against HIV, although promising but with the difficulties as outlined above. Possibly, there exists some realistic hopes of their use for the therapy of topical tumors etc. Ribosome-inactivating proteins definitely exhibit RNA N-glycosidase and polynucleotide: adenosine glycosidase activity. Most biological activities of RIPs undoubtedly rely on their enzymatic activity. RIPs probably plays a role in plant defense. Type 2 RIPs are defense proteins directly targeted against plant consuming organisms, whereas type 1 RIPs are involved in the defense against viruses and microorganisms. To conclude the authors see no reason that the scope for further research is still there in this promising area.

 

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Received on 23.04.2010       Modified on 20.06.2010

Accepted on 06.07.2010      © RJPT All right reserved