N glycosidase that inhibit translation through their activity ...shodhganga.inflibnet.ac.in/bitstream/10603/10001/10/10...One class of proteins with selective toxicity, called ribosome-inactivating

Review of Literature

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Proteins with selective toxicity have been investigated for various biological

properties including increased plant defense against pathogens, cancer therapy and

antiviral agents. One class of proteins with selective toxicity, called ribosome-inactivating

proteins (RIPs), is found in fungi, bacteria and mainly in plant kingdom. RIPs are N-

glycosidase that inhibits translation through their activity against ribosomal RNA. Due to

selective toxicity of RIPs, a primary focus of research has been to use them as the toxic

agent in immunotherapies. As a result, much of the RIPs literature involve isolation and

characterization of RIPs from new plant species and their use as an anticancer therapy,

antiviral agent and antimicrobial agent (Puri et al., 2012). These studies have led

researchers to propose a role of novel RIPs from novel source. Complete review on the

ribosome-inactivating proteins (RIPs) is beyond the purview of this chapter. However,

brief description about various aspects has been provided in the following sections.

2.1 Ribosome-inactivating proteins (RIPs) in plants

2.2 Classification of Ribosome-inactivating proteins (RIPs)

2.3 Distribution in plant

2.4 Overview of RIPs

2.4.1 Type I RIPs

2.4.2 Type II RIPs

2.5 Enzymatic Function

2.5.1 N-glycosidase activity

2.5.2 Inhibition of protein synthesis

2.6 Biological Functions of RIP

2.6.1 Anti-tumour Activity

2.6.2 Antiviral Activity

2.6.2.1 HIV structure and life cycle

2.6.2.2 Effects on Human Immunodeficiency Virus (HIV)


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2.6.2.3 Effects on Herpes Simplex Virus

2.6.2.4 Effects on Hepatitis B Virus

2.6.2.5 Effect on Poliovirus

2.6.2.6 Effects on Kapsoi sarcoma-Associated Herpes Virus

2.6.2.7 Effects on Human T-cell leukemia virus I

2.7 Plasma Half Life of RIPs

2.8 Structure-function relationship of RIPs

2.1 Ribosome-inactivating proteins (RIPs) in plants

Ribosome-inactivating proteins (RIPs) are a group of proteins that inhibit protein

synthesis in many prokaryotic and eukaryotic cells (Stripe, 2004). The inhibition is

caused by the N-glycosidase depurination of rRNA at conserved residues. Ribosome-

inactivating proteins (RIPs) are RNA N-glycosidase which cleave N-glycosidic bond of

adenine A2660 in E.coli 23S rRNA or A4324 in eukaryotic 28S rRNA located in a highly

conserved α-sarcin/ricin (SR) loop on the rRNA. RIPs molecules specifically cleave at an

adenine residue within the GAGA sequence loop in rRNA (Figure 2.1).

In eukaryotic systems, the rRNA N-glycosidase activity leads to the loss of

binding of the eukaryotic elongation factor-2 (eF-2) and subsequent attenuation of protein

translation (Endo et al., 1987). The activities of these proteins probably explain their

putative role in plants as defensive agents against pathogens. The other biological

properties of RIPs include antiproliferative, anti-tumour, immunosuppressive,

abortifacient, antiviral and anti-human immunodeficiency virus (anti-HIV) activities (Ng

et al., 2010; Girbés et al., 2004). The potential applications of RIPs include conjugation

with antibodies to form immunotoxins for cancer therapy (Pirie et al., 2011), used as anti-

HIV agent in AIDS therapy (Uckun et al., 1998).

The RIPs of higher plants have been isolated, purified and characterized from

various plants belong to taxonomically unrelated families. Thus RIPs are very interesting

to a lot of investigation.


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Eukaryotic 28S rRNA

Figure 2.1 The action sites of RIPs on 28S RNA in Eukaryotic ribosomes

2.2 Classification of Ribosome-inactivating proteins (RIPs)

Stripe and his co-workers divided RIPs into two groups based on primary

structure; type I RIPs and type II RIPs (Stripe and Barbieri, 1986). Later, Mundy et al.,

(1994) classified RIPs into three groups based on their physical properties; type I, II and

III RIPs (Figure 2.2).

Type I RIPs or SCRIPs (Single chain ribosome-inactivating proteins) consists of a

single, intact polypeptide of about 24-30 kDa molecular weight with enzymatic activity.

Type II RIPs is a heterodimeric protein that comprises a catalytically active A-chain with

approximately 30 kDa and a galactose-binding B-chain linked by disulfide bond with

molecular weight of approximately 35 kDa (Lord et al., 1994). The A-chain of type II

RIPs is RNA N-glycosidase that hydrolyses a N-C glycosidic bond of a conserved

adenosine in the sarcin/ricin domain of the largest RNA in the ribosome, releasing an

adenine base (Puri et al., 2009). The ribosome depurinated in this manner is unable to

bind elongation factor 1 or elongation factor 2-GTP complex. Thus the protein synthesis

is blocked at the translocation step of the elongation cycle (Ng et al., 2011). The B-chain

of type II RIPs is a lectin that can bind to the galactose-containing receptors on the

mammalian cell surface and facilitate the transport of RIP into the cytosol. When type II

RIPs enters cell, the disulfide bond between A-and B-chain is broken and the A-chain can

exhibit its RNA N-glycosidase activity and strong toxicity to cell (Figure 2.3) (Simpson et

al., 1999). Most of type II RIPs are more toxic to cell than type I RIPs because of the

presence of their B-chains.


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Figure 2.2 Schematic representation of primary structure of type I, type II and type III ribosome-inactivating protein (active chain: ; lectin binding chain: ; unknown activity domain: ; signal peptide: ; C-terminal domain: ). This figure has been reproduced and modified from Stripe and Battelli, (2006).


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Type III RIPs are synthesized as inactive precursor (proRIPs) or zymogens that require proteolytic processing to remove or nick of an NH2-amino acids and/or COOH-amino acids to be active enzymes (Walsh et al., 1991). Type III RIPs are much less prevalent than type I or type II RIPs. Their primary structure is similar to type I RIPs. However, the active form of type III RIPs can also be classified as type I RIPs consisting of single chain polypeptides with rRNA N-glycosidase activity (Mak et al., 2007).

2.3 Distribution in plants

RIPs are synthesized and widely distributed among the plant genera spanning 50 different species and 14 families, including Cucurbitaceae, Euphorbiaceae, Poaceae, Caryophyllaceae and Phytolaccaceae (Stripe and Barbieri, 1986). The most commonly found RIPs are type I ribosome-inactivating proteins. Both types of RIPs are localized to leaves, seeds and roots of the plant. However, single chain type I RIPs are much more abundant than their type II RIPs relatives (Stirpe and Battelli, 2006). Pokeweed antiviral protein (PAP) was the first type I RIP isolated from Phytolacca americana followed by momordin (Momordica charantia L.), luffin (Luffa cylindrica), bryodin (Bryonia dioica), dianthin (Dianthus caryophyllus), trichosanthin (Trichosanthes kirilowii), alpha-and beta momorcharin (Momordica charantia) and saporin (Saponaria officinalis) (Stripe and Barbieri, 1986).

Cucurbitaceae is a major family for economically important species, particularly for edible fruits. In the Cucurbitaceae family, several RIPs have been reported and investigated for their potential medicinal usages, such as trichosanthin and trichokirin from Trichosanthes kirilowii, bryodin from Bryonia dioica, luffin from Luffa cylindrica, momorcharin from Momordica charantia, luffangulin and luffaculin from Luffa acutangula (Kaur et al., 2011b). The RIPs in the same plants are possibly isoforms that have similarities in structural and physiochemical properties and have the same conserved amino acids sequences. RIPs may be present in one or more tissues of a plant, sometimes in more than one form. In some part of plants such as M. charantia seed contain multiform of type I RIPs such as momordin I or alpha-momorcharin, beta-momorcharin, MAP30, γ-momorcharin, δ and ε-momorcharin and charantin are discovered (Puri et al., 2009). Toxic type II RIPs, ricin from the seeds of Ricinus communis, and abrin from Abrus precatorius were identified at the end of the 19th century. The structure and mode of action of these proteins were elucidated and turned out to be first identified ribosome-inactivating proteins (Lin et al., 1970). To date, fewer type II RIPs has been discovered and their recent findings are mentioned in Table 2.1. Type III RIPs have been characterized only from maize and barley (Nielson and Boston, 2001).


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(A)

(B)

Figure 2.3 Structures and mechanism of toxicity of type I (A) and type II (B) ribosome-inactivating proteins (figure has been reproduced and modified from http://cmumt.cmu.edu.tw)


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2.4 Overview of RIPs

Few recently discovered type I and II RIPs are described in the following section.

2.4.1 Type I RIPs

Naturally occurring RIPs abundantly found in some plant families, and higher

levels were found in seeds of Caryophyllaceae, Cucurbitaceae, Euphorbiaceae and

Phytolaccaceae. The most commonly found RIPs are type I. RIPs available in plants are

discussed in the Table 2.1.

2.4.1.1 Amaranthin

Amaranthin isolated from Amaranthus viridis has a molecular weight of

approximately 30 kDa. Amaranthin possesses antiviral activity in tobacco mosaic virus

(TMV) infection test of Nicotiana glutinosa leaves. It also shows in vitro translation

inhibition in cell free assays and displays N-glycosidase activity (Kwon et al., 1997).

2.4.1.2 α and β-Momorcharins

RIPs extracted from M. charantia are referred to as momorcharins (MMCs),

including α- and β-momorcharins that are isolated from seeds. Specifically, the α-MMCs

have a molecular weight of approximately 30 kDa and a neutral sugar content of

approximately 1.6%. Similarly, β-MMCs have a molecular weight of 29 kDa and neutral

sugar content of about 1.3%. Both proteins possess similar structural and biological

properties, but are immunologically distinct (Yeung et al., 1986). Their biological

activities include induction of mid-term abortion (Law et al., 1983), inhibition of tumour

growth, suppression of the immune response (Leung et al., 1987) and inhibition of HIV-1

replication (Lifson et al., 1989). In addition, α-and β-MMCs possess antifungal activity,

with α-MMC showing activity against Fusarium oxysporum and Phythium

aphanidermatum, whilst β-MMCs show antifungal activity against Phythium

aphanidermatum (Wang et al., 2004a).

2.4.1.3 γ, δ- and ε-Momorcharins

γ-Momorcharin is a low molecular weight RIP (11.5 kDa) isolated from the seeds

of M. charantia that possesses RNA N-glycosidase activity. In contrast to α- and β-MMC,

which have a neutral sugar content of 1.6% and 1.3%, respectively, γ-momorcharin


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contains no neutral sugar (Pu et al., 1996). There are also two other momorcharins that

have been recently described, namely, δ-momorcharin (30 kDa) and ε-momorcharin (24

kDa). These RIPs were isolated from the seeds and fruits of M. charantia. Interestingly,

the cell free protein synthesis inhibition by ε-momorcharin is much weaker than other

RIPs isolated from M. charantia; whereas δ-momorcharin possesses similar activity to α-

and β-MMCs (Tse et al., 1999).

2.4.1.4 MAP30

As for the α- and β-MMCs, MAP 30 was first isolated from the seeds of M.

charantia. This RIP is comprised of 263 amino acids with a molecular mass of

approximately 30 kDa and a high proportion of basic residues (11%) (Lee-Huang et al.,

1995). Functionally, MAP30 is demonstrated to possess anti-HIV activity (Lee-Huang et

al., 1990).

2.4.1.5 Charantin

A small “napin-like” ribosome-inactivating peptide charantin (9.7 kDa), was

isolated from M. charantia seeds. Charantin had single peptide chain with no biological

activity yet has been reported till date (Parkash et al., 2002). Contrary to native napin-like

protein, recombinant His-rMcnapin exhibited high antifungal activity against T. humatum

when compared with mature napin-like proteins from M. charantia (Vashishta et al.,

2006).

2.4.1.6 Cochinin

Cochinin B is a single chain ribosome-inactivating protein isolated from the seeds

of M. cochinchinensis. It is a highly basic protein with molecular weight of 28 kDa.

Cochinin B inhibit protein synthesis in a cell free system, exhibit N-glycosidase activity

and cytotoxicity against Vero cell line. It possess broad range of potent anti-tumour

activities against human cervical epithelial carcinoma (HeLa), human embryonic kidney

(HEK 293) and human cell lung cancer (NCI-H187) cell lines (Cheuthong et al., 2007).

2.4.1.7 Momorgrosvin

Momorgrosvin, a single chain ribosome-inactivating protein, isolated from the

seeds of M. grosvenorii has a molecular weight of 27.7 kDa. The molecular weight of

momorgrosvin (27.7 kDa) is slightly lower than that of α-and β-momorcharins and all of


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them are glycoproteins. Momorgrosvin has approximately 50% homology between N-

terminal sequences of α-and β-momorcharins. It acts catalytically on tRNA and has

specific RNase activity compared with other RIPs. Momorgrosvin inhibit cell-free protein

synthesis and displays N-glycosidase activity (Tsang and Ng, 2001).

2.4.1.8 MRK29

A bitter gourd protein (MRK29) isolated from ripe fruits (M. charantia) in

Thailand has ~29 kDa molecular weight. The purified protein inhibited HIV-1 reverse

transcriptase and reduction of viral core protein p24 expression in HIV-infected cells. The

protein was thought to have immunomodulatory role on immune cells, because it

increased 3-fold TNF α cytokine activity (Jiratchariyakul et al., 2001).

2.4.1.9 Saporin

Saporin is a 30 kDa type I RIP, isolated from the seeds of Saponaria officinalis

commonly referred to as soapwort. It possesses unusually high stability, thus making it an

ideal candidate for biotechnological applications (Kuroda et al., 2010). There are several

isoforms of saporin found in different parts of the soapwort plant. These isoforms possess

N-glycosidase activity and also demonstrate inhibition of translation in a variety of cell

types including HeLa, BeWo and NB100 cells (Ferreras et al., 1993). In addition, saporin

(L1 & L2), saporin (R1-R3) and saporin (S5-S9) possess polynucleotide: adenosine

glycosidase activity. Interestingly, saporin is also a potent HIV-1 integrase inhibitor (Au

et al., 2000).

2.4.1.10 PAP (Pokeweed antiviral protein)

PAP isolated from Phytolacca americana found to contain PAP-II (isozymes

from leaves), PAP-S (pokeweed antiviral protein from seeds) and PAP-R (from roots).

PAP (Pokeweed antiviral protein) basic protein has a molecular mass 29 kDa. PAP-II is

slightly larger than PAP, with molecular mass of 30 kDa. PAP-S has molecular weight

29.8 kDa. PAP is a potent inhibitor of protein synthesis in cell free extract (Irvin, 1995)

and exhibit N-glycosidase activity (Kung et al., 1990). PAP has shown antiviral activity

against a number of viruses including influenza virus (Tomilson et al., 1974), polio

(Ussery et al., 1977), herpes simplex virus (Teltow et al., 1983), hepatitis B-virus (He et

al., 2008) and human immunodeficiency virus (Zarling et al., 1990). PAP inhibits

production of p24 in both T cells and in vitro infected macrophages. Anti CD4-PAP


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immunoconjugates exhibit potent anti-HIV activity in zidovudine resistant T-cells. Anti

CD4-PAP immunoconjugate has no cytotoxicity on lymphohematopoietic cell (Erice et

al., 1993).

2.4.1.11 Trichosanthin (TCS)

TCS is a single chain RIP obtained from the root tubers of the Chinese medicinal

herb Trichosanthes kirilowii. TCS has a molecular weight of 27 kDa and possesses

multiple pharmacological properties including N-glycosidase activity and inhibition of

cell free translation in rabbit reticulocyte lysate (Li et al., 2010a). In addition, this RIP

possesses other biological properties, including induction of mid-term abortion, anti-

tumour, anti-HIV and immunosuppressive activity (Shaw et al., 1994). It significantly

inhibits hepatitis B virus, measles and herpes simplex virus (Chen et al., 2006) and was

the first RIP found to possess anti-HIV activity in vitro (McGrath et al., 1989). TCS

induces apoptosis of cervical adenocarcinoma HeLa cell and cervical squamous Caski

cells. It also inhibits the proliferation of breast adenocarcinoma cells in vitro and in vivo

(Li et al., 2010a).

2.4.1.12 Hispin

Hispin is a type I ribosome-inactivating protein and has been isolated from seeds

of Hairy melon. Hispin has a molecular weight of approximate 21 kDa. The N-terminal

amino acid sequence of hispin shows minimal homology to other RIPs like saporin, PAP,

ricin A-chain and abrin A-chain. It exhibits N-glycosidase activity and inhibit protein

synthesis in cell-free rabbit reticulocyte lysate system. Hispin has antifungal activity

against Coprinus comatus, Fusarium oxysporum, Physalospora piricola and

Mycosphaerella arachodicola. Hispin also exhibit ribonuclease activity on tRNA (Ng and

Parkash, 2002).

2.4.1.13 Lagenin

Lagenin is a ribosome-inactivating protein obtained from seeds of Lagenaria

siceraria. The molecular weight of lagenin is 20 kDa which is lower than the range of 25-

32 kDa reported for other RIPs. Lagenin inhibits cell-free translation in a rabbit-

reticulocyte system and possess ribonuclease activity on yeast tRNA (Wang and Ng,

2000).


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2.4.1.14 Luffangulin

Luffangulin is a small molecular weight protein isolated from the seeds of Luffa

acutangula (ridge gourd). This 5.6 kDa peptide inhibits cell-free translation, but lacks

inhibitory activity toward HIV-1 reverse transcriptase (Wang and Ng, 2002).

2.4.1.15 Gelonin

Gelonin is a single chain RIP extracted from Gelonium multiflorum. It has a

molecular weight of approximately 30 kDa and inhibits protein synthesis in reticulocyte

cell lysates (Stripe et al., 1980). Gelonin possesses polynucleotide: adenosine glycosidase

activity that results in the release of adenine from DNA and RNA in vitro (Barbieri et al.,

1997). It also has a unique DNA-glycosylase activity that removes adenine from ssDNA

(Nicolas et al., 2000), and shows significant cytotoxicity to cancer cells (Li et al., 2007).

2.4.2 Type II RIPs

2.4.2.1 Ricin

Ricin is a type II RIP isolated from seeds of the castor bean plant, Ricinus

communis. It is a glycosylated heterodimer comprised of 32 kDa A-chain linked by a

single disulfide bond to a 34 kDa galactose/ N-acetylgalactoseamine-binding lectin B-

chain. Approximately 50% of the A-chain consists of α-helices and β-sheets. The B-chain

of ricin is a bilobal structure composed of two homologous domains (Lord et al., 1994).

2.4.2.2 Mistletoe

Mistletoe lectin is purified from Viscum album, which is classified as type II

ribosome-inactivating protein due to its RNA N-glycosidase activity, immunomodulatory

effects and anticancer activities. The lectins are heterodimeric glycoproteins containing

A-chain with cytotoxicity activity and B-chain with sugar binding properties. Mistletoe

lectins have different sugar binding specificities; which may play an important role

towards the cancer cells (Schöffski et al., 2004). Mishra et al., (2005) solved crystal

structure of Himalayan mistletoe RIP, purified from Viscum album. The structure was

determined by the molecular replacement method and refined at 2.8 Å resolution.

2.4.2.3 Foetidissimin II

Foetidissimin II, another type II ribosome-inactivating protein, isolated from dried

roots of Cucurbita foetidissima and has molecular weight of 61 kDa. In addition to N-

glycosidase and cell free protein synthesis inhibition, it exhibits cytotoxicity towards

adenocarcinoma and erthryoleukemia cancer cells (Zhang and Halaweish, 2007).


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Table 2.1 Recently studied Ribosome-inactivating proteins

Source RIP Mol. wt (kDa) pI Activity Reference

Type I RIP

Amaranthus viridis Amaranthin 30 9.8 N-glycosidase, in vitro translational inhibition, antiviral

Kwon et al., 1997

Benin hispada Hispin 21 - tRNA ribonuclease, N-glycosidase activity, antifungal

Ng and Parkash, 2002

Bougainvillea spectabilis Bouganin 26 9.6 Adenine polynucleotide glycosylase, protein synthesis inhibition

Fermani et al., 2009

Bryonia dioica Bryodin 30 ≥9.5 Antiviral, anti-HIV activity Stripe and Barbieri, (1986); Wachinger et al., (1993)

Dianthus caryophyllus Dianthin 30 & 32 30 & 32 8.65 & 8.55

Antiviral, anti-HIV, anti-HSV, anti-poliovirus, DNase activity

Roncuzzi and Gasperi-Campani (1996); Lee-Huang et al., (1991)

Gelonium multiflorum GAP31 31

Anti-HIV, anti-HSV, anti-tumour activity

Bourinbaiar and Lee-Huang (1996); Lee-Huang et al., 2000

Gelonium multiflorum Gelonin 30 8.15 Deoxyribonuclease, anti-tumour, polynucleotide: adenosine glycosidase activity

Cao et al., (2009); Barbieri et al., (2000)

Jatropha curcas Curcin 28 8.5 N-glycosidase, anti-tumour activity Luo et al., (2006)

Luffa cylindrica Luffin 30 - Anti-HIV, antiproliferative, apoptotic activity

Au et al., (2000); Poma et al., (1999)


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Type I RIP

Lychnis chalcedonica Lychnin ≈30 - Adenine polynucleotide glycosylase, protein synthesis inhibition

Fermani et al., 2009

M. cochinchinensis Cochinin 28 - N-glycosidase, anti-tumour activity, protein synthesis inhibition

Cheuthong et al., 2007

Momordica balsamina Balsamin 28 - N-glycosidase, in vitro translational inhibition

Kaur et al., 2011b

Momordica charantia MAP30 30 - N-glycosidase, anti-HIV, anti-tumour, protein synthesis inhibition, anti-HSV

Lee-Huang et al., 1995

Momordica charantia α-Momorcharin 30 9 Abortifacient, anti-tumour, anti-HIV, immunosuppressive

Zheng et al., 1999

Momordica charantia β-Momorcharin 29 9 Abortifacient, anti-tumour, anti-HIV, immunosuppressive

Zheng et al., 1999

Momordica charantia MRK29 29 - Anti-HIV Jiratchariyakul et al., (2001)

Phytolacca americana PAP 29 - N-glycosidase, anti-HSV, anti-poliovirus, anti-HIV, anti-HCMV, DNase activity

Bolognesi et al., (1990); Uckun et al., (1998); Wang and Tumer (1999)

Saponaria officinalis

Saporin 30 ≥9.5 Anti-HIV, hepatotoxicity activity, anti-tumour activity

Au et al., (2000); Polito et al., (2009)

Trichosanthes kirilowii Trichosanthin 27 9.4 N-glycosidase, cell-free translation inhibition activity, anti-HIV, anti-tumour

Shaw et al., 1994; Li et al., 2010a


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Type II RIP

Adenia lanceolata Lanceolin 61 - Polynucleotide glycosylase activity, cell-free translational inhibition, hemagglutinating activity

Stripe et al., 2007

Adenia stenodactyla Stenodactylin 63 - Polynucleotide glycosylase activity, cell-free translational inhibition, hemagglutinating activity

Stripe et al., 2007

Cinnamomum camphora

Cinnamomin 61 - Anti-tumour, N-glycosidase He and Liu et al., 2003

Cucurbita foetissima Foetidissimin II 61 - N-glycosidase, anti-cancer activity, cell-free protein synthesis inhibition

Zhang and Halaweish, 2007

Sambucus ebulus Ebulin I 56 - N-glycosidase, protein synthesis inhibition

Ferreras et al., 2011

Viscum album Mistletoe 65 - Anti-tumour activity, immunomodulatory activity, N-glycosidase

Pryme et al., 2006

Viscum articulatum Articulatin D 66 5.4 N-glycosidase, hemagglutinating activity, cell-free translational inhibition

Das et al., 2011


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2.5 Enzymatic Function

2.5.1 N-glycosidase activity

Ricin (a globular protein, glycosylated heterodimer joined by a single disulfide

bond) isolated from seeds of castor bean (Ricinus communis) was the first protein whose

biological activity was ascribed to plant protein. Approximately 50% of the A-chain

consists of α-helices and β-sheets. The B-chain of ricin is a bilobal structure composed of

two homologous domains. Endo and Tsurugi, (1987) reported that ricin A-chain removed

a single adenine residue from position 4324 in the 28S rRNA of rat liver ribosomes,

defining RIPs as ribosome specific N-glycosidases. Depurination occurs at a highly

conserved stem-loop structure found in the large RNA of all ribosomes. Ricin recognizes

a highly conserved region in the large 28S rRNA and cleaves a specific N-glycosidic

bond between an adenine and the nucleotide on the RNA whereby the adenine residue is

removed. The depurinated adenine is in the highly conserved sequence context of GAGA,

shown to be involved in ribosome-elongation factor interaction (Endo et al., 1987). After

the removal of adenine, the deadenylated site becomes unstable and a β-elimination

reaction can occur after the RNA is treated with acidic aniline, whereby the 3’-end of the

rRNA is cleaved and can be detected by electrophoresis. This site is usually depicted as

being present in a single-stranded loop, called the sarcin/ricin loop. It is located in domain

VII, some 400 nucleotides from the 3’-end of the rRNA (Endo et al., 1987). This

particular site-specific RNA N-glycosidase activity is a common property of all identified

type I and type II RIPs. RIPs from M. charantia depurinates intact ribosomes in exactly

the same manner as ricin A-chain does.

As for the A-chain of ricin, α- and β-MMC also deactivates eukaryotic ribosomes

using a similar catalytic mechanism (Yeung et al., 1988). Another study showed that the

action of α-and β-MMC on rRNA was very specific with MMCs acting only on the 28S

rRNA, but not on 18S, 5.8S and 5S rRNA, resulting in the release of a RNA fragment

known as “Endo’s fragment” upon acidic aniline treatment of isolated rRNA. The N-

glycosidase activity of the MMCs is not affected by a change in pH from 6.5 to 9.0, but

enzyme activity increases with increasing K+ concentration. By contrast, the enzyme

activity of β-MMC fluctuates slightly with an increasing concentration of NH4+ ions,

whilst a significant inhibitory effect is observed with increasing Mn2+ concentration


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(Fong et al., 1996). A similar effect is also observed on α-sarcin (cytotoxic protein from

Aspergillus giganteus) (Endo et al., 1983).

γ-Momorcharin also exhibits RNA N-glycosidase activity on ribosomes isolated

from rat liver in a dose-dependent manner. To determine the action site of γ-momorcharin

on 28S rRNA, the sequence of 5’-terminal nucleotides of the RNA fragment produced by

γ-momorcharin/aniline treatment were analyzed. By comparing the 5’-terminal nucleotide

sequence of RNA fragment produced by γ-momorcharin with that produced by ricin, it

can be concluded that γ-momorcharin acts on the same active site of 28S rRNA from rat

liver ribosomes (Endo et al., 1987; Pu et al., 1996).

δ-Momorcharin exhibited N-glycosidase activity and released a specific RNA

fragment of ~400 nucleotides from 28S rRNA. ε-Momorcharin exhibited weak N-

glycosidase activity as compared to other existing RIP’s (Tse et al., 1999). Charantin,

reacted positively in the N-glycosidase assay. Charantin produced 470 bp fragment, when

treated with rabbit reticulocyte lysate. This product band was similar to the small RIP’s

like γ-momorcharin and luffin-S (a RIP isolated from Luffa cylindrica) (Parkash et al.,

2002; Gao et al., 1994).

2.5.2 Inhibition of protein synthesis

M. charantia RIP’s were found to be potent inhibitors of protein synthesis in cell

free system. Lectins were first detected in the M. charantia plant in 1978 because of their

ability to inhibit protein synthesis in Ehrlich ascite cells (Lin et al., 1978). M. charantia

lectin is the second example of a non-toxic lectin, inhibiting protein synthesis in-vitro

after Ricinus communis agglutinin (Saltvedt, 1976; Cawley et al., 1978). As for lectins, α-

and β-MMCs also inhibit protein synthesis in rabbit reticulocyte cell free lysates. The

potencies (ID50) of the cell free protein synthesis inhibitory activity by α-MMC and β-

MMC are reported to be as low as 0.12 nM and 0.11 nM, respectively (Yeung et al.,

1988).

As for the lectins and MMCs, MAP30 exhibits a dose dependent inhibition of cell

free translation system. Eukaryotic translation inhibition by MAP30 was assayed in a

rabbit reticulocyte lysate system and the effect on protein biosynthesis expressed as the

incorporation of [3H] labelled leucine into trichloroacetic acid (TCA) insoluble product.

MAP30 exhibited cell-free translation inhibition in a dose dependent manner with an ID50

of 3.3 nM (Lee-Huang et al., 1990). Ribosome-inactivation activity of recombinant


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MAP30 (rec-MAP30) has also been measured by in vitro translation of globin message in

a rabbit reticulocyte lysate system. Rec-MAP30 exhibits similar ID50 (3.3 nM) to that

observed for natural MAP30 (nMAP30). Interestingly, MAP30 could not enter

uninfected (normal) cells and was incapable of activating cellular ribosomes and

inhibiting cellular protein synthesis in these cells (Lee-Huang et al., 1995).

γ-Momorcharin also inhibits protein synthesis in a dose dependent manner in

rabbit reticulocyte lysates with an ID50 of 55 nM. Compared to α-MMC, β-MMC and

MAP30, the higher ID50 value may be due to the small molecular weight of this RIP (Law

et al., 1983). However, the potency of δ-momorcharin is similar to that of α-and β-MMC

with an IC50 of 0.15 nM. By contrast, ε-momorcharin and charantin exhibits much weaker

inhibition of cell free protein synthesis with ID50 values of 170 nM (Tse et al., 1999) and

400 nM (Parkash et al., 2002), respectively.

Musarmins (MU 1, 2 and 3) isoforms from Muscari armeniacum have inhibitory

activity on several cell-free systems from mammals and plants. Three isoforms of MU

showed strong inhibitory activity on rabbit reticulocyte system. MUs 1, 2 and 3 had 7, 9.5

and 4 ng mlˉ1 IC50 values, respectively. In rat liver, MUs 1, 2 and 3 had 79, 95 and 101 ng

mlˉ1 IC50 values. At high concentrations of MU 1, 2 and 3 was not shown protein

synthesis activity against plant-derived cell-free systems (Arias et al., 2003). TRIP, single

chain ribosome-inactivating protein isolated from leaves Nicotiana tabacum inhibited

translation in wheat germ and rabbit reticulocyte system. TRIP inhibited wheat germ

translation system more efficiently than rabbit reticulocyte system. TRIP inhibited protein

synthesis on wheat germ translation system at lower concentration and had 30 ng mlˉ1

IC50 value as compared to 100 ng mlˉ1 for rabbit reticulocyte system (Sharma et al.,

2004). BE (Beetins) inhibited protein synthesis more efficiently on rabbit reticulocyte

lysates as compared to rat liver, Vicia sativa and Triticum aestivum IC50 values for rabbit

reticulocyte lysates, rat liver, Vicia sativa and Triticum aestivum cell free systems were

1.15, 68, 617 and 1318 ng mlˉ1, respectively (Iglsias et al., 2005).

2.6 Biological Functions of RIP

2.6.1 Anti-tumour activity

The RIP’s from many plants have expressed in vitro and in vivo anti-tumour

activity (Lee-Huang et al., 2000; Fan et al., 2008; Mansouri et al., 2009). An overview of

RIPs possessing anti-tumour activity is provided in Table 2. MAP30 exhibits anti-tumour


24

activity against certain human tumour cell lines. These include brain glioblastoma, breast

carcinoma, epidemoid carcinoma, liver hepatoma, melanoma, myeloma, neuroblastoma

and prostate carcinoma (Xiong et al., 2009; Bian et al., 2010). The anti-tumour activity of

rec-MAP30 and nMAP30 were identical with respect to their sensitivity to particular

tumour types. The most sensitive tumour cell lines were breast, CNS, melanoma and

myeloma tumours with EC50 values of 0.21-0.38 nM. Prostate and epidemoid carcinomas

were less responsive with ID50 values of 3.42 and 1.88 nM, respectively (Lee-Huang et

al., 1995).

Dexamethasone is used to treat cancers such as Hodgkin’s disease, non-

Hodgkin’s myeloma and lymphocytic leukemia through inhibition of NF-κB activity (De

Bosscher et al., 2003). Dexamethasone (1μM) treatment of HepG2 cell does not generate

an anti-proliferation effect, but efficiently inhibits TCS-induced degradation of IκB-α

protein and enhanced TCS-induced apoptotic death in HepG2 cells. Dexmethasone may

inhibit dissociation of the NF-κB in the cytoplasm and promote the transcription of the

IκB-α gene, resulting in suppression of NF-κB activation and enhanced TCS-induced

anti-tumour effects (Li et al., 2010a).

In addition, recombinant luffin from Luffa cylindrica displays in vitro cytotoxicity

against various tumour cell lines. Recombinant luffin inhibited proliferation of JEG-3

(human placental choriocarcinoma), HepG2 (human hepatoma) and MCF-7 (human

breast cancer) cell line in a dose and time-dependent manner (Liu et al., 2010). Recently,

Tianhua (TH-R), identified from Trichosanthes kirilowii, inhibits the growth of human

lung cancer A549 cell line. TH-R exhibits inhibition of A549 human lung cancer cell line

by arresting G0/G1 phase of the cell cycle in a dose-and time-dependent manner and

induces apoptosis (Li et al., 2010a).

Fibroblast growth factor-inducible 14 (Fn14) related to TNF receptor superfamily

has been shown to regulate a variety of cellular functions which include cell survival, cell

growth, angiogenesis and inflammation. Although Fn14 expressed at relatively low levels

in normal tissues, but dramatically gets elevated locally in injured and disease tissues

(Han et al., 2010). Recombinant gelonin (rGel), a type I ribosome-inactivating protein,

conjugated to anti-Fn14 monoclonal antibody (ITEM-4) was highly cytotoxic to Fn14

expressing tumour cell line. Upon administration of immunoconjugate, ITEM4-rGel

enhanced long term tumour growth suppression in nude mice bearing T-24 human

bladder cancer cell xenograft (Zhou et al., 2011).


25

2.6.2 Antiviral activity

Ribosome-inactivating proteins possess broad spectrum antiviral properties

against different viruses, which include RNA and DNA viruses, through inhibition of

viral protein synthesis in the infected cells. These observations suggest that ribosome-

inactivating proteins were effective against a number of different viruses coupled with

their ability to inactivate eukaryotic ribosomes in vitro. The anti-HIV mechanism of

ribosome-inactivating proteins is still not clear.

2.6.2.1 HIV structure and life cycle

Structure of Virus

High-resolution electronic microscope techniques help study how mature HIV

virions are organized structurally. HIV particles are roughly spherical and 100-150 nm in

size. The following can be distinguished in the particles (Figure 2.4).

• Envelope: Envelope derived from the host cell membrane. The viral proteins are

designated with numbers reflecting the protein sizes in kilodaltons (kDa). A

mature virus surrounded by a lipid bilayer membrane, on which about 70 trimeric

envelops were embedded (Kuznetsov et al., 2003). The envelope consist of an

external surface glycoprotein, gp120 and a transmembrane glycoprotein gp41,

both derived from a 160 kDa precursor glycoprotein (McCune et al., 1988).

Glycoprotein gp120 and gp41 are responsible for the initial virus-cell interaction,

receptor binding and membrane fusion required for virus entry.

• Gag proteins: Three structural Gag proteins located inside the virus are: matrix

(MA, p17), capsid (CA, p24) and nucleocapsid (NC, p7) (Freed, 1998). The MA

forms an inner shell just inside the viral membrane; recent evidences suggested

that MA might be a regulatory protein involved in enhancing HIV pathogenesis

(Li et al., 2010b). The CA protein constitutes a conical core inside MA, coating

two identical copies of single-stranded RNA. The NC interacts with viral RNA

and are required for RNA splicing and RNA encapsidation. All these Gag proteins

were cleaved from a polyprotein precursor, p55 by the viral protease (PR) (Mervis

et al., 1988; Kohl et al., 1988).

• Nucleocapsid: The two copies of RNA are located inside the capsid (p24) and are

linked together at the 5’ end. The dimerisation initiation site (DIS) on the linkage


26

was a hairpin structure and played a role in virus maturation and recombination

(Balakrishnan et al., 2003). The 5’ end and 3’ end of HIV RNA encode a long

terminal repeat (LTR) sequence, which regulates integration and virus replication.

There are three enzymes closely associated with the viral RNA: reverse

transcriptase (RT, p66, p51), protease (PR, p10) and integrase (IN, p32). RT is

also called RNA-dependent DNA polymerase and played an important role in

viral replication by transcribing the RNA into double-stranded DNA (Carter and

Ehrlich, 2008). PR cleaved viral proteins into their functional forms. IN

incorporate viral DNA into host cell chromosome DNA (Brown et al., 1989). All

the three enzymes cleaved from Pol precursor polyprotein (Jacks et al., 1988).

HIV had two regulatory proteins, transactivator of transcription (Tat, p14) and regulator of virion expression (Rev, p19), which are essential for viral replication. Tat was a major protein that up-regulated HIV replication. It induced T cell apoptosis (Westendrop et al., 1995) and co-receptors expression on cell surfaces (Huang et al., 1998) and blocked natural killer (NK) cell activities (Zocchi et al., 1998). Rev affects viral protein expression by regulating messenger RNA (mRNA) splicing and transporting unspliced mRNA to the cytoplasm of cell for protein translation (Malim et al., 1989).

In addition, there are two accessory proteins closely associated with the core, namely, negative factor (Nef, p27) and virus infectivity factor (Vif, p23). Nef regulated virus replication and activated cellular proteins, while Vif increased virus infectivity (Tokarev and Guatelli, 2011) and cell-to-cell transmission and helped in proviral DNA synthesis and assembly (Borman et al., 1995). Other accessory proteins include viral protein R (Vpr, p15) and viral protein U (Vpu, p16) and viral protein X (Vpx, p15), which mainly helped in virus replication, virus release and viral infectivity, respectively (Greene and Peterlin, 2002).

HIV life cycle

The HIV replication cycle is complex. To simplify and reduce complexity, it is usually divided into different stages (Figure 2.5).

• Virus entry into host cell: This stage involves adhesion, co-receptor interaction, fusion and virus internalization. HIV adhesion with the host cell surface occurs due to a high affinity interaction between the envelope protein gp120 and the host cell CD4 receptor (Magnus and Regoes, 2012). The gp120-CD4 complex interacts with the CCR5 or CXCR4 co-receptor and the glycoprotein gp41 allows


27

membrane fusion and the internalization of the virus into the host cell. After fusion, the nucleocapsid loses its structure and its content is released into the cytoplasm.

• Reverse transcription and transport into the nucleus: Single-stranded RNA is transformed into double-stranded DNA by Reverse transcriptase (RT). The enzyme has a high frequency for misincorporation of nucleotides, contrary to other mammal polymerases, because it lacks proofreading mechanisms. The synthesized DNA is the translocated into the nucleus.

• Viral DNA integration into genomic DNA: Viral DNA, once in the nucleus, is processed and transferred into the host genome by integrase (IN) enzyme activity. When the viral DNA is integrated, the infected cell is “permanently infected”. The provirus can be inactive (latent) in the genome for a long time (months or even years) or it can undergo active viral production, depending on the activation state of the cellular polymerase.

• Transcription and translation: The activation of the provirus occurs through replicative cellular machinery (such as polymerases or selective and constitutive transcriptional factors) and as a result favours the production of mRNAs from the virus, which are later translated into the regulatory proteins Tat, Rev, Vpu and Nef. After this, their expression as polyprotein precursors of structural genes, such as Gag, Gag-Pol and Env occurs.

• New virion production and budding: The assembly of the regulatory proteins, enzymes and viral RNA near the host cell membrane is required to form a new viral core. Budding then occurs by pinching off a part of the cell membrane.

• Virion maturation: The final step of the viral cycle, assembly of new HIV-1 virions, begins at the plasma membrane of the host cell. The Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by protease and processed into the two HIV envelope glycoproteins gp41 and gp120. These are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. The actions of protease (PR) allow the processing of the polyprotein precursors Gag and Gag-Pol into structurally and functionally mature proteins. The Gag and Gag-Pol polyprotein also associated with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell. The various structural components then assemble to produce a mature virion and bud from the host cell. Figure 2.4 represent the structure of mature HIV-1virion.


28

Figure 2.4 Structure of HIV: Trimeric glycoprotein gp120 and gp41 are embedded on HIV surface membrane. From outside to viral centre are structural proteins matrix (MA, p17) and capsid (CA, p24). Inside CA are two copies of single stranded RNA, linked at their 5’ end by dimerisation initiation site (DIS). Structure protein nucleocapsid (NC, p7) and three enzymes reverse transcriptase (RT, p66, p51), protease (PR, p10) and integrase (IN, p32) are inside the viral core (Figure reproduced from http://www.niaid.nih.gov).

Figure 2.5 Summary of HIV-1 replication cycle: The basic steps of the HIV replication cycle: viral entry, reverse transcription, and formation of infectious particles. (Figure reproduced from Scientific American Magazine, December 2008).


29

2.6.2.2 Effects on Human Immunodeficiency Virus (HIV)

RIPs from M. charantia possess broad spectrum antiviral properties against

different viruses through inhibition of viral protein synthesis in the infected cells. This,

coupled to the ability of the RIPs to inactivate eukaryotic ribosomes in vitro, suggests that

the mechanism of antiviral action involves inactivation of host ribosomes in virus-

infected cells (Puri et al., 2009). α-and β-momorcharins were used to inhibit HIV antigen

expression in HIV-infected human T-cells and monocytes/macrophages, as an improved

drug therapy for treating HIV-infections in humans (Yeung et al., 1986). α-MMC showed

complete inhibition of reverse transcriptase (RT) activity in HIV infected cells and this

inhibition was achieved at concentrations of anti-viral protein at which uninfected

(normal) cells were largely unaffected (Lifson et al., 1989). On treatment of HIV-infected

monocytes with MMCs, a complete inhibition of p24 expression was observed. Lower

effective doses of MMCs were determined when infected monocytes were treated with

0.5 or 5 μg mlˉ1 of the antiviral proteins. There was complete inhibition of HIV antigen

expression in infected monocytes at both concentrations. In HIV patients, the anti-HIV

proteins (MMCs) inhibited other events related to the loss of immunological competence

in HIV infected individuals, through general suppression of virus levels and inhibition of

viral protein synthesis in infected cells. The protein (α-MMC) may be administered

parenterally, including liposome-encapsulated form, solution form and attached to a

carrier, such as an anti-macrophage, anti T-cell or anti-HIV antibody for targeting the

protein to HIV infected cells. Intramuscular placement of liposome-encapsulated protein

or protein enmeshed in a collagen matrix provides slow release of the drug into the blood

stream, the possibility of greater viral inhibition and reduced toxicity (Lifson et al., 1989).

MAP30 exhibited dose-dependent inhibition of cell-free HIV-1 infection and

replication. as measured by: (i) quantitative focal syncytium formation on CEM

monolayers; (ii) viral core protein p24 expression; and (iii) viral-associated reverse

transcriptase (RT) activity in HIV-1 infected H9 (human T cell line) cells. The doses

required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM,

respectively. MAP30 showed no inhibition of cellular DNA synthesis and protein

production at 33.4 nM, yet 98% and 87% inhibition of p24 and RT activity, respectively,

was achieved (Lee-Huang et al., 1990). MAP30 could improve the efficacy of anti-HIV

therapy when used in combination with low pharmacological doses of steroidal and anti-

inflammatory drugs like dexamethasone (DEX) and indomethacin (IND), respectively.


30

The combination of non-toxic 1.5 nM dose of MAP30 either with DEX or IND resulted in

reduction in p24 expression in acutely infected MT-4 lymphocytes (Bourinbaiar and Lee-

Huang, 1995). Rec-MAP30 exhibited similar anti-HIV activity as shown by natural

MAP30 (nMAP30) (Lee-Huang et al., 1995). MAP30 was reportedly non toxic to human

sperms at the doses known to inhibit HIV-1 replication. It had no effect on the motility

and vitality of human spermatozoa (Schreiber et al., 1999).

Luffin P1, the smallest RIP from the seeds of Luffa cylindrica, has been observed

to possess potent effect on HIV replication. HIV viral replication require assembly of a

ribonucleoprotein (RNP) which is composed of Rev protein homooligomer and Rev

response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs (Lee

et al., 2008). Rev binds to one specific site in the RRE using a 17 amino acid α-helical

arginine-rich motif (ARM) for interaction with RRE. The α-helical structure of Rev ARM

is important for RRE binding and C-terminus of luffin P1 is similar to the ARM of Rev.

Luffin P1 may inhibit HIV-1 replication by interacting with Rev response element (Ng et

al., 2011).

TCS in Phase I/II clinical trials elicits a moderate increase in circulating CD4+ T

cells and a significant decrease in p24 levels in AIDS patients failing treatment with anti-

retroviral drug such as zidovudine (Byers et al., 1994). TCS recognizes HIV-1 through

viral envelope interactions. The viral envelope is significantly different to host cell

membranes with a rich lipid raft content and a high level of sphingolipid and cholesterol

(Lee et al., 2008). Exogenous TCS taken up by HIV-1 infected cells is associated with the

lipid rafts on HIV-1 budding sites, where the TCS enriched virions are generated. TCS

exploits the sorting strategy to eradicate both budding and prevent the virus dissemination

(Zhao et al., 2009).

According to recent studies, several maize variants were constructed by the

addition of HIV-1 protease recognition sequences to the internal inactivation region. Two

maize-RIP variants activated by recombinant HIV-1 infected cells, and inhibited viral

replication in human T-lymphocytes and enhanced N-glycosidase activity. The first and

last 10aa of Pro-RIP replaced with MA/CA site and Pro-RIP-MA/CA construct were

cleaved completely by HIV-1 protease. Eleven amino acids derived from HIV-1 TAT

protein and fused with N-termini of Pro-RIP and MOD to generate TAT-Pro and TAT-

MOD variants. The substitution of HIV-1 protease recognition sequences (p2/NC site)


31

and (MA/CA site), two TAT fused maize RIP variants were generated TAT-Pro-HIV-

p2/NC and TAT-Pro-HIV-MA/CA. TAT-Pro and TAT-MOD maize-RIP variant have

dose-dependent inhibition of HIV replication in HIV-1IIIB and an HIV-1 protease

inhibitor-resistant virus strain. TAT-Pro has weaker inhibiting effect on p24 antigen

production and syncytium formation as compared to TAT-Pro-HIV-MA/CA, TAT-Pro-

HIV-p2/NC and TAT-MOD (Law et al., 2010).

2.6.2.3 Effects on Herpes Simplex Virus

Momordica charantia inhibitor (MCI) had shown inhibition of herpes simplex

virus type 1 (HSV-1) multiplication (Aron and Irvin, 1980). The yield of HSV-1 was

reduced on incubation of the infected cells with MCI. The inhibition of HSV-1 replication

by MCI implies that it might be acting during the intracellular reproductive cycle and on

protein synthesis, which inhibited more markedly in virus-infected than in control cells

(Foa-Tomasi et al., 1982). MAP30 was found to be effective against HSV-1, HSV-2 as

well as on HSV specific nucleoside analog acyclovir (ACV)-resistant HSV strains.

Symptomatic infection, especially with HSV-2, is common among individuals infected

with HIV. HSV infection causes more mortality and morbidity in AIDS patients than any

other viral pathogen. The ID50 for MAP30 was between 0.1 to 0.3 μM in both ACV-

sensitive and resistant strains of HSV-1 and HSV-2. However, at these levels MAP30

caused no detectable effect on the viability of the WI-38 cells. It is interesting that

MAP30 (EC50 μM) demonstrated an exceptionally high activity against wild type HSV-2

strain, which is about 20-fold more potent than ACV (Bourinbaiar and Lee-Huang, 1996).

2.6.2.4 Effects on Hepatitis B virus

Furthermore, studies show that the RIP, MAP30, inhibits production of Hepatitis

B virus (HBV). The exposure of HepG cells to MAP30 results in inhibition of HBV DNA

replication and HBsAg secretion. More specifically, MAP30 is shown to inhibit the

expression of HBV antigen, decrease the viral DNA replication, down regulate replicative

intermediates, and reduces cDNA synthesis. High doses of MAP30 are also effective in

suppressing viral replication by altering the kinetics of replicative DNA intermediates;

whilst lower doses of MAP30 inhibit the expression of HBsAg and HBeAg. MAP30 thus

inhibits the production of HBV in a dose- and time-dependent manner (Fan et al., 2009).


32

2.6.2.5 Effects on Poliovirus

Poliovirus is a non-enveloped positive-strand RNA virus that infects animal cells

and causes a debilitating neuromuscular disease in humans. The virus does not bud from

the cell; instead they accumulate in the cytoplasm and are released when the cell bursts.

M. charantia inhibitor (MCI) inhibited multiplication of poliovirus I in HEp-2 cells.

Poliovirus was thermostable during 24 h incubation period either in the absence or in the

presence of the ribosome-inactivating protein. This rules out that the reduction in virus

yield in the presence of the inhibitory protein was due to the inactivation of infectivity of

progeny virions. It was demonstrated that the reduced multiplication of poliovirus was not

due to an inhibition of protein synthesis in infected cells (Foa-Tomasi et al., 1982).

2.6.2.6 Effects on Kapsoi sarcoma-associated herpes virus

MAP30, anti-HIV protein inhibited the proliferation of BC-2, an AIDS-related

primary effusion lymphoma (PEL) cell line derived from an AIDS patient. BC-2 cells are

latently infected with Kaposi sarcoma-associated herpes virus (KSHV), also known as

human herpes virus 8 (HHV8). MAP30 caused a dose-dependent inhibition of BC-2 cell

proliferation with an EC50 of 0.3 to 0.6 nM and was equally effective against lytic and

latent states of virus. The HHV8 genes vCD, vFLIP and vIL-6 are among the few viral

genes expressed in BC-2 tumour cells, which play key roles in oncogenesis and cellular

transformation. RT-PCR and northern blot analysis showed that MAP30 blocks the

expression of these viral genes. cDNA microarrays analyze the expression profile of 92

specific cellular genes involved in cell cycle regulation, apoptosis and cytokine signalling

in AIDS-related tumour cells. Out of these, twelve genes were expressed at high levels in

both latent and lytic phases of the viral life cycle: ATF-2, egr-1, hsp27, hsp90, IκBa, IL-2,

mdm2, caspase-6, caspase-10, NIK, TNFR2 and Skp1. Microarray analysis indicates that

MAP30 down-regulates significantly the expression of 11 of the 12 cellular genes

involved in Kaposi’s sarcoma (KS) pathogenesis, while it down regulates slightly the

expression of IL-2. MAP30 up-regulates specific genes related to apoptosis, including

Bax, caspase-3 and CRADD, interfering with the viral program to suppress apoptosis.

Thus, MAP30 was active against both latent and lytic phases of the viral life cycle, it

alters the expression of both viral and cellular genes involved in KS pathogenesis and

may have significant therapeutic potential in the treatment of AIDS-related tumours (Sun

et al., 2001).


33

2.6.2.7 Effects on Human T-cell leukemia virus I

PAP from Pokeweed americana exhibits antiviral activity against a number of

viruses and also inhibits the production of human T-cell leukemia virus I (HTLV-1).

HTLV-1 is a delta retrovirus that is a causative agent of adult T-cell leukemia and

neurological disorder. HTLV-1 has also been associated in myelopathy/tropical spastic

paraparesis. Currently, there is no effective anti-retroviral treatment available to restrict

the development of diseases associated with this virus. PAP depurinated nucleotides

within the Gag open reading frame suppress the synthesis of viral proteins by decreasing

the translational efficiency of HTLV-1 Gag/Pol mRNA. Viral mRNA reduction due to a

decrease in viral transactivator protein, Tax, leads to feedback inhibition of transcription

from the viral promoter. PAP diminishes virus production by suppressing HTLV-1 gene

expression at both translational and transcriptional levels (Mansouri et al., 2009).

2.7 Plasma Half Life of RIPs

In pharmacoproteomics and structural genomics, many newly identified bioactive

proteins are generally unstable in vivo. To overcome this problem, RIPs need conjugation

to some water-soluble polymers like polyethylene glycol (PEG). PEG is a non-toxic, non

immunogenic, non-antigenic, water soluble and FDA approved polymer. Covalent

coupling of PEG to proteins is an effective way to prolong plasma half life and reduce

immunogenicity. This polymer has been extensively used to modify proteins and

PEGylation conducted non-specifically through ε-NH4 site of lysine residue (Abuchowski

et al., 1977). Usually there are more than one residue (eg. lysine) in a protein and its

PEGylation produces different products with variation in structure and activity.

PEGylation of proteins usually results in masking some surface sites, increasing the

molecular size and enhancing stearic hindrance. Therefore, attachment of PEG to proteins

decreases immunogenicity, improves the plasma half life and stabilizes against

proteolytic cleavage (Bonora et al., 1997).

Trichosanthin (TCS) is the first RIP that possesses anti-HIV activity in vitro. TCS

has limited clinical applications because of its major side effects, short plasma half-life,

immunogenicity and neurotoxicity. TCS administration elicited production of specific

antibody in the body and its re-administration resulted severe anaphylactic reaction

leading to death. TCS plasma half-life range is 8.4-12.7 min in the body. This range

requires frequent administration to maintain an effective therapeutic concentration in


34

blood (Byers et al., 1994). PEGylation is shown to reduce immunogenicity and prolongs

circulating half-life of proteins. The specific antibodies neutralizes some TCS, speeds up

its plasma clearance, rapidly clears glomerular filtration which facilitates its lost in urine,

which corresponds to very short plasma half-life of TCS.

Further studies show that TCS has three antigenic sites; Ser (S7), Lys-173 (K173)

and Gln-219 (Q219) that are mutated to a cysteine residue, namely, S7C, K173C and

Q19C. The sulfhydryl group of newly created solitary cysteine residue of S7C, K173C

and Q219C are used for PEG20K attachment. PEG masks antigenic sites to prevent

specific antibodies binding and, therefore, reduces antigenicity, improves plasma half-

life; however in vitro RIP activity is not affected. The anti-HIV activity of PEGylated

TCS is retained and longer plasma half-life can usually compensate for the reduction in

activity (Wang et al., 2004b).

On further investigation of PEGylated TCS, two antigenic sites of TCS; YFF81-83 and KR173-174 mutated by site directed mutagenesis and then PEG-maleimide are coupled with newly created cysteine residue by site-directed PEGylation. The MRT (mean residence time) of the PEGylated TCS mutants increases by 4.5 to 6 fold and the C1P is reduced by about 2-fold as compared to nTCS (natural TCS). The increase in molecular size of the PEGylated-TCS conjugate leads to reduced renal clearance and resistance of PEGylated proteins to proteolysis and contributes to the prolonged plasma half-life (An et al., 2007).

RIPs from bitter melon seeds are subjected to PEG-modifications. Chemical modification of RIP with 20 kDa (mPEG)2-Lys-NHS was done to reduce immunogeneicity by increasing plasma half-life for in vivo application. The inhibitory activity of both non-PEGylated and PEGylated RIP against cancer cells, as measured by the caspase 3-assay (apoptotic pathway), is much stronger than against normal cells. The antigenicity of PEGylated RIP is reduced and plasma half-life in vivo increases (Li et al., 2009).

2.8 Structure-function relationship of type I RIPs

The first three dimensional structure of ricin was solved by X-ray crystallography and refined to 2.8 Ǻ resolution (Montfort et al., 1987). This structure is archetypical for other RIPs. The overall structure of ricin is glycosylated, globular, heterodimer, with the monomer units joined by a single disulfide bond (Figure 2.6A). The ricin A-chain is a 267


35

residue globular protein, structured from three domains, with approximately 50% of the polypeptide arranged into α-helix or β-sheet. Ricin A-chain has a pronounced active site at the interface of the three domains, which is able to recognize and accommodate the rRNA stem-loop which is its main target. The ricin B-chain comprising 262 amino acid residues, is a two domain structure. Each ricin B-chain domain has galactose binding site lying in a pocket formed in part by a kink in the polypeptide chain by the tripeptide Asp-Val-Arg.

α–MMC structure was solved by X-ray crystallography using multiple-isomorphus-replacement methods and refined at 1.6-2.2 Ǻ resolution (Huang et al., 1995). It consists of 250 amino acid residues and has three domains. Domain one and domain two are associated and form a large domain, which consist of residue 1-202 and a small domain, which was near the C-termini. These two domains form an apparent cleft at their interface on the surface of protein. α–MMC consists of eight α-helices and β-sheets. Helix α21 is a very short helix with only seven residues near the C-terminus. Hydrophobic interactions, ion-pairs and hydrogen bonds have been observed between main chain atoms or side chain atoms. These interactions stabilized whole folding and secondary structure of the protein. Active centers residues are from 69-71, 83-85, 108-114 and 155-163. The residues from 189 to 192 form the outer edge of the catalytic centres. These residues have been located near helices α12, α13 and α14 and near β14, β15 and β16, respectively. Conserved residues Tyr-70, Glu-160, Arg-163 and Trp-192 form active centres and the side chain of these two residues form the opening of the binding pocket. Some conserved residues in protein, which are not directly involved in forming the active centres, like Phe-4, Tyr-14, Phe-17, Arg-22, Leu-52 and Arg-122. These residues involved in the stabilization of the geometry of active centres (Figure 2.6B).

β-MMC structure is the first example among RIPs, which gives information about the three-dimensional structure and binding site of the oligosaccharide in the active chains of RIPs (Figure 2.6C). The crystal structure of β-MMC protein was determined using the molecular-replacement method and refined to 2.55 Ǻ resolution (Yuan et al., 1999). β-MMC has 249 amino acid residues and contains nine α-helices, two 310 helices and three β-sheets with a total of ten β-strands. The polypeptide chain folds into two domains, a large N-terminal domain (Asp1-Asn180) and a smaller C-terminal domain (Leu181-Asn 249). Active site residues Tyr-70, Tyr-109, Glu-158 and Arg-161 are expected to be associated with rRNA N-glycosidase activity. β-MMC molecule contained branched hexasaccharide, which are composed of two β-N-acetylglucosamines [GlcNAc(1) and GlcNAc(2)], one α-fucose (Fuc), one β-mannose [Man (1)], one α-mannose [Man (2)]


36

and one β-xylose (Xyl). The oligosaccharide is bound to Asn-51 through N-glycosidic bond [β-GlcNAc-(1-N)-Asn-51] and stretches from the surface of the N-terminal domain far from the active site, suggesting that it should not play a vital role in the enzymatic function. The oligosaccharide molecule interacts with β-MMC through hydrogen bonds, although in the crystals, most of these are intermolecular interactions with the protein atoms in the adjacent unit cell.

A MAP30 structure was solved by NMR (Wang et al., 1999). The N-terminal domain (residues 1-105) contains an extended mixed sheet of six β-strands. Strand β3 contains the N-linked glycosylation site, Asn51-Leu52-Thr53, in which one face of this sheet packs against α-helices in the highly helical central domain i.e residues 108-180 of the molecule. The C-terminal domain, residues from 181-263 contains structured region, in which the long bent helix, residues from 181-200 packs against an antiparallel two-stranded β-sheet (Figure 2.6D). The residues Tyr-70, Tyr-109, Glu-158 and Arg-161 are known to be responsible for RNA N-glycosidase activity and located in a deep pocket that specifically accommodates an extra helical adenine base. MAP30 RNA N-glycosidase pocket is located in the middle of a groove that runs along one side of the protein. The surface of the groove is negatively charged with residues Glu-110, Glu-121 and Glu-187 to the right and residues Asp-43, Asp-65, Glu-85 and Glu-89 to the left of the RNA N-glycosidase pocket. Based on structural homology of MAP30 similar groove was found in ricin A-chain and hypothesized that this groove was a binding site for DNA substrates (Wang et al., 2000).

The three-dimensional structures of bouganin (from Bougainvillea spectabilis) and lychnin (from Lychnis chalcedonica) have been solved and along with their activities were determined together with those of dianthin 30, PAP-R, ricin A chain RTA, saporin-S6 and momordin I. Relative to other RIPs, saporin-S6 has the highest protein synthesis inhibitory activity, and efficiently deadenylates polynucleotides from rat ribosomes, poly(A) and hsDNA. Lychnin showed little increase in protein synthesis inhibitory activity as compare with bouganin. Deadenylation of poly(A) was inefficient for both lychnin and bouganin. Structure analysis of saporin-S6 revealed the presence of several exposed arginine and lysine residues surrounding the active site-cleft. In lychnin, the electrostatic surface potential does not favour adenine removal. Bouganin showed some negative potential at the active site, but the negative charge of some surrounding residues prevented the enzyme-substrate interaction (Fermani et al., 2009).


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Figure 2.6 Ribbon diagram of ribosome-inactivating proteins: (A) Ribbon diagram of ricin backbone. The ricin A-chain is shown in yellow and the B-chain in blue. Galactose is shown in red. The cysteinyl residues involved in the interchain disulfide bond shown in green; (B) Ribbon representation of α-MMC [Protein Data Bank file 1aha]; (C) Ribbon diagram of β-MMC [Protein Data Bank file 1cf5I] and (D) Ribbon structure of MAP [Protein Data Bank file 1d8v]. These figures have been reproduced from www.pdb.org.

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N glycosidase that inhibit translation through their activity ...shodhganga.inflibnet.ac.in/bitstream/10603/10001/10/10...One class of proteins with selective toxicity, called ribosome-inactivating