Purification and characterization of α- and β-benincasins, arginine/glutamate-rich peptides with translation-inhibiting activity from wax gourd seeds

doi:10.1016/S0196-9781(02)00271-1

Peptides

Volume 24, Issue 1, January 2003, Pages 11-16

https://doi.org/10.1016/S0196-9781(02)00271-1 Get rights and content

Abstract

Two peptides, with a molecular mass of about 11 kDa and an N-terminal sequence abundant in arginine and glutamine residues, were isolated from wax gourd seeds. The isolation protocol included affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono-S and gel filtration on Superdex 75. The peptides, designated α- and β-benincasins, inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC₅₀ of 20 and 320 pM, respectively. α-Benincasin exhibited weak antifungal activity toward Coprinus comatus and Physalospora piricola but not toward Mycosphaerella arachidicola.

Introduction

The seeds of cucurbitaceous plants constitute a source of proteins including ribosome inactivating proteins (RIPs) [1], [4], [10], [17], [18], [19], ribonucleases [4], [15], lectins [18], α-amylase inhibitors [22] and trypsin inhibitors [6], [16]. There is evidence suggesting that these proteins play a defensive role in plants. Among these proteins, RIPs have formed a popular subject of research. RIPs manifest a range of activities encompassing antifungal, antiviral, antiproliferative, antitumor and immunomodulatory activities [1], [2], [10], [11], [12], [14], [17], [19]. There are different types of RIPs. Type 1 RIPs consist of a single chain with a molecular mass of about 30 kDa. Type 2 RIPs represent a combination of a Type 1 RIP and a lectin, yielding a molecular mass of about 60 kDa [1]. Small RIPs with a molecular mass of about 10 kDa are by comparison much less well known and have been isolated only from bitter gourd seeds [13] and sponge gourd seeds [5]. Structural information about small RIPs is meager. In addition to the aforementioned RIPs, arginine/glutamate-rich polypeptides (AGRPs) have been reported from sponge gourd seeds [7], [8]. Their relationship to small RIPs is unknown though they demonstrate translation-inhibiting activities [7], [8], [21].

Taking into account the paucity of literature pertaining to small RIPs and AGRPs, we undertook the present investigation to isolate small proteins from wax gourd seeds. The N-terminal sequences of these small proteins disclose that they are AGRPs but not fragments of Type 1 RIPs. In addition, these AGRPs manifest translation-inhibiting activity.

Section snippets

Materials

Seeds were purchased from a local shop. Affi-gel blue gel was purchased from Bio-Rad, DEAE-cellulose was from Sigma Chemical Co. (St. Louis, MO), and CM-Sepharose and Superdex 75 HR 10/30 column were from Amersham Pharmacia Biotech. Chemicals for sequence analysis were obtained from Hewlett-Packard (Palo Alto, CA, USA). All other chemicals used were of reagent grade.

Isolation of proteins

Fresh seeds collected from wax gourds (Benincasa hispida var. dong-gua) were obtained locally. A crude extract was obtained by

Purification

Affinity chromatography of a crude extract of wax gourd seeds (IC₅₀ of cell-free translation inhibition=74 ng/ml) on Affi-gel blue gel yielded a large unadsorbed peak (B1) and a sharp adsorbed peak (B2) (Fig. 1). Fraction B2 from Affi-gel blue gel with higher cell-free protein synthesis-inhibitory activity (IC₅₀=10 ng/ml) than fraction B1 (IC₅₀ of inhibition=750 ng/ml) was chosen for further purification. B2 was fractionated into four major peaks M1–M4 upon FPLC using an FPLC Mono-S column (Fig. 2

Discussion

The peptides isolated from wax gourd seeds, designated α- and β-benincasins, can be considered as AGRPs because of their rich content of arginine, glutamate and glutamine residues in their N-terminal sequences, just like AGRPs from the sponge gourd [7], [8]. The small RIP from the sponge gourd is also characterized by a high content of these residues as reflected in the amino acid composition [5]. Both α- and β-benincasins manifest potent translation-inhibiting activity, but α-benincasin shows

Acknowledgements

We thank the Research Grants Council of Hong Kong for award of an earmarked grant, the Research Committee (The Chinese University of Hong Kong) for award of a direct grant, and Miss Fion Yung for excellent secretarial assistance.

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Ribosome inactivating proteins – An unfathomed biomolecule for developing multi-stress tolerant transgenic plants
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Citation Excerpt :
Antifungal RIPs have been reported from several plant genera belonging to Amaranthaceae (Beta), Arecaceae (Elaeis), Cucurbitaceae (Benincasa, Cucurbita, Luffa, Momordica), Nyctaginaceae (Mirabilis), Phytolaccaceae (Phytolacca), Poaceae (Hordeum), and Solanaceae (Nicotiana) [50]. Of these plant families, Cucurbitaceae members have contributed various RIPs that exhibit significant antifungal properties; for example, α-Benincasin and Hispin from Benincasa hispida controlled Coprinus comatus, and Physalospora piricola, and Fusarium oxysporum, P. piricola and Mycosphaerella arachidicola, respectively (Table 2) [110,111]. Cucurbita moschata RIP inhibits Phytophthora infestans [112], Luffacylin from Luffa cylindrica kills F. oxysporum, M. arachidicola [108], MbRIP-1 from Momordica balsamina controls Aspergillus niger, and Sclerotinia sclerotiorum [113], and α-momorcharin from M. charantia remains effective against Bipolaris maydis, F. graminearum, A. oryzae, and A. niger [114].
Transgenic crops would serve as a tool to overcome the forthcoming crisis in food security and environmental safety posed by degrading land and changing global climate. Commercial transgenic crops developed so far focus on single stress; however, sustaining crop yield to ensure food security requires transgenics tolerant to multiple environmental stresses. Here we argue and demonstrate the untapped potential of ribosome inactivating proteins (RIPs), translation inhibitors, as potential transgenes in developing transgenics to combat multiple stresses in the environment. Plant RIPs target the fundamental processes of the cell with very high specificity to the infecting pests. While controlling pathogens, RIPs also cause ectopic expression of pathogenesis-related proteins and trigger systemic acquired resistance. On the other hand, during abiotic stress, RIPs show antioxidant activity and trigger both enzyme-dependent and enzyme-independent metabolic pathways, alleviating abiotic stress such as drought, salinity, temperature, etc. RIPs express in response to specific environmental signals; therefore, their expression obviates additional physiological load on the transgenic plants instead of the constitutive expression. Based on evidence from its biological significance, ecological roles, laboratory- and controlled-environment success of its transgenics, and ethical merits, we unravel the potential of RIPs in developing transgenic plants showing co-tolerance to multiple environmental stresses.
Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida
2013, Phytochemistry
Citation Excerpt :
Apart from exudates, cucumisin and serine proteases were purified from fruits (Kaneda and Tominaga, 1975; Kaneda et al., 1986; Uchikoba et al., 1993, 1998; Kaneda and Uchikoba, 1994). Moreover, chitinase (Shih et al., 2001) and antimicrobial peptides, benincasins, that have translation-inhibiting activity (Ng et al., 2003), were purified from seeds of wax gourd. Cucurbitacins are known to show cytotoxicity and some biological activities, besides having a bitter taste (Rehm et al., 1957; Fuller et al., 1994; Chen et al., 2005).
Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 μg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.
Differential abilities of the mushroom ribosome-inactivating proteins hypsin and velutin to perturb normal development of cultured mouse embryos
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The teratogenicity of two fungal ribosome-inactivating proteins, hypsin from Hypsizigus mamoreus and velutin from Flammulina velutipes, was examined in this investigation using microinjection and postimplantation whole-embryo culture. The results demonstrated that hypsin induced abnormal embryonic development at 2.5 μM during the organogenesis period from E8.5 to E9.5. As its dosage increased, there was an increase in the total number of abnormal embryos, a drop in the final somite number, and a rise of abnormal structures. Structural abnormalities were detected: open cranial neural tube, abnormal branchial arches, absence of forelimb buds and twisted body axis. The otic and optic placodes were, however, less affected. Histological study of the abnormal embryos revealed a correlation of increased cell death with abnormal structures, suggesting that induction of cell death by hypsin may account for its teratogenicity. In contrast, velutin did not exert any adverse influence on mouse development.
Fungal Ribosome Inactivating Proteins
2006, Handbook of Biologically Active Peptides
Ribosome inactivating proteins (RIPs) have been isolated from a number of fungi including Aspergillus and Trichoderma spp. and several mushrooms. Ribosome inactivating peptides found in Cucurbitaceous seeds have not been detected in fungi. The smallest fungal RIP isolated to date, velutin—from the mushroom (basidiomycete fungus) Flammulina velutipes—has a molecular mass of about 14 kDa. Small RIPs with a molecular mass of approximately 20 kDa, and RIPs about 30 kDa in molecular mass, are found in both fungal and plant RIPs. The Aspergillus RIPs are ribonucleases with high-sequence homology. In contrast, most mushroom RIPs do not show structural resemblance to each other and have little or no ribonuclease activity. All fungal RIPs demonstrate potent inhibitory activity in the cell-free translation system. Other activities of fungal RIPs include mitogenic/antimitogenic activity toward splenocytes and antiproliferative activity toward tumor cells. RIPs probably serve a role of defense in fungi.
Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase
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An antifungal peptide with a molecular mass around 7 kDa and an N-terminal sequence highly homologous to defensin was isolated from ground beans (Vigna sesquipedalis cv. ‘Ground Bean’). The peptide was adsorbed on Affi-gel blue gel and on Mono S. It exerted an antifungal action on Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola; and an antibacterial action on Escherichia coli B, Proteus vulgaris, Mycobacterium phlei and Bacillus megaterium. The antimicrobial activity was inhibited in presence of the 5 mM CaCl₂ and MgCl₂, but no inhibition was observed in 5 mM NaCl. The peptide exerted antiproliferative activity toward breast cancer (MCF-7) cells and leukemia M1 cells, this activity could not be inhibited by the ions mentioned above. It also exhibited some inhibitory activity toward human immunodeficiency virus-type 1 reverse transcriptase.
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Novel effects of naturally occurring peptides are continuing to be discovered, and their mechanisms of actions as well as interactions with other substances, organs, and systems have been elucidated. Synthetic analogs may have actions similar or antagonistic to the endogenous peptides, and both the native peptides and analogs have potential as drugs or drug targets. The journal Peptides publishes many leading articles on the structure–activity relationship of peptides as well as outstanding reviews on some families of peptides. Complementary to the reviews, here we extract information from the original papers published during the past five years in Peptides (1999–2003) to summarize the effects of different classes of peptides, their modulation by other chemicals and various pathophysiological states, and the mechanisms by which the effects are exerted. Special attention is given to peptides related to feeding, pain, and other behaviors. By presenting in condensed form the effects of peptides which are essential for systems biology, we hope that this summary of existing knowledge will encourage additional novel research to be presented in Peptides.

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Peptides

Abstract

Introduction

Section snippets

Materials

Isolation of proteins

Purification

Discussion

Acknowledgements

References (22)

Ribosome-inactivating proteins from plants

Biochem. Biophys. Acta

A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of Chinese medicinal herbs

Life Sci.

RNA N-glycosidase activity of ricin A chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes

J. Biol. Chem.

Luffin S—a small novel ribosome inactivating protein from Luffa cylindrica

FEBS Lett.

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi

Int. J. Biochem. Cell Biol.

Purification and characterization of novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum

Biochem. Biophys. Res. Commun.

Biochemical and molecular characterization of three barley seed proteins with antifungal properties

J. Biol. Chem.

Proteins with abortifacient, ribosome inactivating, immunomodulatory, anti-tumor and anti-AIDS activities from Cucurbitaceae plants

Gen. Pharmacol.

Characterization of the enzymatic machanism of γ-momorcharin, a novel ribosome-inactivating protein with lower molecular weight of 11,500 purified from seeds of the bitter gourd (Momordica charantia)

Biochem. Biophys. Res. Commun.

Isolation and partial characterization of two antifungal proteins from barley

Biochim. Biophys. Acta

Ribosome inactivating protein and lectin from bitter melon (Momordica charantia) seeds: sequence comparison with related proteins

Biochem. Biophys. Res. Commun.

Cited by (22)

Ribosome inactivating proteins – An unfathomed biomolecule for developing multi-stress tolerant transgenic plants

Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida

Differential abilities of the mushroom ribosome-inactivating proteins hypsin and velutin to perturb normal development of cultured mouse embryos

Fungal Ribosome Inactivating Proteins

Sesquin, a potent defensin-like antimicrobial peptide from ground beans with inhibitory activities toward tumor cells and HIV-1 reverse transcriptase

Effects of peptides, with emphasis on feeding, pain, and behavior A 5-year (1999-2003) review of publications in Peptides