Research on ribosome-inactivating proteins from angiospermae to gymnospermae and cryptogamia.

Ribosome-inactivating Proteins (RIPs) are a group of cytotoxin proteins that usually contain a RNA N-glycosidase domain, which irreversibly inactivates ribosome, thus inhibiting protein synthesis. During the past 14 years (1990-2004), the studies conducted in our laboratory had been focusing on the structure and enzymatic mechanism of several PIPs. Herein, we briefly described a summary of the studies conducted mainly in our laboratory on RIPs from angiospermae to gymnospermae and cryptogamia as follows. (1) Cinnamomin is a novel type II RIP isolated from mature seeds of camphor tree. Like ricin, it specifically removes the adenine at A4324 in rat liver 28S rRNA. We systematically studied this low-toxic RIP in term of its enzymatic mechanism, the primary and crystal structure and the nucleotide sequence of its gene, the genetic expression, and its physiological role in the seed cell and the toxicity to human cancer cells and insect larvae. The cleavage of supercoiled double-stranded DNA was its intrinsic property of cinnamomin A-chain, its N- and C-terminal regions were found to be required for deadenylation of rRNA and also necessary for deadenylation of supercoiled double-stranded circular DNA. These results strongly excluded the possibility that cleavage of supercoiled DNA was due to nuclease contamination. (2) Trichosanthin, an abortifacient protein, was purified from the Chinese medicinal herb, Tian-hua-fen, obtained from root tubers of Chinese trichosanthes plant. We proved that trichosanthin was a RNA N-glycosidase, inactivating eukaryotic ribosome by hydrolyzing the N-C glycosidic bond of the adenose at site 4324 in rat 28S rRNA, and inhibited protein synthesis in vitro. (3) A unique Biota orientalis RNase (RNase Bo) was extracted from the mature seeds of the cypress cypress tree (Oriental arborvita), which was gymnospermae plant. It cleaved only a specific phosphodiester bond between C4453 and A4454 of 28S RNA in rat ribosomes, producing a small RNA-fragment (S-fragment), thus inhibiting protein synthesis and belonging to RNase-like RIP, similar to α-sarcin, a special RIP. (4) Lamjapin, the first RIP purified from kelp, the marine cryptogamia algal plant, was shown to be the first single-chained RNA N-glycosidase from marine plant to date. It hydrolyzed rat ribosomal 28S RNA to produce meanly a rather smaller RNA, shorter than the diagnostic R-fragment under the restricted condition. The significance of existence of type I RIP in the lower marine algal plant was briefly discussed.

The three-dimensional structure and X-ray sequence reveal that trichomaglin is a novel S-like ribonuclease.

Trichomaglin is a protein isolated from root tuber of the plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The crystal structure of trichomaglin has been determined by multiple-isomorphous replacement and refined at 2.2 A resolution. The X-ray sequence was established, based on electron density combined with the experimentally determined N-terminal sequence, and the sequence information derived from mass spectroscopic analysis. X-ray sequence-based homolog search and the three-dimensional structure reveal that trichomaglin is a novel S-like RNase, which was confirmed by biological assay. Trichomaglin molecule contains an additional beta sheet in the HV(b) region, compared with the known plant RNase structures. Fourteen cystein residues form seven disulfide bridges, more than those in the other known structures of S- and S-like RNases. His43 and His105 are expected to be the catalytic acid and base, respectively. Four hydrosulfate ions are bound in the active site pocket, three of them mimicking the substrate binding sites.

Both N- and C-terminal regions are essential for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA.

Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. In this report, deadenylation of both rRNA and supercoiled DNA by native and recombinant cinnamomin A-chain expressed in Escherichia coli was demonstrated. However, the mutants of cinnamomin A-chain devoid of N-terminal 52 or/and C-terminal 51 amino acid residues lost both the activity of RNA N-glycosidase and the ability to release adenines from supercoiled DNA. Additionally, supercoiled DNA could not be cleaved into nicked and linear forms by these mutants. These results indicate that both N- and C-terminal regions are essential for the cinnamomin A-chain to deadenylate rRNA and supercoiled DNA. It was suggested that phosphodiester bonds in the extensively deadenylated region of supercoiled DNA would become fragile and liable to be broken spontaneously owing to the existence of tension in the supercoiled DNA.

Fluorescence labeling of short RNA by oxidation at the 3'-end.

In RNA nanotechnology, construction of nanoparticles involves conjugation of functionalities, cross-linking of modules, labeling of RNA subunits, and chemical modification of nucleotides. Efficiency and sensitivity are important for the RNA labeling, which also can be used as probes in microarrays, Northern blotting, and gel-shift assays. Here, we describe a method for fluorescence labeling of short RNA at the 3'-end by oxidation. The 3'-terminus of in vitro-transcribed short RNA is oxidized by sodium periodate, and fluorescein-5-thiosemicarbazide is added after removal of excess oxidant. Purified short RNA with fluorescence is then applied for detection of RNA-protein interaction by gel-shift assay.

Cinnamomin: a multifunctional type II ribosome-inactivating protein.

Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes by specifically removing the conserved adenine base from the "Sarcin/Ricin domain" of the 28S RNA in ribosome. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of camphor tree. Besides site-specific deadenylation of the A4324 in the Sarcin/Ricin domain of rat ribosome, this protein could also release the adenine base from DNA molecules at multiple sites and from AMP, ADP, dAMP and adenosine. Furthermore, cinnamomin displays cytotoxicity to carcinoma cells and insect larvae by modifying their ribosomal RNA. These functions possessed by cinnamomin shed a new light on the possible application of cinnamomin in the field of immunotoxin design and transgenic reagents. In this review, we introduce the major recent results on cinnamomin obtained in our laboratory, including purification of this protein, characterization of its enzymatic mechanism, structure and function, gene pattern, physiological role and its biological implications in cytotoxicity.

Simultaneous existence of cinnamomin (a type II RIP) and small amount of its free A- and B-chain in mature seeds of camphor tree.

Cinnamomin, a type II ribosome-inactivating protein (RIP), was isolated from the mature seeds of camphor tree (Cinnamomum camphora). In this paper, small amount of free A- and B-chain of cinnamomin were found to be present in the mature seed cell of C. camphora besides the intact cinnamomin. Our results demonstrated that camphorin, a type I RIP previously reported to coexist with cinnamomin in the seeds of C. camphora, actually was the A-chain of cinnamomin. The percentage of free A- and B-chain in the total cinnamomin was 2.6-2.8% in the seed extract. Of these free A- and B-chain approximate 80% already existed in the seed cell, only about 20% were produced during the purification operation. As the enzymatic activity to reduce disulfide bond of cinnamomin in the seed extract of C. camphora was detected, we proposed that the free A- and B-chain were derived from the enzymatic reduction of the interchain disulfide bond of cinnamomin. It was demonstrated that the endogenous type II RIPs of several plant species, such as Cinnamomum porrectum, Cinnamomum bodinieri and Ricinus communis, could be enzymatically reduced into the free A- and B-chain in their respective seed cells. The function of the free A-chain in the seed cell and the possibility that metabolic enzymes might be involved in the reduction of the interchain disulfide bond of type II RIPs in vivo are discussed.

In vitro interaction of eukaryotic elongation factor 2 with synthetic oligoribonucleotide that mimics GTPase domain of rat 28S ribosomal RNA.

Eukaryotic elongation factor 2 (eEF2) catalyzed the translocation of peptidyl-tRNA from the ribosomal A site to the P site. In this paper, the interaction between eEF2 and GTD RNA, a synthetic oligoribonucleotide that mimicked the GTPase domain of rat 28S ribosomal RNA, was studied in vitro. The purified eEF2 could bind to GTD RNA, forming a stable complex. Transfer RNA competed with GTD RNA in binding to eEF2, whereas poly(A), poly(U) and poly(I, C) did not interfere with the interaction between eEF2 and GTD RNA, demonstrating that the tertiary structure of RNA might be necessary for the recognition of and binding to eEF2. The complex formation of eEF2 with GTD RNA was inhibited by SRD RNA, a synthetic oligoribonucleotide mimic of Sarcin/Ricin domain RNA of rat 28S RNA. Similarly, GTD RNA inhibited the interaction between eEF2 and SRD RNA. This fact implies that these small oligoribonucleotides probably share similar recognition or binding identity elements in their tertiary structures. In addition, the binding of eEF2 to GTD RNA could be obviously weakened by the ADP-ribosylation of eEF2 with diphtheria toxin. These results indicate that eEF2 behaves differently from prokaryotic EF-G in binding to ribosomal RNA.

Bioactivities of ricin retained and its immunoreactivity to anti-ricin polyclonal antibodies alleviated through pegylation.

Ricin has long been employed to construct immunotoxins, whose efficacy was often undermined by immunogenicity. Pegylation (modification of proteins with polyethylene glycol, PEG) was one of those recently developed approaches to circumvent immunogenicity of legions of drugs. Herein, pegylation of ricin was found to have barely changed the RNA N-glycosidase activity and protein synthesis inhibiting activity of ricin, but remarkably altered the cytotoxicity of ricin on hepatoma cell line (BEL7404) or the immunoreactivity with polyclonal anti-ricin antibodies. This result suggested that the attached PEG or monomethyloxyl polyethylene glycol (mPEG) groups did not hinder ricin from hydrolyzing ribosomal RNA, but indeed covered some areas on the surface of ricin molecule, including those involved in the interaction with cellular receptors and epitopes recognized by polyclonal antibodies. Pegylation, masking certain epitopes of ricin, might contribute to alleviate the immunogenicity of the toxin. Approach in this work, if applied to thereby constructed immunotoxins, would help improve the prospective efficacy of these toxins.

Studies of three genes encoding Cinnamomin (a type II RIP) isolated from the seeds of camphor tree and their expression patterns.

Cinnamomin, which has three isoforms, is a type II ribosome-inactivating protein (RIP) purified from the mature seeds of camphor tree (Cinnamomum camphora). In a previous study, an incomplete cDNA that encoded the A- and B-chain of Cinnamomin but lacked signal peptide sequence was cloned. In the present paper, its full-length cDNA was obtained by 5' rapid amplification of cDNA ends (5'RACE). Subsequently, polymerase chain reaction (PCR) amplification of its genomic DNA was performed. Unexpectedly, sequence analysis of the PCR products revealed three cinnamomin genes with >98.0% sequence identity. One of them corresponded to the published cDNA and was designated as cinnamomin I, whereas the other two genes were named as cinnamomin II and cinnamomin III, respectively. RT-PCR amplification of the cDNAs of cinnamomin II and III manifested that these two genes were functional. The three genes have no intron. Three Cinnamomin precursors that were inferred from the cDNA sequence of three cinnamomin genes exhibited relatively high sequence homology with other type II RIPs. Northern blot analysis demonstrated that the cinnamomin genes only expressed in cotyledons of C. camphora seeds and the acmes of expression emerged at 75-90 DAF when seeds were close to maturity. It is proposed that the three cinnamomin genes may encode three isoforms of Cinnamomin. The physiological function of Cinnamomin in C. camphora seeds is briefly discussed.

Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on different nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs differ in their depurinating activity on the different substrates with differences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

The role of tyrosine residues in the RNA N-glycosidase activity of cinnamomin A-chain.

Cinnamomin is a type II ribosome-inactivating protein (RIP) and its A-chain (CTA) is a RNA N-glycosidase. It is observed that modification of tyrosine residues by N-acetylimidazole (N-AI) causes almost complete loss of CTA activity. Adenine partially protects tyrosine residues from modification by N-AI. It is proposed that tyrosine residues are involved in the active site of CTA and they are crucial in recognition and binding of ribosomal RNA. Tryptophan residues of CTA are also studied by NBS modification.

Comparative study of interaction of two type II ribosome-inactivating proteins and their A-chains with model membrane.

Both cinnamomin and ricin are type II ribosome-inactivating proteins. Cinnamomin is less cytotoxic compared with ricin. In order to clarify the mechanism of their different cytotoxicities, the interaction of cinnamomin and its A-chain with model membrane was investigated and compared with that of ricin and its A-chain. It was revealed that cinnamomin is less effective than ricin in interacting with model membrane. Cinnamomin A-chain interacts with model membrane much less violently than ricin A-chain. The differences in the interaction of cinnamomin, ricin or their A-chains with model membrane might at least in part indicate the different cytotoxicity between cinnamomin and ricin.

Low content of protein S29 in ribosomes of human lung cancer cell line a549: detected by two-dimensional electrophoresis.

This study revealed that the content of protein S29 in ribosomes of cancer cell line A549 was distinctly low (equivalent to about 30% of that of 2BS). The conclusion was acquired based on the ratios of spot volume of ribosomal protein S29 to that of several other ribosomal proteins (S29/L37a, S29/L38, S29/S27 and S29/S28) in the same gel plate. The possible biological roles of ribosomal protein S29 in malignant transformation and translation regulation are briefly discussed.

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain.

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

Cinnamomin--a versatile type II ribosome-inactivating protein.

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

Refolding of partially thermo-unfolded cinnamomin A-chain mediated by B-chain.

The pure cinnamomin A-chain is unstable compared to that in the mixture of A- and B-chain or in intact cinnamomin molecule either being stored at 4 degrees C or being heated. When being heated at 45 degrees C for 20min, the A-chain generates partially unfolded intermediate and loses its tertiary structure as monitored by circular dichroism (CD) and tryptophan fluorescence, thus resulting in the inactivity of its RNA N-glycosidase albeit it retains most of its secondary structures. This partially unfolded intermediate is sensitive to protease, exhibiting property of a molten globule. The changes in conformation and activity are irreversible upon cooling. The partially unfolded intermediate can fully restore its RNA N-glycosidase activity in the presence of cinnamomin B-chain. The phenomenon, that the cinnamomin B-chain mediates the refolding of partially unfolded A-chain, probably plays an important role in the intracellular transport of the cytotoxic protein, i.e., keeping the structural stability of A-chain and refolding partially unfolded A-chain that occasionally appeared in the process of intracellular transport, to avoid the destiny of proteolysis that occurs in most denatured proteins in cell.

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora.

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

A novel ribotoxin with ribonuclease activity that specifically cleaves a single phosphodiester bond in rat 28S ribosomal RNA and inactivates ribosome.

A unique ribonuclease named Biota orientalis ribonuclease (Biota orientalis RNase) is purified to homogeneity from mature seeds of oriental arborvitae (Biota orientalis). The molecular mass of Biota orientalis RNase is about 13 kDa. When the concentration of Mg(2+) is 25 mM in the incubation buffer, the ribonuclease specifically cleaves the phosphodiester bond between C4453 and A4454 in region K (a region in domain VII) of 28S RNA in rat ribosome, resulting in inactivation of ribosome. Thus, it is a ribotoxin similar to alpha-sarcin. The region around C4453-A4454 in rat 28S rRNA is named "Biota orientalis RNase region." Rat ribosome treated by Biota orientalis RNase produces a small RNA fragment (S-fragment) that contains 333 nucleotides from the 3'-terminus of rat 28S rRNA. The distance between the cleavage-sites of alpha-sarcin (G4325) and Biota orientalis RNase (C4453) is 128 nucleotides. Under restricted conditions (25 mM Mg(2+)), the substrate specificity of Biota orientalis RNase is extremely high: it acts only on the "Biota orientalis RNase region" of the largest RNA in ribosomes from certain eukaryotes. The ribosome specifically damaged by Biota orientalis RNase is unable to EF-1alpha-dependently bind aminoacyl-tRNA, whereas the formation of the EF-2/GDP/ribosome complex is not affected. It is proposed that Biota orientalis RNase inactivates ribosome at least partially by interfering with the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosome. Biota orientalis RNase might be a useful tool in studying the structure/function of ribosome.

Chromium (III) enhanced diamine silver staining of proteins and DNA in gels.

Chromium (III) enhanced the sensitivities of diamine silver staining of four proteins between 6- and 50-fold over that of the Coomassie Brilliant Blue (CBB)-chromium modified thiosulfate-silver staining method (Zhou et al. Biotechnology Letters, 2002, 24: 1561-1567). Using six dsDNA fragments, the detection limits of this new method was 10 to 30 pg per band, being 10- to 25-fold more sensitive than previous methods.