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Cinnamomin: a multifunctional type II ribosome-inactivating protein
The International Journal of Biochemistry & Cell Biology 35 (2003) 1021–1027
Molecules in focus
Cinnamomin: a multifunctional type II ribosome-inactivating protein Wen-Jun He, Wang-Yi Liu∗ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
Abstract 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. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cinnamomin; Deadenylation; Ribosome-inactivating protein (RIP); RNA N-glycosidase
1. Introduction Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes and thus inhibit protein synthesis (Barbieri, Battelli, & Stirpe, 1993). Most RIPs are widely distributed in the higher plants and contain an RNA N-glycosidase domain that can specifically remove an adenine from the highly conserved loop (Sarcin/Ricin domain) of the largest RNA in eukaryotic ribosomes (Endo, Mitsui, Motizuki, & Tsurugi, 1987; Zhang & Liu, 1992). Another enzymatic activity of RIP is a unique ribonuclease possessed only by ␣-sarcin hith∗ Corresponding author. Tel.: +86-21-64374430; fax: +86-21-64338357. E-mail address: [email protected] (W.-Y. Liu).
erto investigated. Alpha-sarcin is capable of selectively hydrolyzing a single phosphodiester bond between G4325 and A4326 of 28S RNA in rat ribosome as shown in Fig. 1 (Endo & Wool, 1982). This Sarcin/ Ricin domain is responsible for the interaction of elongation factors with ribosomes (Xu & Liu, 2000). On the basis of structural diversity, plant RIPs have been classified into three types as indicated in Fig. 2 (Van Damme et al., 2001). Type I RIP is composed of a single, intact polypeptide of around 11–30 kDa that, in some cases, is processed proteolytically into two shorter polypeptides held together by non-covalent interaction. While type II RIP consists of two polypeptides (A- and B-chain) linked by a disulfide bond. Both type I RIP and the A-chain of type II RIP consist exclusively of a single RNA N-glycosidase domain. The B-chain of type II RIP is a galactose-specific lectin
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Fig. 1. The action sites of RIPs on 28S RNA in rat ribosome.
Fig. 2. Comparison of the primary structures of three types of RIPs.
that is responsible for the recognition of d-galactose of galactose-terminated receptors on the cell surface, and facilitates the internalization of the toxic A-chain. Additionally, a type III RIP is identified and this protein is composed of a type I RIP-like N-terminal domain covalently linked to a C-terminal domain with unknown function (Reinbothe et al., 1994). RIPs have received wide attention from researchers in both theoretical and applied fields owing to their increasing application in probing the structure of the ribosome, potential drugs in the clinic trials and transgenic reagents in the agriculture. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of the camphor tree (Cinnamomum camphora) (Ling, Liu, & Wang, 1995a). Its purification, enzymatic mechanism, structure, gene pattern, physiological function and possible applications have been intensively investigated. In this review, we have summarized our major results related to cinnamomin in recent years.
2. Purification and characterization of cinnamomin The camphor tree belongs to the Lauraceae family. It is resistant to viral infection and insect disease. Cinnamomin, a new type II RIP, was purified from the mature seeds of one species of camphor tree (C. camphora) (Ling et al., 1995a). It has been investigated in detail with respect to the biochemical and biophysical properties of cinnamomin. This 61-kDa protein has three isoforms. The A- and B-chains derived from cinnamomin isoforms have similar mobility on the SDS-polyacrylamide gel. The significant phylogenetic finding presents the co-existence of the three isoforms of a type II RIP in the same organ of a higher plant. Cinnamomin A-chain was demonstrated to be a specific RNA N-glycosidase. Among the four rat ribosomal RNAs, only the 28S RNA was modified by cinnamomin A-chain. The cleavage site was
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N–C-glycosidic bond of the A4324 embedded in the highly conserved “Sarcin/Ricin domain” in rat ribosome (Lin & Liu, 1996; Ling et al., 1995a). Interestingly, the intact cinnamomin also exhibits the RNA N-glycosidase activity. Meanwhile, an improved method for large-scale preparation has been developed taking advantage of the lectin properties of its B-chain (Li, Chen, Liu, & Wang, 1997). Instead of gel filtration, cinnamomin could be purified to high yield by a single step of acid-treated Sepharose 4B affinity chromatography followed by elution with lactose. Thus, 620 mg of cinnamomin was obtained from 500 g of wet seeds, instead of only 10.6 mg by the previous method. In addition, a simplified method for purification of the A- and B-chains of cinnamomin on a large scale was also developed (Pu, Xie, Wang, & Liu, 1998). Urea was introduced to weaken the non-covalent interaction that holds the A- and B-chains together very tightly. In the presence of 4 M urea, the Aand B-chains of the reduced cinnamomin were separated effectively by DEAE-cellulose chromatography. The purified A-chain still displayed the RNA N-glycosidase activity and the B-chain lost its lectin activity. After refolding in vitro in the presence of lactose, the active B-chain with lectin activity could be further purified by acid-treated Sepharose 4B affinity chromatography. From 80 mg of cinnamomin, 10 mg of A-chain and 38 mg of B-chain were obtained. However, it is noteworthy that the purified A-chain is unstable in solution due to its liability to precipitate and thus loses its RNA N-glycosidase activity in the absence of the B-chain.
3. Structural and functional studies of cinnamomin The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the cDNAs of its Aand B-chains. It has 549 amino-acid residues: 271 residues in the A-chain, 14 residues in the linker and 264 residues in the B-chain (Xie et al., 2001). The functional roles of five conserved amino-acid residues (Y75, Y115, E167, R170 and W201) in the predicted active site of cinnamomin A-chain were investigated by site-directed mutagenesis (Xie et al.,
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2001). Single mutations of these amino-acid residues led to 8–50 folds decrease of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of cinnamomin A-chain. Also, the strong electric charge introduced at the single Cys251 in A-chain was involved in the substrate binding. And the results of deletion showed that both the N-terminal region (52 amino-acid residues) and the C-terminal region (51 amino-acid residues) of cinnamomin A-chain were vital for its enzymatic activity. Cinnamomin is also a glycoprotein. Its A- and B-chains contain 0.3 and 3.9% sugar, respectively (Ling et al., 1995a). After the sugar chains of cinnamomin were oxidized with periodate and then fluorescence-labeled with FTSC, the RNA N-glycosidase activity of its A-chain and the lectin activiy of its B-chain decreased three folds (Xie, Hou, Liu, & Wang, 2000). However, the physiological function of the sugar chains is still unclear. Three major glycopeptides have been purified from cinnamomin B-chain by gel filtration chromatography, anion-exchange chromatography and HPLC. Their primary structures have been determined by two-dimensional NMR (Fig. 3) (Pu et al., 2000).
4. Location of cysteine residues and proposed position of disulfide bridges in cinnamomin A total of 10 cysteine residues were titrated with DTNB in the cinnamomin under denatured condition, one in the A-chain and the other nine in the B-chain (Fig. 3) (Xie et al., 2000). This result was confirmed by the primary structure deduced from its cDNA (Xie et al., 2001). In cinnamomin, all the cysteine residues formed the disulfide bonds, which accounted for its highly structural stability. Based on the simulated three-dimensional structure of cinnamomin A-chain, the N-terminal region of the A-chain had a ␣-helix structure of around 50 amino-acid residues. This ␣-helix was juxtaposed closely to the central region where a cluster of ␣-helixes formed the globular module. And the central region had key amino-acid residues and the active site cleft. The C-terminal region of cinnamomin A-chain consisted of a random coil about 50 amino-acid residues. A Cys251 in the random coil forms the interchain disulfide bond
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Fig. 3. Schematic structure of cinnamomin. The precursor polypeptide chain is cleaved proteolytically to remove the signal peptide and the 14 amino-acid linker. The structure of glycan chains of the B-chain and the positions of disulfide bridges as well as the conserved amino-acid residues in the active site of the A-chain are indicated. (䊊) Mannose; (䉫) GlcNAc; () Xylulose; (䉲) Tyr 75 and Tyr 115, respectively; (䊉) Glu167; (䉱) Arg170; (䉬) Trp201.
with the Cys4 of B-chain (Xie et al., 2001). Meanwhile, the B-chain was also stabilized by four intrachain disulfide bridges: Cys20–Cys39, Cys63–Cys80, Cys151–Cya164 and Cys194–Cys209 (Xie et al., 2001; Liu et al., 1998).
5. Three genes coding for cinnamomin and their expression patterns (Yang, Liu, Gong, & Liu, 2002) After the full-length cDNA of cinnamomin was obtained by 5 rapid amplification of cDNA ends (5 RACE), polymerase chain reaction (PCR) amplification of its genomic DNA was performed. Unexpectedly, sequence analysis of the PCR products reveals three cinnamomin genes with >98.0% identity. One of them was the cDNA of cinnamomin mentioned above and designated as cinnamomin I, whereas the other two genes were named as cinnamomin II and III, respectively. RT-PCR amplification of the cDNAs of cinnamomin II and III showed that these two genes were also functional. The three genes have no introns. Three cinnamomin precursors that were inferred from the cDNA sequence of three cinnamomin genes exhibited relatively high sequence homology with other type II RIPs. It suggested that the three genes for cinnamomin might encode the three isoforms (of cinnamomin) already characterized.
6. Physiological function of cinnamomin Previous studies revealed that many RIPs are involved in defence mechanisms in plant cells and terminate protein synthesis under appropriate physiological conditions and thus are involved in metabolic regulation. However, cinnamomin could not inactivate its own (autologous) ribosome in vitro (Zhang, Tang, & Liu, 2001). Northern and Western blotting revealed that cinnamomin was expressed exclusively in cotyledons of C. camphora seeds and the acmes of expression emerged at 75–90 days after flower (DAF) when seeds were close to maturity (Yang et al., 2002; Liu, Wei, Yang, He, & Liu, 2002; Lin & Liu, 1995). It accumulated in large amounts (accounting for about 11% of total seed proteins) simultaneously with other proteins at the post-stages of seed development. Cinnamomin was degraded rapidly by the endopeptidase during the early stages of seed germination. Moreover, the amino-acid composition of cinnamomin was very close to that of other storage proteins. All of these properties suggest that cinnamomin functioned as a storage protein providing sources of carbon, nitrogen and sulphur during seedling growth and development (Liu et al., 2002). Besides the intact cinnamomin, small amount of its free A- and B-chains were identified to be present in the mature seed cells. It was proposed that the free A- and B-chains were derived from the enzymatic
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reduction of the interchain disulfide bond of cinnamomin. In the seed extract, the percentage of free A- and B-chains to the intact cinnamomin was 2.6–2.8 and approximately 80% of these free A- and B-chains were produced by enzymatic reduction in the seed cells. Also, a small protein of about 46 kDa in the mature seeds was eluted together with cinnamomin by lactose from the acid-treated Sepharose 4B column. Like cinnamomin, this small protein consists of two chain connected by the disulfide bond since it could be reduced with 2-mercaptoethanol to produce a miniature A-chain and a normal B-chain, the latter is completely identical to the B-chain of cinnamomin. The miniature A-chain exhibited the RNA N-glycosidase activity and strong inhibitory activity to protein synthesis in vitro almost the same as the normal A-chain. Furthermore, the N-terminal 10 amino acids of this miniature A-chain are entirely identical sequence with that of cinnamomin A-chain. The structural homology of these two similar type II RIPs were further confirmed by Western blotting using the antibody of cinnamomin. However, RT-PCR revealed that there was no corresponding mRNA of this small protein in vivo. Northern blotting also showed that the mRNA of cinnamomin exists as only one normal form in the cotyledons of seeds of C. camphora. Based on these facts, it is proposed that the miniature A-chain is composed of all essential amino-acid residues related to the RNA N-glycosidase activity of cinnamomin A-chain and probably it is a splicing product of cinnamomin A-chain. If it is the case, cinnamomin will be interesting as a good model to study the splicing process of proteins.
7. Cytotoxicity of cinnamomin to tumor cells and insect larvae Inhibition of protein synthesis in the target cells was considered as the main function of RIPs although RIPs did exhibit various effects, such as inhibiting infection and replication of viruses, causing lesions in cancer cells and affecting the immune response. Cinnamomin also inhibited the growth of cultured carcinoma cells (Lin & Liu, 1996). The IC50 of cinnamomin to human hepatocarcinoma cell-line 7721 and the melanoma M21 were 18.8 and 11.7 nmol,
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respectively. And cinnamomin remarkably retarded the growth of solid melanoma in the skin of the nude mouse. Furthermore, the toxicity of cinnamomin to bollworm (Helicoverpa armigera), mosquito (Culex pipines pallens) and silkworm (Bombyx mori) during larval stage was tested (Zhou, Li, Yuan, Tang, & Liu, 2000). The IC50 of cinnamomin to bollworm larvae fed on diet containing cinnamomin was 1839 ppm and the IC50 to larvae of mosquito was 168 ppm. The inhibition of protein synthesis by cinnamomin was tested in in vitro translation system of bollworm larvae and its IC50 was determined to be approximately 14 nM. An R-fragment could be isolated from ribosomes of cinnamomin-treated carcinoma cells and cinnamomin-treated bollworm larvae ribosomes after incubation with acidic aniline, indicating that the cytotoxicity of cinnamomin to carcinoma cells or bollworm larvae was caused by the modification of the ribosome (Lin & Liu, 1996; Zhou et al., 2000). In addition, it is found that cinnamomin could form a cation-specific channel in a bilayer membrane and its B-chain may be responsible for the action (Zhang, Shi, Wang, & Liu, 1999). Moreover, lowering the pH from 7.5 to 5.0 evoked conformational changes of cinnamomin and unmasked its hydrophobic areas, facilitating the penetration of cinnamomin into the lipid bilayer (Hu, Tang, & Liu, 2000). These findings further elucidate the mechanism for the entry of cinnamomin into integral cells to act on the cytosolic target.
8. Other enzymatic activities of cinnamomin Besides the site-specific RNA N-glycosidase activity, adenine nucleotide N-glycosidase activity of cinnamomin A-chain has been characterized by 1 H NMR (Xu et al., 2000). Cinnamomin A-chain could cleave the N-glycosidic bond and release an adenine base from AMP, ADP, dAMP and adenosine. However, it cannot act on GMP, CMP and UMP, indicating a base preference of the hydrolysis of the N–C-glycosidic bonds. Cinnamomin A-chain could also deadenylate DNA molecules at multiple sites. Furthermore, our work has revealed that the native and the recombinant cinnamomin both can cleave the supercoiled double-stranded DNA instead of the linear double-stranded DNA, excluding the possibility of nuclease contamination during the RIP preparation
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(Ling, Liu, & Wang, 1994; Ling, Li, Wu, & Liu, 1995b). It was proposed that the conversion of the supercoiled DNA to the nicked or the linear forms by cinnamomin was due to the spontaneous breakage of the phosphodiester bonds in the AT-rich region because of the weakening produced by removing adenines.
9. Distribution of the counterpart of cinnamomin in genus Cinnamomum Except the camphora, the counterparts of cinnamomin, porrectin and bodinierin were identified and isolated from the seeds of other two species of genus Cinnamomum (Porrectum and Bodinieri), respectively (Hou, Wang, & Liu, 2001; Li, Liu, & Niu, 1996). Interestingly, a miniature counterpart of cinnamomin also existed in the seeds of these two species of cinnamomum. These three proteins have similar structure and biological activities, which provided phylogenetic evidence to the three species (Hou et al., 2001). References Barbieri, L., Battelli, M. G., & Stirpe, F. (1993). Ribosomeinactivating proteins from plants. Biochimica et Biophysica Acta, 1154(3–4), 237–283. Endo, Y., Mitsui, K., Motizuki, M., & Tsurugi, K. (1987). The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes: The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. Journal of Biological Chemistry, 262(12), 5908–5912. Endo, Y., & Wool, I. G. (1982). The site of action of alpha-sarcin on eukaryotic ribosomes: The sequence at the alpha-sarcin cleavage site in 28S ribosomal ribonucleic acid. Journal of Biological Chemistry, 257(15), 9054–9060. Hou, F. J., Wang, B. Z., & Liu, W. Y. (2001). Comparative studies of three type II ribosome-inactivating proteins from the seeds of three species of the genus Cinnamomum. Protein Peptide Letters, 8(3), 193–200. Hu, R., Tang, S., & Liu, W. Y. (2000). The pH-dependent interaction of cinnamomin with lipid membrane investigated by fluorescence methods. Biological Chemistry, 381(7), 567–573. Li, X. D., Liu, W. Y., & Niu, C. I. (1996). Purification of a new ribosome-inactivating protein from the seeds of Cinnamomum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein. Biological Chemistry, 377(12), 825–831. Li, X. D., Chen, W. F., Liu, W. Y., & Wang, G. H. (1997). Largescale preparation of two new ribosome-inactivating proteins:
Cinnamomin and camphorin from the seeds of Cinnamomum camphora. Protein Expression and Purification, 10(1), 27–31. Lin, J., & Liu, W. Y. (1995). The dynamic characterization of ribosome-inactivating protein during the process of maturation of the seeds of camphor tree. Chinese Journal of Biochemistry, 11(3), 286–291. Lin, J., & Liu, W. Y. (1996). Cytotoxicity of two new ribosome-inactivating proteins, cinnamomin and camphorin, to carcinoma cells. Cell Biochemistry and Function, 14(3), 157– 161. Ling, J., Liu, W. Y., & Wang, T. P. (1994). Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro. FEBS Letters, 345(2–3), 143–146. Ling, J., Liu, W. Y., & Wang, T. P. (1995a). Simultaneous existence of two types of ribosome-inactivating proteins in the seeds of Cinnamomum camphore: Characterization of the enzymatic activities of these cytotoxic proteins. Biochimica et Biophysica Acta, 1252(1), 15–22. Ling, J., Li, X. D., Wu, X. H., & Liu, W. Y. (1995b). Topological requirements for recognition and cleavage of DNA by ribosome-inactivating proteins. Biological Chemistry, 376(11), 637–641. Liu, W. Y., Chen, H. Y., Chen, W. F., Li, X. D., Ulbrich, N., & Xchroder, W. (1998). A proposed position of a disulfide bridge in the molecules of cinnamomin and porrectin: Two new type II ribosome-inactivating proteins isolated from the seeds of camphor trees. Biochemistry and Molecular Biology International, 44(5), 1045–1049. Liu, R., Wei, G., Yang, Q., He, W., & Liu, W. Y. (2002). Cinnamomin: A type II ribosome-inactivating protein, is a storage protein in the seed of the camphor tree (Cinnamomum camphora). Biochemical Journal, 362(3), 659–663. Pu, Z., Xie, L., Wang, E. D., & Liu, W. Y. (1998). Purification and activity study of the A- and B-chains of cinnamomin: A type II ribosome-inactivating protein. Biological Chemistry, 379(12), 1413–1418. Pu, Z., Li, Y., Hou, F., He, F., Zhang, N., Wu, H., & Liu, W. Y. (2000). Structural studies of the glycopeptides of B-chain of cinnamomin: A type II ribosome-inactivating protein by nuclear magnetic resonance. Glycoconjugate Journal, 17(11), 749–759. Reinbothe, S., Reinbothe, C., Lehmann, J., Becker, W., Apel, K., & Parthier, B. (1994). JIP60: A methyl jasmonate-induced ribosome-inactivating protein involved in plant stress reactions. Proceedings of the National Academy of Sciences of the United States of America, 91(15), 7012–7016. Van Damme, E. J. M., Hao, Q., Chen, Y., Barre, A., Vandenbussche, F., Desmyster, S., Rouge, P., & Peumans, W. J. (2001). Ribosome-inactivating proteins: A family of plant proteins that do more than inactivate ribosomes. Critical Reviews in Plant Science, 20(5), 395–465. Xie, L., Hou, F., Liu, W. Y., & Wang, E. (2000). Location of cysteine residues and the biological activities of the fluorencence-labeled cinnamomin. Protein Peptide Letters, 7(4), 265–272. Xie, L., Wang, B. Z., Hu, R. G., Ji, H., Zhang, L., & Liu, W. Y. (2001). Structural and functional studies of cinnamomin: A new type II ribosome-inactivating protein isolated from the seeds of
W.-J. He, W.-Y. Liu / The International Journal of Biochemistry & Cell Biology 35 (2003) 1021–1027 the camphor tree. European Journal of Biochemistry, 268(22), 5723–5733. Xu, Y. Z., & Liu, W. Y. (2000). Effects of the active aldehyde group generated by RNA N-glycosidase in the Sarcin/Ricin domain of rat 28S ribosomal RNA on peptide elongation. Biological Chemistry, 381(2), 113–119. Xu, Y. Z., Li, Y. J., Hu, H. Y., Hu, R., Wu, H., & Liu, W. Y. (2000). Adenine nucleotide N-glycosidase activity of the A-chain of cinnamomin characterized by 1 H nuclear magnetic resonance. Biological Chemistry, 381(5–6), 447–451. Yang, Q., Liu, R., Gong, Z., & Liu, W. Y. (2002). Studies of three genes encoding cinnamomin (a type II RIP) isolated from the seeds of camphor tree and their expression patterns. Gene, 284(1–2), 215–223.
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Zhang, J. S., & Liu, W. Y. (1992). The mechanism of action of trichoxanthin on eukaryotic ribosomes: RNA N-glycosidase activity of the cytotoxin. Nucleic Acids Research, 20(6), 1271– 1275. Zhang, G., Shi, Y., Wang, W., & Liu, W. Y. (1999). Cation channel formed at lipid bilayer by cinnamomin: A new type II ribosome-inactivating protein. Toxicon, 37(9), 1313–1322. Zhang, A. H., Tang, S., & Liu, W. Y. (2001). Substrate-structure dependence of ribotoxins on cleaving RNA in C. camphora ribosome. Journal of Natural Toxins, 10(2), 119–125. Zhou, X., Li, X. D., Yuan, J. Z., Tang, Z. H., & Liu, W. Y. (2000). Toxicity of cinnamomin: A new type II ribosome-inactivating protein to bollworm and mosquito. Insect Biochemistry and Molecular Biology, 30(3), 259–264.
Molecules in focus
Cinnamomin: a multifunctional type II ribosome-inactivating protein Wen-Jun He, Wang-Yi Liu∗ State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
Abstract 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. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cinnamomin; Deadenylation; Ribosome-inactivating protein (RIP); RNA N-glycosidase
1. Introduction Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes and thus inhibit protein synthesis (Barbieri, Battelli, & Stirpe, 1993). Most RIPs are widely distributed in the higher plants and contain an RNA N-glycosidase domain that can specifically remove an adenine from the highly conserved loop (Sarcin/Ricin domain) of the largest RNA in eukaryotic ribosomes (Endo, Mitsui, Motizuki, & Tsurugi, 1987; Zhang & Liu, 1992). Another enzymatic activity of RIP is a unique ribonuclease possessed only by ␣-sarcin hith∗ Corresponding author. Tel.: +86-21-64374430; fax: +86-21-64338357. E-mail address: [email protected] (W.-Y. Liu).
erto investigated. Alpha-sarcin is capable of selectively hydrolyzing a single phosphodiester bond between G4325 and A4326 of 28S RNA in rat ribosome as shown in Fig. 1 (Endo & Wool, 1982). This Sarcin/ Ricin domain is responsible for the interaction of elongation factors with ribosomes (Xu & Liu, 2000). On the basis of structural diversity, plant RIPs have been classified into three types as indicated in Fig. 2 (Van Damme et al., 2001). Type I RIP is composed of a single, intact polypeptide of around 11–30 kDa that, in some cases, is processed proteolytically into two shorter polypeptides held together by non-covalent interaction. While type II RIP consists of two polypeptides (A- and B-chain) linked by a disulfide bond. Both type I RIP and the A-chain of type II RIP consist exclusively of a single RNA N-glycosidase domain. The B-chain of type II RIP is a galactose-specific lectin
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Fig. 1. The action sites of RIPs on 28S RNA in rat ribosome.
Fig. 2. Comparison of the primary structures of three types of RIPs.
that is responsible for the recognition of d-galactose of galactose-terminated receptors on the cell surface, and facilitates the internalization of the toxic A-chain. Additionally, a type III RIP is identified and this protein is composed of a type I RIP-like N-terminal domain covalently linked to a C-terminal domain with unknown function (Reinbothe et al., 1994). RIPs have received wide attention from researchers in both theoretical and applied fields owing to their increasing application in probing the structure of the ribosome, potential drugs in the clinic trials and transgenic reagents in the agriculture. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of the camphor tree (Cinnamomum camphora) (Ling, Liu, & Wang, 1995a). Its purification, enzymatic mechanism, structure, gene pattern, physiological function and possible applications have been intensively investigated. In this review, we have summarized our major results related to cinnamomin in recent years.
2. Purification and characterization of cinnamomin The camphor tree belongs to the Lauraceae family. It is resistant to viral infection and insect disease. Cinnamomin, a new type II RIP, was purified from the mature seeds of one species of camphor tree (C. camphora) (Ling et al., 1995a). It has been investigated in detail with respect to the biochemical and biophysical properties of cinnamomin. This 61-kDa protein has three isoforms. The A- and B-chains derived from cinnamomin isoforms have similar mobility on the SDS-polyacrylamide gel. The significant phylogenetic finding presents the co-existence of the three isoforms of a type II RIP in the same organ of a higher plant. Cinnamomin A-chain was demonstrated to be a specific RNA N-glycosidase. Among the four rat ribosomal RNAs, only the 28S RNA was modified by cinnamomin A-chain. The cleavage site was
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N–C-glycosidic bond of the A4324 embedded in the highly conserved “Sarcin/Ricin domain” in rat ribosome (Lin & Liu, 1996; Ling et al., 1995a). Interestingly, the intact cinnamomin also exhibits the RNA N-glycosidase activity. Meanwhile, an improved method for large-scale preparation has been developed taking advantage of the lectin properties of its B-chain (Li, Chen, Liu, & Wang, 1997). Instead of gel filtration, cinnamomin could be purified to high yield by a single step of acid-treated Sepharose 4B affinity chromatography followed by elution with lactose. Thus, 620 mg of cinnamomin was obtained from 500 g of wet seeds, instead of only 10.6 mg by the previous method. In addition, a simplified method for purification of the A- and B-chains of cinnamomin on a large scale was also developed (Pu, Xie, Wang, & Liu, 1998). Urea was introduced to weaken the non-covalent interaction that holds the A- and B-chains together very tightly. In the presence of 4 M urea, the Aand B-chains of the reduced cinnamomin were separated effectively by DEAE-cellulose chromatography. The purified A-chain still displayed the RNA N-glycosidase activity and the B-chain lost its lectin activity. After refolding in vitro in the presence of lactose, the active B-chain with lectin activity could be further purified by acid-treated Sepharose 4B affinity chromatography. From 80 mg of cinnamomin, 10 mg of A-chain and 38 mg of B-chain were obtained. However, it is noteworthy that the purified A-chain is unstable in solution due to its liability to precipitate and thus loses its RNA N-glycosidase activity in the absence of the B-chain.
3. Structural and functional studies of cinnamomin The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the cDNAs of its Aand B-chains. It has 549 amino-acid residues: 271 residues in the A-chain, 14 residues in the linker and 264 residues in the B-chain (Xie et al., 2001). The functional roles of five conserved amino-acid residues (Y75, Y115, E167, R170 and W201) in the predicted active site of cinnamomin A-chain were investigated by site-directed mutagenesis (Xie et al.,
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2001). Single mutations of these amino-acid residues led to 8–50 folds decrease of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of cinnamomin A-chain. Also, the strong electric charge introduced at the single Cys251 in A-chain was involved in the substrate binding. And the results of deletion showed that both the N-terminal region (52 amino-acid residues) and the C-terminal region (51 amino-acid residues) of cinnamomin A-chain were vital for its enzymatic activity. Cinnamomin is also a glycoprotein. Its A- and B-chains contain 0.3 and 3.9% sugar, respectively (Ling et al., 1995a). After the sugar chains of cinnamomin were oxidized with periodate and then fluorescence-labeled with FTSC, the RNA N-glycosidase activity of its A-chain and the lectin activiy of its B-chain decreased three folds (Xie, Hou, Liu, & Wang, 2000). However, the physiological function of the sugar chains is still unclear. Three major glycopeptides have been purified from cinnamomin B-chain by gel filtration chromatography, anion-exchange chromatography and HPLC. Their primary structures have been determined by two-dimensional NMR (Fig. 3) (Pu et al., 2000).
4. Location of cysteine residues and proposed position of disulfide bridges in cinnamomin A total of 10 cysteine residues were titrated with DTNB in the cinnamomin under denatured condition, one in the A-chain and the other nine in the B-chain (Fig. 3) (Xie et al., 2000). This result was confirmed by the primary structure deduced from its cDNA (Xie et al., 2001). In cinnamomin, all the cysteine residues formed the disulfide bonds, which accounted for its highly structural stability. Based on the simulated three-dimensional structure of cinnamomin A-chain, the N-terminal region of the A-chain had a ␣-helix structure of around 50 amino-acid residues. This ␣-helix was juxtaposed closely to the central region where a cluster of ␣-helixes formed the globular module. And the central region had key amino-acid residues and the active site cleft. The C-terminal region of cinnamomin A-chain consisted of a random coil about 50 amino-acid residues. A Cys251 in the random coil forms the interchain disulfide bond
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Fig. 3. Schematic structure of cinnamomin. The precursor polypeptide chain is cleaved proteolytically to remove the signal peptide and the 14 amino-acid linker. The structure of glycan chains of the B-chain and the positions of disulfide bridges as well as the conserved amino-acid residues in the active site of the A-chain are indicated. (䊊) Mannose; (䉫) GlcNAc; () Xylulose; (䉲) Tyr 75 and Tyr 115, respectively; (䊉) Glu167; (䉱) Arg170; (䉬) Trp201.
with the Cys4 of B-chain (Xie et al., 2001). Meanwhile, the B-chain was also stabilized by four intrachain disulfide bridges: Cys20–Cys39, Cys63–Cys80, Cys151–Cya164 and Cys194–Cys209 (Xie et al., 2001; Liu et al., 1998).
5. Three genes coding for cinnamomin and their expression patterns (Yang, Liu, Gong, & Liu, 2002) After the full-length cDNA of cinnamomin was obtained by 5 rapid amplification of cDNA ends (5 RACE), polymerase chain reaction (PCR) amplification of its genomic DNA was performed. Unexpectedly, sequence analysis of the PCR products reveals three cinnamomin genes with >98.0% identity. One of them was the cDNA of cinnamomin mentioned above and designated as cinnamomin I, whereas the other two genes were named as cinnamomin II and III, respectively. RT-PCR amplification of the cDNAs of cinnamomin II and III showed that these two genes were also functional. The three genes have no introns. Three cinnamomin precursors that were inferred from the cDNA sequence of three cinnamomin genes exhibited relatively high sequence homology with other type II RIPs. It suggested that the three genes for cinnamomin might encode the three isoforms (of cinnamomin) already characterized.
6. Physiological function of cinnamomin Previous studies revealed that many RIPs are involved in defence mechanisms in plant cells and terminate protein synthesis under appropriate physiological conditions and thus are involved in metabolic regulation. However, cinnamomin could not inactivate its own (autologous) ribosome in vitro (Zhang, Tang, & Liu, 2001). Northern and Western blotting revealed that cinnamomin was expressed exclusively in cotyledons of C. camphora seeds and the acmes of expression emerged at 75–90 days after flower (DAF) when seeds were close to maturity (Yang et al., 2002; Liu, Wei, Yang, He, & Liu, 2002; Lin & Liu, 1995). It accumulated in large amounts (accounting for about 11% of total seed proteins) simultaneously with other proteins at the post-stages of seed development. Cinnamomin was degraded rapidly by the endopeptidase during the early stages of seed germination. Moreover, the amino-acid composition of cinnamomin was very close to that of other storage proteins. All of these properties suggest that cinnamomin functioned as a storage protein providing sources of carbon, nitrogen and sulphur during seedling growth and development (Liu et al., 2002). Besides the intact cinnamomin, small amount of its free A- and B-chains were identified to be present in the mature seed cells. It was proposed that the free A- and B-chains were derived from the enzymatic
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reduction of the interchain disulfide bond of cinnamomin. In the seed extract, the percentage of free A- and B-chains to the intact cinnamomin was 2.6–2.8 and approximately 80% of these free A- and B-chains were produced by enzymatic reduction in the seed cells. Also, a small protein of about 46 kDa in the mature seeds was eluted together with cinnamomin by lactose from the acid-treated Sepharose 4B column. Like cinnamomin, this small protein consists of two chain connected by the disulfide bond since it could be reduced with 2-mercaptoethanol to produce a miniature A-chain and a normal B-chain, the latter is completely identical to the B-chain of cinnamomin. The miniature A-chain exhibited the RNA N-glycosidase activity and strong inhibitory activity to protein synthesis in vitro almost the same as the normal A-chain. Furthermore, the N-terminal 10 amino acids of this miniature A-chain are entirely identical sequence with that of cinnamomin A-chain. The structural homology of these two similar type II RIPs were further confirmed by Western blotting using the antibody of cinnamomin. However, RT-PCR revealed that there was no corresponding mRNA of this small protein in vivo. Northern blotting also showed that the mRNA of cinnamomin exists as only one normal form in the cotyledons of seeds of C. camphora. Based on these facts, it is proposed that the miniature A-chain is composed of all essential amino-acid residues related to the RNA N-glycosidase activity of cinnamomin A-chain and probably it is a splicing product of cinnamomin A-chain. If it is the case, cinnamomin will be interesting as a good model to study the splicing process of proteins.
7. Cytotoxicity of cinnamomin to tumor cells and insect larvae Inhibition of protein synthesis in the target cells was considered as the main function of RIPs although RIPs did exhibit various effects, such as inhibiting infection and replication of viruses, causing lesions in cancer cells and affecting the immune response. Cinnamomin also inhibited the growth of cultured carcinoma cells (Lin & Liu, 1996). The IC50 of cinnamomin to human hepatocarcinoma cell-line 7721 and the melanoma M21 were 18.8 and 11.7 nmol,
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respectively. And cinnamomin remarkably retarded the growth of solid melanoma in the skin of the nude mouse. Furthermore, the toxicity of cinnamomin to bollworm (Helicoverpa armigera), mosquito (Culex pipines pallens) and silkworm (Bombyx mori) during larval stage was tested (Zhou, Li, Yuan, Tang, & Liu, 2000). The IC50 of cinnamomin to bollworm larvae fed on diet containing cinnamomin was 1839 ppm and the IC50 to larvae of mosquito was 168 ppm. The inhibition of protein synthesis by cinnamomin was tested in in vitro translation system of bollworm larvae and its IC50 was determined to be approximately 14 nM. An R-fragment could be isolated from ribosomes of cinnamomin-treated carcinoma cells and cinnamomin-treated bollworm larvae ribosomes after incubation with acidic aniline, indicating that the cytotoxicity of cinnamomin to carcinoma cells or bollworm larvae was caused by the modification of the ribosome (Lin & Liu, 1996; Zhou et al., 2000). In addition, it is found that cinnamomin could form a cation-specific channel in a bilayer membrane and its B-chain may be responsible for the action (Zhang, Shi, Wang, & Liu, 1999). Moreover, lowering the pH from 7.5 to 5.0 evoked conformational changes of cinnamomin and unmasked its hydrophobic areas, facilitating the penetration of cinnamomin into the lipid bilayer (Hu, Tang, & Liu, 2000). These findings further elucidate the mechanism for the entry of cinnamomin into integral cells to act on the cytosolic target.
8. Other enzymatic activities of cinnamomin Besides the site-specific RNA N-glycosidase activity, adenine nucleotide N-glycosidase activity of cinnamomin A-chain has been characterized by 1 H NMR (Xu et al., 2000). Cinnamomin A-chain could cleave the N-glycosidic bond and release an adenine base from AMP, ADP, dAMP and adenosine. However, it cannot act on GMP, CMP and UMP, indicating a base preference of the hydrolysis of the N–C-glycosidic bonds. Cinnamomin A-chain could also deadenylate DNA molecules at multiple sites. Furthermore, our work has revealed that the native and the recombinant cinnamomin both can cleave the supercoiled double-stranded DNA instead of the linear double-stranded DNA, excluding the possibility of nuclease contamination during the RIP preparation
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(Ling, Liu, & Wang, 1994; Ling, Li, Wu, & Liu, 1995b). It was proposed that the conversion of the supercoiled DNA to the nicked or the linear forms by cinnamomin was due to the spontaneous breakage of the phosphodiester bonds in the AT-rich region because of the weakening produced by removing adenines.
9. Distribution of the counterpart of cinnamomin in genus Cinnamomum Except the camphora, the counterparts of cinnamomin, porrectin and bodinierin were identified and isolated from the seeds of other two species of genus Cinnamomum (Porrectum and Bodinieri), respectively (Hou, Wang, & Liu, 2001; Li, Liu, & Niu, 1996). Interestingly, a miniature counterpart of cinnamomin also existed in the seeds of these two species of cinnamomum. These three proteins have similar structure and biological activities, which provided phylogenetic evidence to the three species (Hou et al., 2001). References Barbieri, L., Battelli, M. G., & Stirpe, F. (1993). Ribosomeinactivating proteins from plants. Biochimica et Biophysica Acta, 1154(3–4), 237–283. Endo, Y., Mitsui, K., Motizuki, M., & Tsurugi, K. (1987). The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes: The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. Journal of Biological Chemistry, 262(12), 5908–5912. Endo, Y., & Wool, I. G. (1982). The site of action of alpha-sarcin on eukaryotic ribosomes: The sequence at the alpha-sarcin cleavage site in 28S ribosomal ribonucleic acid. Journal of Biological Chemistry, 257(15), 9054–9060. Hou, F. J., Wang, B. Z., & Liu, W. Y. (2001). Comparative studies of three type II ribosome-inactivating proteins from the seeds of three species of the genus Cinnamomum. Protein Peptide Letters, 8(3), 193–200. Hu, R., Tang, S., & Liu, W. Y. (2000). The pH-dependent interaction of cinnamomin with lipid membrane investigated by fluorescence methods. Biological Chemistry, 381(7), 567–573. Li, X. D., Liu, W. Y., & Niu, C. I. (1996). Purification of a new ribosome-inactivating protein from the seeds of Cinnamomum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein. Biological Chemistry, 377(12), 825–831. Li, X. D., Chen, W. F., Liu, W. Y., & Wang, G. H. (1997). Largescale preparation of two new ribosome-inactivating proteins:
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