Three new ribosome-inactivating protein (RIP; EC 3.2.2.22) isoforms that we have named musarmins (MUs) 1, 2 and 3 have been isolated from the bulbs of Muscari armeniacum L. and Miller by ion-exchange chromatography and gel filtration. Analysis by electrophoresis revealed that they are single-chain proteins and mass spectrometry analysis afforded Mr values of 28,708,30,003 and 27,626 for MUs 1, 2 and 3, respectively. Musarmins strongly inhibited protein synthesis carried out by mammalian ribosomes, with IC50 values in the 0.14-0.24nM range but not that carried out by plant cell-free systems or HeLa cells. MUs promote the single depurination of rabbit reticulocyte 28S rRNA. cDNA cloning of genes coding for musarmins revealed that they contain open reading frames of 298, 294 and 295 aminoacids for MU1, MU2 and MU3, respectively. Mature MU1, MU2 and MU3 contain 277, 273 and 273 aminoacids, respectively suggesting post-translational C-terminal processing. An untranslated mRNA coding for an ORF very similar to that of MU3 was detected in leaves. Each of the four MU genes contains an intron. In contrast to other RIPs, MUs are present only in bulbs and are not induced in leaves either by senescence, or by treatment of leaves with H2O2 or salicylic acid, or by growth in darkness. Therefore, these proteins could play a non-vital role in plants; for instance, as anti-pathogens and protective agents only in some stages of the plant life cycle (237). © 2002 Elsevier Science Ltd. All rights reserved.

Cited By :10Export Date: 16 July 2015CODEN: IJBBFCorrespondence Address: Girbés, T.; Depto. de Bioquim. y Biol. Molecular, Facultad de Ciencias, Universidad de Valladolid, E-47005 Valladolid, Spain; email: girbes@bio.uva.esMolecular Sequence Numbers: GENBANK: AF289116, AF289117, AF289118, AF289119, AF289120, X86085;Chemicals/CAS: hydrogen peroxide, 7722-84-1; salicylic acid, 63-36-5, 69-72-7; Hydrogen Peroxide, 7722-84-1; N-Glycosyl Hydrolases, EC 3.2.2.-; Plant Proteins; Protein Isoforms; Protein Synthesis Inhibitors; Salicylic Acid, 69-72-7References: Hartley, M.R., Chaddock, J., Bonness, M., The structure and function of ribosome-inactivating proteins (1996) Trends Plant Sci., 1, pp. 254-260;Barbieri, L., Battelli, M.G., Stirpe, F., Ribosome-inactivating proteins from plants (1993) Biochim. Biophys. Acta, 1154, pp. 237-282; Girbés, T., Ferreras, J.M., Ribosome-inactivating proteins from plants (1998) Recent Res. Dev. Agric. Biol. Chem., 2, pp. 1-16; Wang, H.X., Ng, T.B., Isolation of pleuturegin, a novel ribosome-inactivating protein from fresh sclerotia of the edible mushroom pleurotus tuber-regium (2001) Biochem. Biophys. Res. Commun., 288, pp. 718-721; Suh, J.K., Hovde, C.J., Robertus, J.D., Shiga toxin attacks bacterial ribosomes as effectively as eukaryotic ribosomes (1998) Biochemistry, 37, pp. 9394-9398; Barbieri, L., Valbonesi, P., Bondioli, M., Alvarez, M.L., Dal Monte, P., Landini, M.P., Stirpe, F., Adenine glycosylase activity in mammalian tissues: An equivalent of ribosome-inactivating proteins (2001) FEBS Lett., 505, pp. 196-197; Hudak, K.A., Wang, P., Tumer, N.E., A novel mechanism for inhibition of translation by pokeweed anti-viral protein: Depurination of the capped RNA template (2000) RNA, 6, pp. 369-380; Fong, W.P., Mock, W.Y., Ng, T.B., Intrinsic ribonuclease activities in ribonuclease and ribosome-inactivating proteins from the seeds of bitter gourd (2000) Int. J. Biochem. Cell Biol., 32, pp. 571-577; Lord, J.M., Roberts, L.M., Robertus, J.D., Ricin: Structure, mode of action, and some current applications (1994) FASEB J., 8, pp. 201-208; Ferreras, J.M., Citores, L., De Benito, F.M., Arias, F.J., Rojo, A., Muñoz, R., Iglesias, R., Girbés, T., Ribosome-inactivating proteins and lectins from Sambucus (2000) Curr. Top. Phytochem., 3, pp. 113-128; Muñoz, R., Arias, Y., Ferreras, J.M., Jimenez, P., Rojo, M.A., Girbés, T., Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein Nigrin b (2001) Cancer Lett., 167, pp. 163-169; Bonness, M., Ready, M.P., Irvin, J.D., Mabry, T., Pokeweed anti-viral protein inactivates pokeweed ribosomes; Implication for the anti-viral mechanism (1994) Plant J., 5, pp. 173-183; Girbés, T., De Torre, C., Iglesias, R., Ferreras, J.M., Méndez, E., RIP for viruses (1996) Nature, 379, pp. 777-778; Lodge, J.K., Kaniewski, W., Tumer, N., Broad-spectrum virus resistance in transgenic plants expressing pokeweed anti-viral protein (1993) Proc. Natl. Acad. Sci. U.S.A., 90, pp. 7089-7093; Stirpe, F., Barbieri, L., Gorini, P., Valbonesi, P., Bolognesi, A., Polito, L., Activities associated with the presence of ribosome-inactivating proteins increase in senescent and stressed leaves (1996) FEBS Lett., 382, pp. 309-312; De Benito, F.M., Iglesias, R., Ferreras, J.M., Citores, L., Camafeita, E., Mendez, E., Girbés, T., Constitutive and inducible type 1 ribosome-inactivating proteins (RIPs) in elderberry (Sambucus nigra L.) (1998) FEBS Lett., 428, pp. 75-79; Girbés, T., Citores, L., Iglesias, R., Ferreras, J.M., Muñoz, R., Rojo, M.A., Arias, F.J., Calonge, M., Ebulin l, a non-toxic novel type 2 ribosome-inactivating protein from Sambucus ebulus L. leaves (1993) J. Biol. Chem., 268, pp. 18195-18199; Ferreras, J.M., Barbieri, L., Girbés, T., Batelli, M.G., Rojo, M.A., Arias, F.J., Rocher, M.A., Stirpe, F., Distribution and properties of major ribosome-inactivating proteins (28S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae) (1993) Biochim. Biophys. Acta, 1216, pp. 31-42; Pascal, J.M., Day, P.J., Monzingo, A.F., Ernst, S.R., Robertus, J.D., Iglesias, R., Perez, Y., Girbés, T., 2.8 Å crystal structure of a non-toxic type 2 ribosome-inactivating protein, ebulin l (2001) Proteins, 43, pp. 319-326; Murray, M.G., Thompson, W.F., Rapid isolation of high molecular weight plant DNA (1990) Nucleic Acids Res., 8, pp. 4321-4325; Girbés, T., Citores, L., Ferreras, J.M., Rojo, M.A., Iglesias, R., Muñoz, R., Arias, F.J., Méndez, E., Isolation and partial characterization of Nigrin b, a non-toxic novel type 2 ribosome-inactivating protein from the bark of Sambucus nigra L. (1993) Plant Mol. Biol., 22, pp. 1181-1186; Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-685; Citores, L., De Benito, F.M., Iglesias, R., Ferreras, J.M., Argüeso, P., Jiménez, P., Testera, A., Girbés, T., Characterization of a new non-toxic two-chain ribosome-inactivating protein and a structurally-related lectin from rhizomes of dwarf elder (Sambucus ebulus L.) (1997) Cell. Mol. Biol., 43, pp. 485-499; Citores, L., De Benito, F.M., Iglesias, R., Ferreras, J.M., Argüeso, P., Jiménez, P., Méndez, E., Girbés, T., Presence of polymerized and free forms of the non-toxic type 2 ribosome-inactivating protein ebulin and a structurally-related new heterodimeric lectin in fruits of Sambucus ebulus L. (1998) Planta, 204, pp. 310-317; Merino, M.J., Ferreras, J.M., Muñoz, R., Iglesias, R., Girbés, T., Plant species containing inhibitors of eukaryotic polypeptide synthesis (1990) J. Exp. Bot., 41, pp. 67-70; Kozak, M., Initiation of translation in prokaryotes and eukaryotes (1999) Gene, 234, pp. 187-208; Nielsen, H., Engelbrecht, J., Brunak, S., Von Heijne, G., Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites (1997) Protein Eng., 10, pp. 1-6; Van Damme, E.J., Barre, A., Barbieri, L., Valbonesi, P., Rouge, P., Van Leuven, F., Stirpe, F., Peumans, W.J., Type 1 ribosome-inactivating proteins are the most abundant proteins in iris (Iris hollandica var. Professor Blaauw) bulbs: Characterization and molecular cloning (1997) Biochem. J., 324, pp. 963-970; Leah, R., Tommerup, H., Svendsen, I., Mundy, J., Biochemical and molecular characterization of three barley seed proteins with anti-fungal properties (1991) J. Biol. Chem., 266, pp. 1564-1573; Habuka, N., Kataoka, J., Miyano, M., Tsuge, H., Ago, H., Noma, M., Nucleotide sequence of a genomic gene encoding tritin, a ribosome-inactivating protein from Triticum aestivum (1993) Plant Mol. Biol., 22, pp. 171-176; Lamb, F.I., Roberts, L.M., Lord, J.M., Nucleotide sequence of cloned cDNA coding for preproricin (1985) Eur. J. Biochem., 148, pp. 265-270; Munishkin, A., Wool, I.G., Systematic delection analysis of ricin A-chain function (1995) J. Biol. Chem., 270, pp. 30581-30587; Lorkovic, Z.J., Wieczorek, D.A., Lambermon, M.H.L., Filipowicz, W., Pre-mRNA splicing in higher plants (2000) Trends Plant Sci., 5, pp. 160-167; Kataoka, J., Miyano, M., Habuka, N., Masuta, C., Koiwai, A., A genomic gene for MAP, a ribosome-inactivating protein from Mirabilis jalapa, contains an intron (1993) Nucleic Acids Res., 21, p. 1035; Wong, R.N.S., Mak, N.K., Choi, W.T., Law, P.T.W., Increased accumulation of trichosanthin in Trichosanthes kirilowii induced by microorganisms (1995) J. Exp. Bot., 46, pp. 355-358; Liu, R.S., Wei, G.G., Yang, Q., He, W.J., Liu, W.Y., Cinnamomin, a type 2 ribosome-inactivating protein, is a storage protein in the seed of the Camphor tree (Cinnamomun camphora) (2002) Biochem. J., 362, pp. 659-663