Transient occurrence of an ebulin-related D-galactose-lectin in shoots of Sambucus ebulus L. q Lucı ´a Citores a , Marı ´a A. Rojo a , Pilar Jime ´nez a , Jose ´ M. Ferreras a , Rosario Iglesias a , Isabel Aranguez b , Toma ´s Girbe ´s a, * a Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultad de Ciencias, Universidad de Valladolid, E-47005 Valladolid, Spain b Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultad de Farmacia, Universidad Complutense, E-28040 Madrid, Spain Received 17 July 2007; received in revised form 9 October 2007 Abstract Young shoots of Sambucus ebulus L. contain a monomeric D-galactose binding lectin (SELlm), which disappears upon shoot devel- opment, and was previously undetected since it co-purifies with the non-toxic type 2 ribosome-inactivating protein ebulin l and the dimeric lectin SELld. Molecular cloning of cDNA coding for SELlm and mass spectrometry analysis revealed a protein with a molecular mass of 34,239 Da, which displays 80%, 77% and 45% of amino acid sequence identity with the ebulin l-B chain, SELld and ricin-B chain, respectively. Furthermore, the cloned precursor, with respect to the ebulin l precursor is truncated and contains the signal peptide, a piece of the A chain, a piece of the connecting peptide and the B chain. Further processing yields the lectin protein, which contains only the B chain. Despite the fact that SELlm displays the same D-galactose-binding sites than ricin, it was found that the lectin has different binding properties to D-galactose-containing matrix than ricin. Notably, and unlike ricin, the binding of SELlm and other Sambucus lectins to such matrix was maximum in range of 0–10 °C and abolished at 20 °C. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Sambucus ebulus L.; Dwarf elder; Lectin; SELlm; Ricin; Ebulin; Ribosome-inactivating protein (RIP) 1. Introduction Lectins are a heterogeneous group of sugar-binding pro- teins found in microorganisms, plants, algae, fungi and ani- mals (Sharon and Lis, 2004). They differ with respect to their biochemical and physicochemical properties, interac- tion with sugar and oligosaccharides, molecular structure and biological activity. Based on structural analysis, plant lectins can be grouped into families of structurally and evo- lutionary-related proteins (Van Damme et al., 1998, 2004). One of these families is the type 2 ribosome-inactivating proteins (RIPs), a group of lectins which share a large structural homology, characterized by displaying two sepa- rated activities: lectin activity in one chain and inhibitory activity of protein synthesis in the other. RIPs, in general, are protein synthesis inhibitors that trigger the catalytic inactivation of mammalian, plant, yeast and bacterial sen- sitive ribosomes (Barbieri et al., 1993; Girbes et al., 1993a; Iglesias et al., 1993; Stirpe, 2004). The mechanism of the action of RIPs is the release of an adenine from the rRNA loop responsible for the interaction of elongation factor 2 in eukaryotes and G in prokaryotes (Girbes et al., 2004). RIPs have been classified into two categories: types 1 and 2. Type 1 RIPs consist of one polypeptide chain with the enzymatic activity. The type 2 RIPs consist of two polypep- tide chains, an A chain and a B chain, linked by a disulfide bridge. The A chain is similar to the type 1 enzymic poly- peptide chain. The B-chain, a carbohydrate-binding lectin, binds to glycoproteins and glycolipids with terminal galact- ose from the cell surface facilitating delivery of the toxin to the cytosol, where the A chain inactivates the ribosomes 0031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2007.10.014 q The nucleotide sequence reported in this paper has been submitted to the GenBank nucleotide database under accession number AM491634. * Corresponding author. Tel./fax: +34 983 423082. E-mail address: girbes@bio.uva.es (T. Girbe ´s). www.elsevier.com/locate/phytochem Available online at www.sciencedirect.com Phytochemistry 69 (2008) 857–864 PHYTOCHEMISTRY