Amino-acid sequence alignment of the short regions with those of other type 2 RIPs indicates a high homology with the A and B chains of volkensin, the structure of which is usually unknown

Amino-acid sequence alignment of the short regions with those of other type 2 RIPs indicates a high homology with the A and B chains of volkensin, the structure of which is usually unknown. lectin activity and is able to bind to glycosylated receptors around the cell surface. Stenodactylin is a type 2 RIP from the caudex ofAdenia stenodactylafrom the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high ASP1126 resolution (up to 2.15 ). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space groupC2 and ASP1126 contained two molecules in the asymmetric unit. == 1. Introduction == Ribosome-inactivating proteins (RIPs) have a broad distribution in nature, especially in the herb kingdom (Girbeset al., 2004). They are specific RNA N-glycosidases (EC 3.2.2.22) that depurinate a conserved adenine in an exposed loop of rRNA in the largest subunit of the ribosome (Endo & Tsurugi, 1987). RIPs have been classified into two main groups, type 1 and type 2 RIPs, with the first group being larger than the second. Type 1 RIPs are strongly basic single-chain enzymes with a molecular weight of about 30 kDa. Type 2 RIPs, or toxic lectins, are proteins with a molecular weight of about 6065 kDa that consist of an A chain with the same enzymatic activity as a type 1 RIP and a B chain linked together by a disulfide bond. The B chain is usually a lectin specific for galactose,N-acetyl galactosamine orN-acetyl neuraminic acid (van Dammeet al., 2001). The enzymatic activity of RIPs causes protein-synthesis arrest, targeting the rRNA loop involved in elongation-factor binding. ASP1126 All RIPs also remove adenine from DNA and other polynucleotides; the name polynucleotide adenine glycosylases has therefore been proposed (Barbieriet al., 1997, 2001). The toxicity of RIPs to cells and consequently to animals is usually highly variable and depends on their structure: in general, type 1 RIPs have a lower toxicity than type 2 RIPs (Stirpe & Battelli, 2006). The B chain facilitates the entry of the molecule into the cell by binding to sugars around the cell membrane, Mouse monoclonal to CRTC2 whereupon the A chain can damage ribosomes and possibly other structures, finally causing cell death. Several type 2 RIPs are potent toxins, some of which, such as ricin, have been known for a long time (Olsnes, 2004). RIPs have been used for criminal and even warfare or bioterrorism purposes (Bigalke & Rummel, 2005; Audiet al., 2005). However, they have also found medical applications in cancer therapy when converted into very potent and specific drugs by conjugation with antibodies or other molecules that act as site-directing carriers (Bolognesi & Polito, 2004; Pastanet al., 2007). Despite the similarities in their structures, type 2 RIPs show differences in the lesions that they cause in experimental animals, including their effects around the nervous system. All the toxic lectins tested, namely ricin, abrin, modeccin and viscumin, are retrogradely transported when injected into peripheral nerves, whereas only modeccin, volkensin and stenodactylin are retrogradely transported when injected into the central nervous system ASP1126 (Wiley & Kline, 2000; Montiet al., 2007). Stenodactylin is usually a recently characterized type 2 RIP (Stirpeet al., 2007). It was purified from the caudex ofAdenia stenodactyla, a herb belonging to the Passifloraceae family (Pelosiet al., 2005). Stenodactylin has a high enzymatic activity towards ribosomes and hsDNA substrates and is probably the most cytotoxic type 2 RIP described to date (Stirpeet al., 2007). In the rat central nervous system, stenodactylin is usually taken up and retrogradely transported from the terminal field of nerve cells to the cell bodies. Its toxic action results in a significant depletion of medial septum cholinergic neurons that project into the injected area of the hippocampus (Montiet al., 2007). Stenodactylin is usually highly toxic to mice and also shows a strong systemic toxicity, causing death in 100% of mice injected with 1.21 g kg1within 7 d (Stirpeet al., 2007). This activity is comparable to or even higher than that observed with volkensin, the most toxic lectin of herb origin known to date (Stirpeet al., 1985). The astonishingly high biological activity of stenodactylin makes the study of its structurefunction relationship exceedingly important. In this paper, crystallization conditions and preliminary X-ray diffraction data analysis of stenodactylin are reported. == 2. Protein purification == Stenodactylin was extracted and purified from the caudices ofA. stenodactylaas described in Pelosiet al.(2005). Briefly, the protein was precipitated in 100% saturated ammonium sulfate and the precipitate was dissolved in and dialyzed against PBS.