Abstract: Volkensin, isolated from Adenia volkensii, is one of the most toxic type 2 ribosome-inactivating protein (RIP), exerting its biological function by inhibiting protein synthesis. Despite the high sequence identity with type 2 RIPs, including ricin, volkensin shows interesting peculiar properties. In this work a computational model building of volkensin was performed. The volkensin electrostatic potential charge distribution, the hydrophobic profile and the surface topology analyses were also carried out to aid the understanding of structure-function relationships of this potent toxin. Volkensin surface topology was probed by applying a limited proteolysis approach with the aim to gain insights into volkensin conformational features.

Abstract: The structure of the highly glycosylated type 1 ribosome inactivating protein PD-L1 was determined by x-ray crystallography. This protein belongs to a group of four PD-Ls (PD-L1-4) expressed in Phytolacca dioica leaves. Of these, PD-L1 and PD-L2 are endowed with the ability to cleave double strand DNA, a property which is not shared by the other two components of the family. Single crystals of native PD-L1, the most glycosylated, were obtained using seeding techniques and phase determination was achieved using molecular replacement. To investigate the role of glycosylation in the different functionality of these proteins, we performed DNA cleavage assays on the E. coli plasmid pBR322. These experiments revealed that DNA cleaving ability does not depend on the level of glycosylation of PD-L1, since there is no difference in the activities displayed by native PD-L1 and a recombinant non-glycosylated form. Beside confirming that DNA cleavage by PD-L1 cannot be attributed to contaminations, these data unambiguously show that functional changes between PD-L1 and PD-L4 are solely to be attributed to their sequence differences. Based on the comparison of PD-L1 and PD-L4 crystal structures, we propose possible structural determinants responsible for their different functional behaviour. (c) 2009 Wiley Periodicals, Inc. Biopolymers, 2009.

Abstract: Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography-mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Abstract: The gonadotropin-releasing hormone (GnRH) family includes several hypophysiotropic peptides occupying a central position in the regulatory loop controlling reproduction. Studies are still under way to clarify its biological role and evolutionary implication. Although sequencing of multiple genomes is bringing further advances in the understanding of the evolution of GnRH, there is still a need for biochemical studies aiming to identify GnRH from different species. Using a hybrid quadrupole-time-of-flight (Q-TOF) instrument, a new method for selective and sensitive GnRH detection and characterization from tissue extracts has been developed. The method uses the "precursor ion discovery" mode based on the capability of the Q-TOF analyzer to quickly record alternate mass spectra at low and high collision energy of precursor and product ion spectra, respectively, following liquid chromatographic separation of complex biological mixtures. The method exploits the selective detection of a specific b(2) product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine, highly conserved among nearly all species (22 of 24), and deriving from the preferential fragmentation of GnRHs carrying the dipeptide. Importantly, the method also includes acquisition of the product ion spectra from any candidate precursor ion, thereby allowing the determination of sequence information to confirm the GnRH identity or to isolate new ones.

Abstract: Type 2 ribosome inactivating proteins (RIPs) include some potent plant toxins, among which ricin from Ricinus communis and abrin from Abrus precatorius seeds, have been known for more than a century. Two other type 2 RIPs belong to this class of proteins, both isolated from plants of the same family (Passifloraceae), modeccin and volkensin, from Adenia digitata and Adenia volkensii roots, respectively. Volkensin is probably the most potent plant toxin known, with an LD50 for rats of 50-60 ng/kg. Here we report the cloning, expression and renaturation of recombinant volkensin B chain. Furthermore, starting from separately expressed A and B chains, a co-association procedure was set-up, leading to in vitro heterodimeric volkensin reconstitution. The recombinant heterodimer was characterized by N-terminal sequence analysis and its hemagglutinating activity assessed. In parallel, we have explored the carbohydrate-binding properties of native volkensin with the aim to correlate toxin-specific properties (i.e., axonal transport along neurons) to lectin's sugar-binding preferences.