Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin - a Novel Feature in Lys49-PLA2s Protein Class

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys. © 2013 Salvador et al.

Effect of ionic strength solution on the stability of chitosan-DNA nanoparticles

Chitosan-DNA nanoparticles employed in gene therapy protocols consist of a neutralised, stoichiometric core and a shell of the excess of chitosan which stabilises the particles against further coagulation. At low ionic strength, these nanoparticles possess a high stability; however, as the ionic strength increases, it weakens the electrostatic repulsion which can play a decisive part in the formation of highly aggregated particles. In this study, new results about the effect of ionic strength on the colloidal stability of chitosan-DNA nanoparticles were obtained by studying the interaction between chitosans of increasing molecular weights (5, 10, 16, 29, 57 and 150 kDa) and calf thymus DNA. The physicochemical properties of polyplexes were investigated by means of dynamic light scattering, static fluorescence spectroscopy, optic microscopy, transmission electronic microscopy and gel electrophoresis. After subsequent addition of salt to the nanoparticles solution, secondary aggregation increased the size of the polyplexes. The nanoparticles stability decreased drastically at the ionic strengths 150 and 500 mM, which caused the corresponding decrease in the thickness of the stabilising shell. The morphologies of chitosan/DNA nanoparticles at those ionic strengths were a mixture of large spherical aggregates, toroids and rods. The results indicated that to obtain stable chitosan-DNA nanoparticles, besides molecular weight and N/P ratio, it is quite important to control the ionic strength of the solution. © 2013 Copyright Taylor and Francis Group, LLC.

PEGylation of SPIONs by polycondensation reactions: A new strategy to improve colloidal stability in biological media

In this study, we report on a new route of PEGylation of superparamagnetic iron oxide nanoparticles (SPIONs) by polycondensation reaction with carboxylate groups. Structural and magnetic characterizations were performed by X-ray diffractometry (XRD), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and vibrating sample magnetometry (VSM). The XRD confirmed the spinel structure with a crystallite average diameter in the range of 3.5-4.1 nm in good agreement with the average diameter obtained by TEM (4.60-4.97 nm). The TGA data indicate the presence of PEG attached onto the SPIONs' surface. The SPIONs were superparamagnetic at room temperature with saturation magnetization (M S) from 36.7 to 54.1 emu/g. The colloidal stability of citrate- and PEG-coated SPIONs was evaluated by means of dynamic light scattering measurements as a function of pH, ionic strength, and nature of dispersion media (phosphate buffer and cell culture media). Our findings demonstrated that the PEG polymer chain length plays a key role in the coagulation behavior of the Mag-PEG suspensions. The excellent colloidal stability under the extreme conditions we evaluated, such as high ionic strength, pH near the isoelectric point, and cell culture media, revealed that suspensions comprising PEG-coated SPION, with PEG of molecular weight 600 and above, present steric stabilization attributed to the polymer chains attached onto the surface of SPIONs. © 2013 Springer Science+Business Media Dordrecht.

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s. © 2013 Elsevier Ltd.

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