Adrijana Leonardi

Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes

Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolytic as well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the Aalpha-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala(14) and Leu(15). Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.

Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.

Conus consors snail venom proteomics proposes functions, pathways, and novel families involved in its venomic system.

For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fish-hunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process, echotoxins and hyaluronidases, were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step toward identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation, and function.

Recruitment of Glycosyl Hydrolase Proteins in a Cone Snail Venomous Arsenal: Further Insights into Biomolecular Features of Conus Venoms

Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms (“injectable venom” stands for the venom variety obtained by milking of the snails. This is in contrast to the “dissected venom”, which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.

Two coagulation factor X activators from Vipera a. ammodytes venom with potential to treat patients with dysfunctional factors IXa or VIIa.

Two activators of coagulation factor X, 58kDa VAFXA-I and 70kDa VAFXA-II, were purified from the venom of long-nosed viper (Vipera ammodytes ammodytes) by chromatography on gel filtration, affinity, ion-exchange and hydroxyapatite media. Both enzymes are glycoproteins composed of a heavy chain and two C-type lectin-like light chains all joined by disulphide bonds. LC-MS and LC-MS/MS analysis of their tryptic fragments demonstrated that the heavy chain consists of three domains, metalloproteinase, disintegrin-like and cysteine-rich domains. The partial amino acid sequences of VAFXAs are very similar to those of the known factor X activators, RVV-X from Vipera russelli and VLFXA from Vipera lebetina venoms, as well as to other members of the reprolysin family of metalloproteinases. The VAFXAs activate factor X in a Ca(2+)-dependent manner with the same specificity as physiological activators. The activators weakly hydrolyzed insulin B-chain, fibrinogen and some components of the extracellular matrix in vitro, but did not activate prothrombin or plasminogen. VAFXAs inhibit collagen-induced platelet aggregation in vitro. They activate coagulation factor X to Xa without toxic effects. Their application in treating patients with dysfunctional factors IXa or VIIa to restore the normal blood coagulation process is thus promising.

Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona

Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored—the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs.

Ammodytagin, a heterodimeric metalloproteinase from Vipera ammodytes ammodytes venom with strong hemorrhagic activity

Ammodytagin, a hemorrhagic Zn(2+)-dependent metalloproteinase from Vipera ammodytes ammodytes (Vaa) venom, is a glycosylated heterodimer of 108 kDa, as determined by MALDI mass spectrometry. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by Na(2)EDTA, Zn(2+) and DTT. It cleaves insulin B-chain, hydrolysing it at positions Gln(4)-His(5), His(10)-Leu(11) and Tyr(16)-Leu(17). Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of a substrate specificity revealed that the hemorrhagic activity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective Vaa specific antivenom should be directed.

The role of antibodies specific for toxic sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom

The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy. We further revealed that Atxs and structurally very similar but non-toxic AtnI2 from the venom are not immuno cross-reactive.

Structural insight into LexA–RecA* interaction

RecA protein is a hallmark for the bacterial response to insults inflicted on DNA. It catalyzes the strand exchange step of homologous recombination and stimulates self-inactivation of the LexA transcriptional repressor. Importantly, by these activities, RecA contributes to the antibiotic resistance of bacteria. An original way to decrease the acquisition of antibiotic resistance would be to block RecA association with LexA. To engineer inhibitors of LexA–RecA complex formation, we have mapped the interaction area between LexA and active RecA–ssDNA filament (RecA*) and generated a three-dimensional model of the complex. The model revealed that one subunit of the LexA dimer wedges into a deep helical groove of RecA*, forming multiple interaction sites along seven consecutive RecA protomers. Based on the model, we predicted that LexA in its DNA-binding conformation also forms a complex with RecA* and that the operator DNA sterically precludes interaction with RecA*, which guides the induction of SOS gene expression. Moreover, the model shows that besides the catalytic C-terminal domain of LexA, its N-terminal DNA-binding domain also interacts with RecA*. Because all the model-based predictions have been confirmed experimentally, the presented model offers a validated insight into the critical step of the bacterial DNA damage response.

Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: Therapeutic implications.

UNLABELLED Vipera berus berus (Vbb) is the most widely distributed and Vipera ammodytes ammodytes (Vaa) the most venomous viper in Europe. In particular areas of the Old continent their toxic bites constitute a considerable public health problem. To make the current envenomation therapy more effective we have analysed the proteome of Vbb venom and compared it with that of Vaa. We found the proteome of Vbb to be much less complex and to contain smaller levels of particularly snaclecs and sPLA2s. Snaclecs are probably responsible for thrombocytopenia. The neurotoxic sPLA2s, ammodytoxins, are responsible for the most specific feature of the Vaa venom poisoning - induction of signs of neurotoxicity in patients. These molecules were not found in Vbb venom. Both venoms induce haemorrhage and coagulopathy in man. As Vaa and Vbb venoms possess homologous P-III snake venom metalloproteinases, the main haemorrhagic factors, the severity of the haemorrhage is dictated by concentration and specific activity of these molecules. The much greater anticoagulant effect of Vaa venom than that of Vbb venom lies in its higher extrinsic pathway coagulation factor-proteolysing activity and content of ammodytoxins which block the prothrombinase complex formation. BIOLOGICAL SIGNIFICANCE Envenomations by venomous snakes constitute a considerable public health problem worldwide, and also in Europe. In the submitted work we analysed the venom proteome of Vipera berus berus (Vbb), the most widely distributed venomous snake in Europe and compared it with the venom proteome of the most venomous viper in Europe, Vipera ammodytes ammodytes (Vaa). We have offered a possible explanation, at the molecular level, for the differences in clinical pictures inflicted by the Vbb and Vaa venoms. We have provided an explanation for the effectiveness of treatment of Vbb envenomation by Vaa antiserum and explained why full protection of Vaa venom poisoning by Vbb antiserum should not be always expected, especially not in cases of severe poisoning. The latter makes a strong case for Vaa antiserum production as we are faced with its shortage due to ceasing of production of two most frequently used products.