The Disulfide Bonds in Glycoprotein E2 of Hepatitis C Virus Reveal the Tertiary Organization of the Molecule

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change

Reappraisal of Vipera aspis Venom Neurotoxicity

BACKGROUND: The variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA(2) neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA(2) composition of the snakes captured in the same areas. [br/] METHODOLOGY/PRINCIPAL FINDINGS: We conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA(2)s. We used SELDI technology to study the diversity of PLA(2) in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA(2)s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA(2) venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains. [br/] CONCLUSIONS/SIGNIFICANCE: The association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA(2) genome and transcriptome data

Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts

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A 92-kDa human immunostimulatory protein.

We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety

An agarose-based gel-concentration system for microsequence and mass spectrometric characterization of proteins previously purified in polyacrylamide gels starting at low picomole levels.

An agarose-based concentration gel system is described for eluting and concentrating proteins previously purified either in one-dimensional or two-dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 microliters. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in-solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on-line electrospray mass spectrometry, allowing their identification by peptide mass fingerprinting

Protective effect of Water bug saliva against M. ulcerans lesion development

International audienceAt this day, no specific vaccine against Buruli ulcer is available. BCG vaccination has first been suggested to have incomplete but significant short-term protective effects. Subsequent studies have provided highly controversial results with some showing partial protection against M. ulcerans osteomyelitis. The only study that did not solely rely on BCG scar for assessing BCG vaccination status (a procedure that has been shown to be unreliable when BCG is given under the age of three months which is the case is most African countries), but used vaccine booklet examination, did not find any significant protective effect of routine BCG vaccination against Buruli ulcer. Vaccination based on mycobacterial antigens (Hsp65, Ag85A) showed a short delay in lesion development and borderline impact on bacterial loads. These results demonstrated that the most efficient vaccine for Buruli ulcer does not solely rely on the Mycobacterium alone but is a combination of bacilli- and vector-based molecules as already shown for other microbial agents such as Borrelia and Leishmania. Recently, we have demonstrated that repeated water bug bites free of M. ulcerans could protect against M. ulceransinduced lesions.&nbsp; To test whether vector salivary proteins can protect against M. ulcerans lesion, a mouse model was developed involving intradermal inoculation in the tail of 1000 bacilli together with Water bug salivary gland homogenate to mimic a potential natural transmission. Before M. ulcerans inoculation, mice were immunized by immunogenic salivary proteins purified by affinity chromatography. &nbsp;We showed that salivary proteins confer protection against the M. ulcerans lesion development in mouse model. Moreover, two candidate proteins (isolated form salivary gland homogenate) that are able to bound M. ulcerans surface, were identified and detected in saliva fluid. These proteins were detected in several water bugs families.&nbsp; This work reinforces the concept of using components of arthropod saliva in vaccine strategies against M. ulcerans lesions.</p