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The neurotoxins produced by the various strains of the anaerobic bacterium Clostridium botulinum naturally occur associated with complexing proteins which serve to protect the neurotoxins from the harsh environment of the mammalian gastrointestinal tract during bacterial invasion of the host. Three different complex species with the discrete sizes 19S (900 kDa, LL complex), 16S (500 kDa, L complex) and 12S (300 kDa, M complex) may be isolated from C. botulinum type A cultures. However, to affect their target cells these complexes must dissociate releasing the free 150 kDa neurotoxin. This study assesses the stability of these Clostridium botulinum neurotoxin serotype A (BoNT/A) complexes and identifies factors which influence their dissociation. The knowledge gained with purified toxin complexes was subsequently employed to analyze the presence of such complexes in the freeze or spray-dried commercial BoNT/A products Botox and Dysport in comparison to the complexing protein free product Xeomin. Purified 900 kDa and 500 kDa toxin complex preparations show a pH and time dependent release of the 150 kDa neurotoxin with a half-life of less than a minute at pH values >7.0. At pH values of 6.25 or less, the complexes are stable. Furthermore, dilution of concentrated 900 kDa complexes leads to dissociation into 500 kDa, neurotoxin containing complexes. Addition of sodium chloride as contained in isotonic saline leads to further disruption of these complexes resulting in the release of the free 150 kDa neurotoxin. Examination of the commercial botulinum neurotoxin products Botox and Dysport using the same analytical procedures leads to the same conclusion: the dilution, drying and reconstitution processes of these products lead to a complete dissociation of 900 kDa complexes and 85% or more of neurotoxin are present in free form. Conclusion BoNT A toxin complexes have evolved to quickly respond to specific environmental changes by efficient release of the neurotoxin. During pharmaceutical production and reconstitution of BoNT A products, the same principles effect the quantitative dissociation of 900 kDa complexes and release of free neurotoxin prior to injection into target tissues.

An Expert Meeting on The Current Scientific and Legal Status of Alternative Methods to the LD50 Test for Botulinum Neurotoxin (BoNT) Potency Testing was organised by the Centre for Documentation and Evaluation of Alternatives to Animal Experiments (ZEBET) at the Federal Institute for Risk Assessment (BfR), and took place on 27-28 April 2009, in Berlin, Germany. The goal of ZEBET is to promote primarily the replacement of legally required animal experiments with valid alternative test methods, to reduce the numbers of test animals used to the absolutely necessary level, and to alleviate their pain and suffering. ZEBET's tasks are the documentation, assessment and promotion of alternative methods to animal experiments on a national and international level, and the initiation of research and development, including the funding of smaller research projects.

The hemagglutinin of influenza virus A/FPV/Rostock/34 (H7) was altered at its multibasic cleavage site by site-directed mutagenesis and assayed for proteolytic activation after expression in CV-1 cells. The results indicated that the cellular protease responsible for activation recognizes the tetrapeptide motif R-X-K/R-R that must be presented in the correct sequence position. Studies on plaque variants of influenza virus A/fowl/Victoria/75 (H7N7) showed that alteration of the consensus sequence resulted in a loss of pathogenicity for chickens.

Inhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatmentof infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R × K/R R. Uncleaved gB precusor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone.

The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.

We have cloned a bovine cDNA encoding the trans-Golgi network (TGN) protease furin and expressed it via recombinant vaccinia viruses to investigate intracellular maturation. Pulse-chase labeling reveals that the 104-kD pro-furin bearing high mannose N-glycans is rapidly processed into the 98-kD protease whose N-glycans remain sensitive to endoglycosidase H for a certain period of time. Furthermore, in the presence of brefeldin A, pro-furin cleavage occurs. From these data we conclude that the ER is the compartment of propeptide removal. Studies employing the ionophore A23187 and DTT show that autocatalysis is Ca2+ dependent and that it does not occur under reducing conditions. Pro-furin produced under these conditions never gains endo H resistance indicating that it is retained in the ER. Coexpression of furin with the fowl plague virus hemagglutinin in the presence of brefeldin A and monensin reveals that furin has to enter the Golgi region to gain substrate cleaving activity. N-glycans of furin are sialylated proving its transit through the trans-Golgi network. A truncated form of furin is found in supernatants of cells. Truncation is inhibited in the absence of Ca2+ ions and in the presence of acidotropic agents indicating that it takes place in an acidic compartment of cells. Comparative analysis with furin expressed from cDNA reveals that the truncated form prevails in preparations of biologically active, endogenous furin obtained from MDBK cells. This observation supports the concept that secretion of truncated furin is a physiological event that may have important implications for the processing of extracellular substrates.

PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays., PLoS ONE, 3 (12), ISSN:1932-6203

Summary Background The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrP Sc ), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrP Sc display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively. Methods We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrP Sc . Findings We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrP Sc -rich brain areas, with a typical PrP Sc type 1 migration pattern. Interpretation The regular coexistence of multiple PrP Sc types in patients with CJD casts doubts on the validity of electrophoretic PrP Sc mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications.

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt–Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP Sc ) used limited proteolysis to digest the precursor cellular PrP (PrP C ). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP Sc , we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP Sc in all 10–24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, ≈90% of the total PrP Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP C . Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.

Like many other viral glycoproteins, the hemagglutinin (HA) of influenza viruses is activated by proteolytic cleavage. Cleavage which is necessary for the fusion activity of the hemagglutinin and thus for the infectivity of the virus is exerted by host cell proteases, and the presence of an appropriate enzyme determines whether infectious virus is made in a given cell. Proteolytic activation is therefore indispensable for effective virus spread in the infected host and has been found to be a prime determinant for virus pathogenicity. This concept has been derived mainly from studies on avian influenza viruses. The pathogenic strains of these viruses are activated by ubiquitous proteases and cause therefore systemic infection mostly leading to rapid death of the animal, whereas activation of the apathogenic strains occurs only in epithelial cells of the respiratory or the enteric tract resulting in local infection of these organs. The mammalian influenza viruses, including the human ones, resemble the apathogenic avian strains in possessing also hemagglutinins of restricted cleavability and in causing usually local infection of the respiratory tract.1