A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)–specific monoclonal antibody (mAb), the Escherichia coli–expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli–expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient–derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. Key points • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells

Development and application of a novel solid-phase assay for the study of the enzymatic degradation of carrier-fixed peptides

Proteasome sind zelluläre Proteasen, involviert in den Abbau von einer Vielzahl zellulärer Proteine. Das 20S Proteasom ist ein zylindrischer 28-mer Protein-Komplex, welcher aus zwei außenständigen sieben-Ringen (α-Ringe; Substrateingang) und zwei innenliegenden sieben-Ringen (β-Ringe; katalytisches Zentrum) besteht. Zahlreiche Studien haben den Beweis erbracht, dass das 20S Proteasom Peptide in verschiedenen Längen sowie komplette denaturierte Polypeptidketten abbaut. Jedoch konnte bis jetzt nicht der Beweis erbracht werden, dass das 20S Proteasom fähig ist, ein oberflächengebundenes, immobilisiertes Peptid zu prozessieren. Im Rahmen dieser Arbeit wurde eine Methode entwickelt, die erstmalig den proteasomalen Verdau trägerfixierter Peptide über den massenspektrometrischen Nachweis in Lösung gegangener Peptidfragmente charakterisiert. Initial wurden unterschiedliche Träger (Gold, Silizium und glass-beads) getestet und neuartige Funktionalisierungsmethoden für eine optimale Peptidanbindung etabliert. Hierbei wurde jede Funktionalisierungsstufe mittels verschiedener optischer und elektrochemischer Verfahren charakterisiert. Schließlich wurde der proteolytische Verdau der kovalent fixierten Peptide mittels LC-MS/MS analysiert und kartographiert. Während der Methodenentwicklung zeigte sich, dass die glass-beads aufgrund der signifikant geringer einzusetzenden Peptidmenge und der resultierenden absoluten Peptidstoffmenge am besten geeignet sind. Mit diesem optimierten Versuchsaufbau konnte erstmalig das Abbauverhalten verschiedener fixierter Peptide durch das Proteasom mittels nanoLC-MS/MS nachgewiesen werden. Zum ersten Mal konnte gezeigt werden, dass das Proteasom N-Terminal fixierte Substrate mit einer erheblich beschleunigten Abbaurate prozessieren kann im Vergleich zum gelösten Substrat. Des Weiteren konnte ein verändertes Schnittmuster im Vergleich zum gelösten Substrat beobachtet werden, das jedoch teilweise mit dem bekannten, in Lösung verdauten Substrats, übereinstimmt. Zusammenfassend weisen die Ergebnisse darauf hin, dass auch die in den Lösungsraum orientierten Termini membranständiger Proteine ubiquitin- unabhängig durch das 20S Proteasom degradiert werden könnten. Die von uns etablierte Methode legt außerdem den Grundstein für die Entwicklung von semi- automatischen Verfahren für die schnelle Identifizierung von Abbauprodukten oder anderen enzymatischen Prozessen.Proteasomes are cellular proteases involved in the degradation of numerous cellular proteins. The 20S proteasome is a cylindrical 28-mer protein complex composed of two outer heptameric α-rings forming the entrance for the protein substrate and two inner heptameric β-rings carrying the catalytic sites. Numerous in vitro studies have provided evidence that the 20S proteasome may degrade peptides of various lengths and even unfolded full-length polypeptide chains. However, a direct demonstration that the 20S proteasome may also cleave surface-attached immobilized peptides is lacking so far. In the present work a method was developed which characterized the proteasomale digestion of solid-phase bound peptides by peptide fragments in the aqueous phase. Initially different surfaces (gold, silicon and glass-beads) were selected and novel functionalization methods for an optimal peptide fixation were established. During this procedure, every functionalization step was characterized by different optical and electrochemistry techniques. Finally, the protolytical fragments of the proteasome digestion of the covalent fixed peptides were analyzed by LC-MS/MS and map. Based on the preliminary results glass-beads showed to be the eligible candidate for the further experiments due to the significant fewer amounts of peptides required for the surface fixation and yield of coupled peptides. With this optimized setup we were for the first time able to demonstrate by means of nanoLC-MS/MS the proteasomal digestion of different surface fixed peptides. Our results clearly demonstrated that the proteasome is able to degrade N-terminal fixed peptides with a much higher degradation rate as compared to the soluble substrate. Furthermore, a modified cleavage pattern could be observed between surface- fixed or in solution proteasomale digestion. However, some digestion products are detectable in both approaches. In summary, this finding supports our initial hypothesis that proteasomes may be able to directly degrade segments of membranebound proteins protruding into the aqueous phase. Furthermore, this study lays the foundation for the development of semi-automatic methods to identify very rapidly proteasomale digestion products

A new strategy for the preparation of maleimide-functionalised gold surfaces

We have developed and investigated a new route to functionalise Au surfaces using maleimide groups. This functionalisation has been performed by grafting aminophenyl (AP) via an electrochemical reduction of 4-aminophenyldiazonium salt in acetonitrile solution and the subsequent chemical binding of N-(2-carboxyethyl) maleimide (NCEM). The resulting maleimide functionalised surface was interacted with a cysteine-modified peptide. The grafting of AP was monitored by the occurrence of NH2 and aryl ring vibrations, whereas the binding of the NCEM led to a strong and sharp peak because of the C═O stretching mode. The immobilisation of the peptide was identified by the appearance of the amide I band. Half of the maleimide surface groups reacted with the peptide because of steric hindrance. The charge efficiency for the AP layer formation was about 45% at a thickness of about 6–8 nm. Keywords: Functionalisation, Electrochemical grafting, Aniline, Aminophenyl, Maleimide, IR spectroscop