Next-Generation Phage Display to Identify Peptide Ligands of Deubiquitinases.

Phage display (PD) is a powerful method and has been extensively used to generate monoclonal antibodies and identify epitopes, mimotopes, and protein interactions. More recently, the combination of next-generation sequencing (NGS) with PD (NGPD) has revolutionized the capabilities of the method by creating large data sets of sequences from affinity selection-based approaches (biopanning) otherwise challenging to obtain. NGPD can monitor motif enrichment, allow tracking of the selection process over consecutive rounds, and highlight unspecific binders. To tackle the wealth of data obtained, bioinformatics tools have been developed that allow for identifying specific binding sequences (binders) that can then be validated. Here, we provide a detailed account of the use of NGPD experiments to identify ubiquitin-specific protease peptide ligands.

A Simple Whole-Plasmid PCR Method to Construct High-Diversity Synthetic Phage Display Libraries.

Phage display technology utilises peptide and antibody libraries with very high diversities to select ligands with specific binding properties. The production of such libraries can be labour intensive and technically challenging and whilst there are commercial sources of libraries, the exploitation of the resulting binders is constrained by ownership of the libraries. Here, a peptide library of ~ 1 × 109 variants for display on gene VIII was produced alongside three VHH antibody libraries with similar diversity, where 12mer, 16mer or 21mer CDR3s were introduced into the highly stable cAbBCII10 scaffold displayed on gene III. The cloning strategy used a simple whole-plasmid PCR method and type IIS restriction enzyme assembly that facilitate the seamless insertion of diversity into any suitable phage coat protein or antibody scaffold. This method reproducibly produced 1 × 109 variants from just 10 transformations and the four libraries had relatively low bias with 82 to 86% of all sequences present as single copies. The functionality of both peptide and antibody libraries were demonstrated by selection of ligands with specific binding properties by biopanning. The peptide library was used to epitope map a monoclonal antibody. The VHH libraries were pooled and used to select an antibody to recombinant human collagen type 1.

Discovery of peptide ligands targeting a specific ubiquitin-like domain-binding site in the deubiquitinase USP11.

Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 µm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro Furthermore, a crystal structure of a USP11-peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket-deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination-mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

A Trematode Parasite Derived Growth Factor Binds and Exerts Influences on Host Immune Functions via Host Cytokine Receptor Complexes.

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allowing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-ß RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-ß RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory receptor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is dependent on TGF-ß RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody-dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages-again dependent on TGF-ß RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for dampened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targeting juvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection.

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells.

BACKGROUND: Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells. METHODS: We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins. RESULTS: Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling. CONCLUSION: Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines.

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections.