Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 \u3b2-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called "domainome", will be well expressed and soluble, making them suitable for structural/functional studies. By cloning and displaying the "domainome" directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification. The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation

The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma

Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori-infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4(+) (13.9%) gastric clones from MALT lymphoma and three of 179 CD4(+) (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-gamma) and Interleukin-17 (IL-17) secretion by gastric CD4(+) T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas

InteractomeSeq: a web server for the identification and profiling of domains and epitopes from phage display and next generation sequencing data

High-Throughput Sequencing technologies are transforming many research fields, including the analysis of phage display libraries. The phage display technology coupled with deep sequencing was introduced more than a decade ago and holds the potential to circumvent the traditional laborious picking and testing of individual phage rescued clones. However, from a bioinformatics point of view, the analysis of this kind of data was always performed by adapting tools designed for other purposes, thus not considering the noise background typical of the 'interactome sequencing' approach and the heterogeneity of the data. InteractomeSeq is a web server allowing data analysis of protein domains ('domainome') or epitopes ('epitome') from either Eukaryotic or Prokaryotic genomic phage libraries generated and selected by following an Interactome sequencing approach. InteractomeSeq allows users to upload raw sequencing data and to obtain an accurate characterization of domainome/epitome profiles after setting the parameters required to tune the analysis. The release of this tool is relevant for the scientific and clinical community, because InteractomeSeq will fill an existing gap in the field of large-scale biomarkers profiling, reverse vaccinology, and structural/functional studies, thus contributing essential information for gene annotation or antigen identification. InteractomeSeq is freely available at https://InteractomeSeq.ba.itb.cnr.it/

Defining the Helicobacter pylori Disease-Specific Antigenic Repertoire

The analysis of the interaction between Helicobacter pylori (HP) and the host in vivo is an extremely informative way to enlighten the molecular mechanisms behind the persistency/latency of the bacterium as well as in the progression of the infection. An important source of information is represented by circulating antibodies targeting the bacteria that define a specific \u201cdisease signature\u201d with prospective diagnostic implications. The diagnosis of some of the HP induced diseases such as gastric cancer (GC), MALT lymphoma (MALT), and autoimmune gastritis (AIG) is not easy because patients do not show symptoms of illness in early-onset stages, at the same time they progress rapidly. The possibility of identifying markers able to provide an early diagnosis would be extremely beneficial since a late diagnosis results in a delay in undergoing active therapy and reduces the survival rate of patients. With the aim to identify the HP antigens recognized during the host immune-response to the infection and possibly disease progression, we applied a discovery-driven approach, that combines \u201cphage display\u201d and deep sequencing. The procedure is based on the selection of ORF phage libraries, specifically generated from the pathogen\u2019s genome, with sera antibodies from patients with different HP-related diseases. To this end two phage display libraries have been constructed starting from genomic DNA from the reference HP 26695 and the pathogenic HP B128 strains; libraries were filtered for ORFs by using an ORF selection vector developed by our group (Di Niro et al., 2005; Soluri et al., 2018), selected with antibodies from patients affected by GC, MALT, and AIG and putative HP antigens/epitopes were identified after Sequencing and ranking. The results show that individual selection significantly reduced the library diversity and comparison of individual ranks for each condition allowed us to highlight a pattern of putative antigens specific for the different pathological outcomes or common for all of them. Within the putative antigens enriched after selection, we have validated protein CagY/Cag7 by ELISA assay as a marker of HP infection and progression. Overall, we have defined HP antigenic repertoire and identified a panel of putative specific antigens/epitopes for three different HP infection pathological outcomes that could be validated in the next future

Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases

Osteopontin (OPN) regulates the immune response at multiple levels. Physiologically, it regulates the host response to infections by driving T helper (Th) polarization and acting on both innate and adaptive immunity; pathologically, it contributes to the development of immune-mediated and inflammatory diseases. In some cases, the mechanisms of these effects have been described, but many aspects of the OPN function remain elusive. This is in part ascribable to the fact that OPN is a complex molecule with several posttranslational modifications and it may act as either an immobilized protein of the extracellular matrix or a soluble cytokine or an intracytoplasmic molecule by binding to a wide variety of molecules including crystals of calcium phosphate, several cell surface receptors, and intracytoplasmic molecules. This review describes the OPN structure, isoforms, and functions and its role in regulating the crosstalk between innate and adaptive immunity in autoimmune diseases

Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells

The interaction between the enzyme transglutaminase 2 (TG2) and fibronectin (FN) is involved in the cell-matrix interactions that regulate cell signaling, adhesion, and migration and play central roles in pathologic conditions, particularly fibrosis and cancer. A precise definition of the exact interaction domains on both proteins could provide a tool to design novel molecules with potential therapeutic applications. Although specific residues involved in the interaction within TG2 have been analyzed, little is known regarding the TG2 binding site on FN. This site has been mapped to a large internal 45-kDa protein fragment coincident with the gelatin binding domain (GBD). With the goal of defining the minimal FN interacting domain for TG2, we produced several expression constructs encoding different portions or modules of the GBD and tested their binding and functional properties. The results demonstrate that the I8 module is necessary and sufficient for TG2-binding in vitro, but does not have functional effects on TG2-expressing cells. Modules I7 and I9 increase the strength of the binding and are required for cell adhesion. A 15-kDa fragment encompassing modules I7-9 behaves as the whole 45-kDa GBD and mediates signaling, adhesion, spreading, and migration of TG2+ cells. This study provides new insights into the mechanism for TG2 binding to FN.-Soluri, M. F., Boccafoschi, F., Cotella, D., Moro, L., Forestieri, G., Autiero, I., Cavallo, L., Oliva, R., Griffin, M., Wang, Z., Santoro, C., Sblattero, D. Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells

Thrombin Cleavage of Osteopontin Modulates Its Activities in Human Cells In Vitro and Mouse Experimental Autoimmune Encephalomyelitis In Vivo

Osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), by recruiting autoreactive T cells into the central nervous system. Osteopontin functions are modulated by thrombin cleavage generating N- and C-terminal fragment, whose individual roles are only partly known. Published data are difficult to compare since they have been obtained with heterogeneous approaches. Interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with α4β1 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. We produced recombinant osteopontin and its N- and C-terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. We investigated, in vitro, their effect on human cells and in vivo in EAE. We found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. These findings suggest that drugs targeting each fragment may be used to fine-tune the pathological effects of osteopontin in several diseases

The -346T polymorphism of the SH2D1A gene is a risk factor for development of autoimmunity/lymphoproliferation in males with defective Fas function

Inherited defects decreasing function of the Fas death receptor cause autoimmune lymphoproliferative syndrome (ALPS) and its variant Dianzani autoimmune lymphoproliferative disease (DALD). Since a deleterious mutation of the SH2D1A gene protects MRLIpr/Ipr mice from ALPS development, we investigated the role of SH2D1A, located in the X chromosome, in 51 patients with ALPS or DALD by mutational screening of coding and regulative sequences. Allelic frequency of the -346C>T polymorphism was different in male patients and controls (-346T: 61% vs 36%, p = 0.01), with similar frequencies in ALPS and DALD. By contrast, no differences were found among females or between the controls and patients with multiple sclerosis (229 males, 157 females). Further analyses showed that -346C was a methylation site in CD8(+) T and natural killer cells, and SH2D1A expression was higher in -346T than in -346C males. Finally, in vitro-activated T cells from -346T males produced lower amounts of interferon-gamma than those from -346C males. These data suggest that -346T is a predisposing factor for ALPS and DALD in males possibly because of its effect on SAP expression influencing the T-cell response. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved

Defective Fas function in the ALPS and DALD patients carrying the UNC13D variations.

<p>[A] Fas-induced cell death in T cells from the ALPS and DALD patients carrying the <i>UNC13D</i> variations. Activated T cells were treated with anti-Fas mAb and survival was assessed after 18 hours. The results are expressed as specific cell survival %. The dotted line indicates the upper limit of the normal range calculated as the 95^th percentile of data obtained from 200 healthy controls; two or more were run in each experiment as positive controls; each patient was evaluated at least twice with the same result. [B] Fas expression and caspase-8 activity in lysates of 293T cells transfected with the wild-type (WT) or mutated form of <i>FAS</i> (Pt.1: p.Gln273His, Pt.2: p.Glu261Lys); cells were lysed 24 hours after transfection. <i>Upper panels</i>: Western blot analysis of the transfected Fas performed using anti-FLAG and anti-β-actin antibodies<i>. Lower panels</i>: fluorimetric enzyme assay for caspase-8 activity. Data are relative to those displayed by mock-transfected cells and are expressed as the mean and SE of the results from 4 experiments performed in duplicate. *p<0.05; **p<0.01 vs. Fas^wt transfected cells.</p