A novel IEF peptide fractionation method reveals a detailed profile of N-terminal Acetylation in chemotherapy-responsive and -resistant ovarian cancer cells

Although acetylation is regarded as a common protein modification, a detailed proteome wide profile of this posttranslational modification may reveal important biological insight regarding differential acetylation of individual proteins. Here we optimised a novel peptide IEF fractionation method for use prior to LC-MS/MS analysis in order to obtain a more in depth coverage of N-terminally acetylated proteins from complex samples. Application of the method to the analysis of the serous ovarian cancer cell line OVCAR-5 identified 341 N-terminally acetylated proteins, 23 of which are previously un-reported. The protein peptidyl-prolyl cis-trans isomerase A (PPIA) was detected in both the N-terminally acetylated and un-modified forms, and was further analysed by data independent acquisition in Carboplatin responsive parental OVCAR-5 cells and Carboplatin resistant OVCAR-5 cells. This revealed a higher ratio of un-acetylated to acetylated N-terminal PPIA in the parental compared to the Carboplatin resistant OVCAR-5 cells, and a 4.1-fold increase in PPIA abundance overall in the parental cells relative to Carboplatin-resistant OVCAR-5 cells (P = 0.015). In summary, the novel IEF peptide fractionation method presented here is robust, reproducible, and can be applied to the profiling of N-terminally acetylated proteins. All mass spectrometry data is available as a ProteomeXchange repository (PXD003547).Florian Weiland, Georgia Arentz, Manuela Klingler-Hoffmann, Peter McCarthy, Noor A. Lokman, Gurjeet Kaur, Martin K. Oehler, and Peter Hoffman

Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels

Extent: 13p.Introduction: Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker. Methods: Protein from laser microdissected tumor and normal mucosa was analysed by two dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry to determine differentially over expressed tumor proteins. Tumor over-expression of one such protein, desmin, was quantified using immunofluorescence staining in a larger cohort. Dual staining for desmin and vimentin, or desmin and von Willebrand factor, was performed to determine the cell type of interest. Results: Desmin expression was significantly increased between stage I and III tumors, (P < 0.0001), and stage II and III tumors, (P < 0.0001). Strong focal desmin expression was found in stroma directly adjacent to carcinomatous glands and microvessels. These cells showed co-localisation of desmin and vimentin in close association with cells expressing VWF, indicating they were pericytes. Significantly higher levels of desmin-positive pericytes were observed in late stage tumors, consistent with increased angiogenesis. Conclusion: Pericyte coverage of vasculature is a marker of vessel maturation, hence desmin expression may have use as a marker for microvessel maturation. Clinical trials will be needed to determine its use in identifying tumors that will be less responsive to anti-angiogenic therapy.Georgia Arentz, Tim Chataway, Timothy J Price, Zaipul Izwan, Gemma Hardi, Adrian G Cummins and Jennifer E Hardingha

Increased phospho-keratin 8 isoforms in colorectal tumors associated with EGFR pathway activation and reduced apoptosis

Extent: 8p.Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (P < 0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.Georgia Arentz, Tim Chataway, Mark R. Condina, Timothy J. Price, Peter Hoffmann, and Jennifer E. Hardingha

MALDI mass spectrometry imaging reveals decreased CK5 levels in vulvar squamous cell carcinomas compared to the precursor lesion differentiated vulvar intraepithelial neoplasia

Published: 8 July 2016Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC.Chao Zhang, Georgia Arentz, Lyron Winderbaum, Noor A. Lokman, Manuela Klingler-Hoffmann, Parul Mittal, Christopher Carter, Martin K. Oehler and Peter Hoffman

Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules

Celiac disease is caused by an abnormal intestinal T-cell response to gluten proteins of wheat, barley and rye. Over the last few years, a number of gluten T-cell epitopes restricted by celiac disease associated HLA-DQ molecules have been characterized. In this work, we give an overview of these epitopes and suggest a comprehensive, new nomenclature

To respond or not to respond - a personal perspective of intestinal tolerance

For many years, the intestine was one of the poor relations of the immunology world, being a realm inhabited mostly by specialists and those interested in unusual phenomena. However, this has changed dramatically in recent years with the realization of how important the microbiota is in shaping immune function throughout the body, and almost every major immunology institution now includes the intestine as an area of interest. One of the most important aspects of the intestinal immune system is how it discriminates carefully between harmless and harmful antigens, in particular, its ability to generate active tolerance to materials such as commensal bacteria and food proteins. This phenomenon has been recognized for more than 100 years, and it is essential for preventing inflammatory disease in the intestine, but its basis remains enigmatic. Here, I discuss the progress that has been made in understanding oral tolerance during my 40 years in the field and highlight the topics that will be the focus of future research

Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide

13 pages, 8 figures.-- PMID: 18509534 [PubMed].-- PMCID: PMC2386552.[Background and Aims] Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.[Methods] Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.[Results] The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.[Conclusions] The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.This work was supported by the Asociación de Celiacos de Madrid (to Carolina Sousa), by the CTA (Corporación Tecnológica de Andalucía) and IDEA (Agencia de Innovación y Desarrollo de Andalucía) (to Angel Cebolla) and by grants BFU2007-64999 from Plan Nacional de Investigación científica, Desarrollo e Innovación tecnológica (Miniterio de Educación y Ciencia) and RICET-RD06/0021-0014, Spain (to Manuel C. López). Belén Morón was supported by a fellowship from Consejo Andaluz de Colegios Oficiales de Farmacéuticos.Peer reviewe

Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease

Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of ‘low CD toxic’ as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD

A catalogue of Triticum monococcum genes encoding toxic and immunogenic peptides for celiac disease patients

The celiac disease (CD) is an inflammatory condition characterized by injury to the lining of the small-intestine on exposure to the gluten of wheat, barley and rye. The involvement of gluten in the CD syndrome has been studied in detail in bread wheat, where a set of “toxic” and “immunogenic” peptides has been defined. For wheat diploid species, information on CD epitopes is poor. In the present paper, we have adopted a genomic approach in order to understand the potential CD danger represented by storage proteins in diploid wheat and sequenced a sufficiently large number of cDNA clones related to storage protein genes of Triticum monococcum. Four bona fide toxic peptides and 13 immunogenic peptides were found. All the classes of storage proteins were shown to contain harmful sequences. The major conclusion is that einkorn has the full potential to induce the CD syndrome, as already evident for polyploid wheats. In addition, a complete overview of the storage protein gene arsenal in T. monococcum is provided, including a full-length HMW x-type sequence and two partial HMW y-type sequences

Celiac disease: how complicated can it get?

In the small intestine of celiac disease patients, dietary wheat gluten and similar proteins in barley and rye trigger an inflammatory response. While strict adherence to a gluten-free diet induces full recovery in most patients, a small percentage of patients fail to recover. In a subset of these refractory celiac disease patients, an (aberrant) oligoclonal intraepithelial lymphocyte population develops into overt lymphoma. Celiac disease is strongly associated with HLA-DQ2 and/or HLA-DQ8, as both genotypes predispose for disease development. This association can be explained by the fact that gluten peptides can be presented in HLA-DQ2 and HLA-DQ8 molecules on antigen presenting cells. Gluten-specific CD4+ T cells in the lamina propria respond to these peptides, and this likely enhances cytotoxicity of intraepithelial lymphocytes against the intestinal epithelium. We propose a threshold model for the development of celiac disease, in which the efficiency of gluten presentation to CD4+ T cells determines the likelihood of developing celiac disease and its complications. Key factors that influence the efficiency of gluten presentation include: (1) the level of gluten intake, (2) the enzyme tissue transglutaminase 2 which modifies gluten into high affinity binding peptides for HLA-DQ2 and HLA-DQ8, (3) the HLA-DQ type, as HLA-DQ2 binds a wider range of gluten peptides than HLA-DQ8, (4) the gene dose of HLA-DQ2 and HLA-DQ8, and finally,(5) additional genetic polymorphisms that may influence T cell reactivity. This threshold model might also help to understand the development of refractory celiac disease and lymphoma