Identification of Critical Amino Acids in an Immunodominant IgE Epitope of Pen c 13, a Major Allergen from Penicillium citrinum

Background: Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Methodology/Principal Findings: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A 148 –E 166) and S22 (A 243 –K 274) were recognized by sera from 90 % and 100 % of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T 261 –K 274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity

Proteome Mining for Novel Ige-Binding Proteins from the German Cockroach ( Blattella Germanica) and Allergen Profiling of Patients

Although cockroaches are known to produce allergens that can cause IgE- mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N- terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE- binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE -binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders

Part I: Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients. Part II: Identification and analysis of the allergenic determinants on the allergen Bla g 9: epitope mapping by human IgE.

第一部份： 蟑螂，目前已知可引起許多由人類免疫球蛋白E（IgE）所媒介的過敏性反應，包含有過敏性鼻炎、氣喘…等，但至目前為止，並非所有的致敏原都己被研究透澈，許多來自蟑螂的蛋白，都顯現出IgE結合活性，但僅有少部分有被進一歩分析與研究。為了釐清這一類的蟑螂過敏原，本研究中利用了二維電泳技術並配合N端定序、質譜儀等蛋白質學的方式進行分析。再進一步結合了生物資訊學中過敏原資料庫的運算，來輔助並找尋具潛力的新德國蟑螂過敏原。利用上述的方式，本篇研究中，共鑑定出10種來自德國蟑螂新的IgE結合蛋白而其中aldolase、 arginine kinase、enolase、Hsp70、TIM及vitellogenin蛋白都已在其它可導致過敏的物種，例如：塵蟎、蝦、植物花粉或真菌中，曾被鑑定為該物種的過敏蛋白。但都未曾在德國蟑螂上有過相類似的報告。另一方面，更進一步利用FARRp過敏源資料庫分析的結果也指出：arginine kinase、enolase及TIM這三個來自德國蟑螂的IgE結合蛋白，個別在各個不同致敏物種間的同源致敏蛋白，均存在著相當高的可能性是具有免疫交互作用。在本篇研究中，同時以人體皮膚試驗方式，驗証了vitellogenin蛋白在人體上，確實能引起典型的過敏反應。進一步分析個人化過敏蛋白IgE結合圖譜時也發現：在不同過敏病人上存在著個人化獨特的結合圖譜：不只在人體IgE與測試蛋白間結合的頻度，另外在結合強度上也存在著個人化的差異。這些發現，除了提供更多的蟑螂過敏病研究材料也同時提供了一個可能性，為將來對此類過敏疾病進行個人化醫療或臨床診斷提供更正確的資訊。 第二部份： 德國蟑螂是一種常見的室內吸入性過敏原，並被認為與過敏性氣喘疾病有關。由於其重要性，故常可見於現行過敏血清醫療檢驗項目中。近幾年來，由於分子生物學技術的進步，有許多的過敏原蛋白都已被基因選殖，並提供良好的平台以進行過敏原特性的分析，例如過敏原決定點的分析。本篇的主角，Bla g 9過敏原蛋白，是由德國蟑螂上，藉由蛋白質體學配合免疫學方式，新發現的過敏蛋白。並已完成基因解序及重組表現。利用陰離子交換管柱，我們也成功由德國蟑螂蟲體中，順利純化到均質化的德國蟑螂Bla g 9蛋白。由於Bla g 9蛋白在蛋白序列分析時，被認為是屬於精胺酸激酶酵素家族的一員，經活性鑑定，純化得到的Bla g 9，也的確具有精胺酸激酶的酵素活性。在後續以蟑螂過敏病人血清所進行的ELISA篩檢試驗中，也再次證實了Bla g 9確實具有結合病人血清中IgE抗體的活性，並且具有約五成的盛行率。 利用純化的美洲蟑螂精胺酸激酶過敏原（Per a 9）對Bla g 9所做的免疫抑制實驗證實了，精胺酸激酶是美洲及德國蟑螂中共通的過敏蛋白，並很有可能具有共通的過敏原決定點。為了找尋在Bla g 9上可能的過敏原決定點，我們利用了分子生物學的方式，成功將Bla g 9蛋白利用大腸桿菌分段表現，並檢測了各片段對於人類IgE抗體的結合活性。在後續研究中也發現，當Bla g 9 胜肽片段不包含氨基酸序列280-300時，對IgE抗體結合的能力會受到明顯的影響。但在利用合成胜肽分別合成兩段，包含280-295及290-305的序列時，卻無法保有IgE的結合活性。這結果暗示了在Bla g 9過敏蛋白上重要的過敏原決定點可能是屬於構型性的過敏原決定點。Part I: Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach (B. germanica) show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins was separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase (TIM), and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the FARRp allergen database indicated that arginine kinase, enolase, and TIM showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders. Part II: The German cockroach （B. germanica） is a well-known indoor aeroallergen, considered as one the causative agents of extrinsic bronchial asthma and frequently included in serum test panels for allergic diagnosis. Molecular characterization of allergens by recombinant DNA technology progressed rapidly during the last few years. The Bla g 9 protein, a novel allergen from B. germanica was identified by two-dimensional immunoblotting followed by the molecular cloning, and expression of this allergen as a 6 × His-tagged fusion protein in Escherichia coli. A purified Bla g 9 was obtained by anion exchange chromatography and had arginine kinase activity. It reacted with serum IgE from cockroach-allergic patients and shown high prevalence recognition. In ELISA inhibition assay, we observed that P. americana arginine kinase （Per a 9） inhibited the IgE-binding to B. germanica arginine kinase （Bla g 9）, which indicated the same IgE epitope between arginine kinase from different cockroach species. In order to identify the allergenic determinants of Bla g 9, recombinant （r）Bla g 9 and fragments were also generated, and IgE-binding epitopes were assessed by ELISA. Recombinant fragmented Bla g 9 proteins not containing 280-300 amino acid residues were significantly decrease the reactivity to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 9, the pooled serum from 10 cockroach-sensitized patients did not show IgE reactivity to two synthetic peptides consisting of 15 residues covering 280-305 amino acids. These results suggest the possibility that Bla g 9 may have a conformational epitope in the C-terminal region

VCP Phosphorylation-Dependent Interaction Partners Prevent Apoptosis in <em>Helicobacter pylori</em>-Infected Gastric Epithelial Cells

<div><p>Previous studies have demonstrated that valosin-containing protein (VCP) is associated with <em>H. pylori</em>-induced gastric carcinogenesis. By identifying the interactome of VCP overexpressed in AGS cells using a subtractive proteomics approach, we aimed to characterize the cellular responses mediated by VCP and its functional roles in <em>H. pylori</em>-associated gastric cancer. VCP immunoprecipitations followed by proteomic analysis identified 288 putative interacting proteins, 18 VCP-binding proteins belonged to the PI3K/Akt signaling pathway. <em>H. pylori</em> infection increased the interaction between Akt and VCP, Akt-dependent phosphorylation of VCP, levels of ubiquitinated proteins, and aggresome formation in AGS cells. Furthermore, phosphorylated VCP co-localized with the aggresome, bound ubiquitinated proteins, and increased the degradation of cellular regulators to protect <em>H. pylori-</em>infected AGS cells from apoptosis. Our study demonstrates that VCP phosphorylation following <em>H. pylori</em> infection promotes both gastric epithelial cell survival, mediated by the PI3K/Akt pathway, and the degradation of cellular regulators. These findings provide novel insights into the mechanisms of <em>H. pylori</em> infection induced gastric carcinogenesis.</p> </div

Molecular models and the immunodominant IgE-binding epitope of Pen c 13.

<p>A, Surface and ribbon diagrams of the Pen c 13 model. The different strengths of IgE-binding epitopes are colored green (weak), yellow (intermediate), and red (strong). Different levels of IgE-binding intensity are defined as: weak (10–20%), intermediate (50–70%), and strong (90–100%). B, Stick diagram of Pen c 13 residues 269–274.</p

IgE-reactivity of human sera against rPen c 13.

<p>The binding of rPen c 13 to serum IgE from 80 patients allergic to molds and 30 nonatopic subjects was tested by ELISA. Each data point represents an individual patient or healthy control. The horizontal dotted line shows the cutoff of the reaction, defined as two times the mean value of the healthy control group. Percentage values represent the overall frequency of positive responders among mold-allergic patients. The mean values are indicated by horizontal bars. Significance for unpaired comparisons of mean values between two groups was calculated using a two-tailed Mann-Whitney <i>U</i> test. The significance level was specified as α=0.05. *<i>P</i><0.0001.</p

Ingenuity Pathway Analysis (IPA) prediction and analysis of signal transduction networks for the 288 proteins in the VCP interactome in AGS cells.

<p><i>(A)</i> AGS cells overexpressing VCP-Flag or mock infected were pretreated 10 µM MG-132 for 3 hr and co-cultured with <i>H. pylori</i> for 6 hr, then immunoprecipitation was performed using anti-Flag antibody affinity gel and the precipitated proteins examined on silver-stained SDS gels (top panel) and immunoblots using anti-Flag antibodies (bottom panel). The lines on the right of the gel indicate where the gel was cut and the sections digested with trypsin and the peptides analyzed by mass spectrometry analysis; the identified VCP-binding proteins are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055724#pone.0055724.s005" target="_blank">Table S2</a>. <i>(B)</i> Proportionately drawn Venn diagram analysis of the total proteins in Mock and VCP co-immunoprecipitates. <i>(C)</i> The networks were generated by the shortest path algorithm of the Ingenuity Pathway Analysis (IPA) software using the list of 288 proteins in VCP co-immunoprecipitates identified by LC-MS/MS analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055724#pone.0055724.s005" target="_blank">Table S2</a>). The lines connecting molecules indicate molecular relationships.</p

Schematic model of the hypothetical mechanism for VCP anti-apoptotic pathways in gastric epithelial cells infected with <i>H. pylori</i>.

<p>The solid arrows indicate pathways that have been previously demonstrated, while the dashed arrows indicate hypothetical links.</p

Characterization of sera from patients with positive IgE binding to rPen c 13.

<p>AD, atopic dermatitis; AR, atopic rhinitis; AS, asthma; UR, urticaria.</p

Top 10 list of canonical pathways activated by VCP-interacting proteins from Ingenuity Pathway Analysis.

<p>Top 10 list of canonical pathways activated by VCP-interacting proteins from Ingenuity Pathway Analysis.</p