Song-Nan Su

Mold Allergen, Pen c 13, Induces IL-8 Expression in Human Airway Epithelial Cells by Activating Protease-Activated Receptor 1 and 2

Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca2+ mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca2+-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca2+-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium.

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.

Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin‐binding activities, and the Lysyl‐C endopeptidase digested peptides were determined by N‐terminal sequencing. The cDNA sequence was analyzed by RACE PCR‐based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI‐TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18 411 Da by MALDI‐TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen‐related protein family. Carbohydrate analysis showed that the most abundant N‐linked glycan is a α(3)‐fucosylated pauci‐mannose (Man3GlcNAc2) structure, without a Xyl β‐(1,2)‐linked to the branching β‐Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis‐related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis‐related protein 1.

Purification and characterization of a novel isoallergen of a major Bermuda grass pollen allergen, Cyn d 1

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.