Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation. METHODS: Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates. RESULTS: All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high M(r )proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at M(r )~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1. CONCLUSION: One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1

Pollen proteolytic enzymes degrade tight junctions

Background and objective: Asthma and allergic rhinitis are significant, increasing causes of morbidity worldwide. Pollen, a major cause of seasonal rhinitis/conjunctivitis, carries proteolytic enzymes on its surface. We showed previously that peptidase allergens from house dust mites compromise epithelial barrier function by degrading the extracellular domains of the tight junction proteins, occludin and claudin, thus facilitating allergen delivery across epithelial layers. In this study, we aimed to determine whether peptidases from allergenic pollens should similarly be considered to have a role in disrupting tight junctions.\ud \ud Methods: Diffusates from stored pollen of Giant Ragweed, White Birch and Kentucky Blue Grass, and fresh pollen from Easter Lily were applied to confluent monolayers of Madin-Darby canine kidney (MDCK) and Calu-3 cells in serum-free medium. Immunofluorescence was performed for the tight junction proteins, occludin, claudin-1 and ZO-1. The effect of pollen diffusate on occludin was studied by Western blotting, and enzymatic activity in the diffusates was demonstrated by zymography. The ability of protease inhibitors to block the action of the diffusate on tight junctions was investigated.\ud \ud Results: Diffusates from all four allergenic pollens caused loss of immunofluorescence labelling for tight junction proteins on MDCK and Calu-3 cells. The effect was blocked by inhibitors of serine and cysteine proteases. Degradation of occludin was demonstrated by Western blotting and zymography indicated that diffusates contain proteolytic activity.\ud \ud Conclusions: Pollen peptidases directly or indirectly disrupt epithelial tight junctions, and this activity should be considered as a possible mechanism for facilitating allergen delivery across epithelia.\u