(from the application): When grass pollen lands on the receptive surface of the stigma, it begins to hydrate and secrete various proteins. Among these is an antigenic protein known as the group-I grass pollenallergen. This protein is a major cause of hay fever and asthma, afflicting ca. 25 percentof the US population. We recently discovered that the group-I allergen from maize pollen (called Zea ml) loosens the extracellular matrix (wall) of the grass stigma. In this proposal, we outline several experimental approaches to elucidate the structure, function, and evolutionary distribution of the group-I allergens and related proteins, which we hypothesize aid pollen tube penetration of the stigma and style. RNA and protein analyses will be used to determine the temporal and spatial pattern of Zea ml expression and secretion. Using a reverse genetics approach we have identified four maize lines with transposon insertions in the Zea ml gene; these lines will be studied to learn the phenotypic consequences of Zea ml gene disruption. We will take advantage of the unusual physical properties of Zea ml to study how it acts to loosen the cell wall, still an enigmatic problem. By X-ray analysis of Zea ml crystals, we propose to solve its structure and to use this structure to investigate how the protein interacts with the polymers of the extracellular matrix. By binding assays we will define the substrates to which this protein binds. We will test these ideas by site-directed mutagenesis of recombinant protein. We will also attempt to identify peptides, monoclonal antibodies and oligosaccharides that interfere with Zea ml activity, to be used as inhibitors of the biological function of these proteins. To date, it appears that group-I grass pollen allergens are found only in grasses. To evaluate the evolutionary distribution of this protein, we will test pollen extracts from grasses, close relatives, and more distant plant groups by means of activity and immunoblot assays. Finally, we will leverage the insights gained from analysis of Zea ml to study the function of group-Il and group-Ill grass pollen allergens, which show significant sequence similarity with the carboxy terminus of group-I allergens.
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