The dorsoventral axis of the Drosophila embryo is established by ventral activation of the Toll signaling pathway, resulting in the graded nuclear uptake of the Dorsal transcription factor. Pathway activation relies on a ventral cue synthesized during oogenesis through the action of Pipe, a putative heparan sulfate 2-0- sulfotransferase, on an unknown carbohydrate substrate. In the embryo, the ligand for the receptor Toll is generated by a proteolytic processing reaction involving 4 members of a serine protease cascade, Nudel, Gastrulation Defective (GD), Snake and Easter. Published work has shown that early cascade events occur independently of pipe activity and are not ventrally restricted, while new data shows that activation of the Easter protease requires pipe and is probably ventrally restricted. These observations establish that spatial control occurs within the protease cascade. To investigate the mechanism by which pipe regulates the cascade, three aims are proposed to test the hypothesis that glycosaminoglycans (GAGs) modified by Pipe serve as cofactors promoting the ventral activity of GD and/or Snake. These include: (1) determining where spatial regulation occurs in the protease cascade in vivo, by Western blotting and localization of active proteases; (2) characterizing the functional consequences of GAG interactions with GD and Snake in vitro using purified proteases, and determining whether the specificity of these interactions is consistent with that expected for Pipe-modified GAGs; and (3) identifying GAGs and proteoglycans involved in protease regulation by pipe in vivo, using novel selection strategies to isolate species structurally altered in the absence of pipe. This work is relevant to understanding the regulation of protease cascades, such as those involved in the innate immune response, hemostasis, wound healing, and synaptic plasticity. Additionally, knowledge of how glycosaminoglycan microheterogeneity contributes to patterning is important to understanding the many emerging roles of heparan sulfate proteoglycans in vertebrate and invertebrate development, where, for example, a knock-out of a mouse gene related to pipe results in renal agenesis and skeletal and eye defects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067738-05
Application #
7484258
Study Section
Pathobiochemistry Study Section (PBC)
Program Officer
Haynes, Susan R
Project Start
2004-04-01
Project End
2012-08-31
Budget Start
2008-09-01
Budget End
2012-08-31
Support Year
5
Fiscal Year
2008
Total Cost
$244,062
Indirect Cost
Name
Georgia Regents University
Department
Biology
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
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Sukumari-Ramesh, Sangeetha; LeMosy, Ellen K (2009) Gastrulation defective protease interacts with anionic components of the Drosophila ovary extracellular matrix. Protein Pept Lett 16:437-43
Park, Youmie; Zhang, Zhenqing; Linhardt, Robert J et al. (2008) Distinct heparan sulfate compositions in wild-type and pipe-mutant eggshell matrix. Fly (Austin) 2:175-9
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Dinkins, Michael B; Fratto, Victoria M; Lemosy, Ellen K (2008) Integrin alpha chains exhibit distinct temporal and spatial localization patterns in epithelial cells of the Drosophila ovary. Dev Dyn 237:3927-39
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