Biophysical Studies of the Type 1 Repeats of Human Thrormbospondin-1 to Characterize the Structural Basis of Its Angiostatic Effect.
Annual rept. 1 Aug 96-31 Jul 97
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Thrombospondin-1 TSP1 is a modular trimeric protein wit several documented functions, including its role as an angiogenic inhibitor. TSP1, TSP1 fragments, and certain TSP1 conserved peptide sequences have been shown to exert an endothelial-specific inhibition of growth and migration. Peptides derived from conserved sequences within the type 1 repeats of TSP1 have been shown to block neo-vascularization in vivo, inhibit DNA synthesis, and migration of cultured endothelial cells in vitro. Our laboratory has shown that recombinantly expressed human TSP1 hTSP1 type 1 repeats inhibit migration of bFGF stimulated bovine adrenal microvascular endothelial cells. This study seeks to define the structural basis for the angiostatic effect of the hTSP1 type 1 repeats. I will employ biophysical methods in a comparative study of TSP 1 type 1 repeats and active peptides based on type 1 sequences. I have successfully generated recombinant baculoviruses that express the three type 1 repeats in tandem P123 and the third type 1 repeat P3 as histidine-tagged fusion proteins. A purification scheme for the recombinant proteins including removal of the histidine-tag has been established. To date, N-terminal sequencing, carbohydrate analysis, and circular dichroism have been performed.
- Anatomy and Physiology