Biophysical Studies of the Type 1 Repeats of Human Thrombospondin-1 to Characterize the Structural Basis of its Angiostatic Effect

Thrombospondin-1 (TSP1) is a disulfide bonded trimer of 450kD; each monomer contains three type 1 repeats (T1Rs). TSP1 and fragments that include the T 1Rs have several documented functions including a role as an inhibitor of endothelial cell growth and migration, as well as an inducer of apoptosis. The purpose of this study was to express, purify, and biophysically characterize the T1Rs. Recombinant baculoviruses were generated that express the three T1Rs in tandem (P123) and the third T1R (P3) as secreted histidine-tagged fusion proteins. Protein purity, molecular mass determination, and di sulfide-bond content was determined using mass spectroscopy. Micro-heterogenity arising from differential glycosylation was determined by western blotting, HPLC, and mass spectroscopy. Circular dichroism was used to access secondary and tertiary structure as well as thermal stability. Fluorescence spectroscopy revealed that the conserved tryptophans were quenched and in a partial polar environment in the native state. The effect of TSP1 and the T1Rs on endothelial cell (EC) morphology and death was determined. The results indicate the T1R encodes an independently folding protein module with spectral properties dominated by the conserved tryptophans. The protein is heterogeneously glycosylated with C-linked and 0-linked sugars. The T1Rs are not responsible for the apoptotic effect on Ecs.