Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells

Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombos pondin 1 has been implicated in antiangiogenic activity. Procollagen module s are found in a number of extracellular proteins and are identifiable by 1 0 cysteines with characteristic spacing. We expressed and studied the proco llagen module (C) of human TSP1, both by itself and in the context of the a djoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal sign al sequence and C-terminal poly-histidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatograph y. NoC is a disulfide bonded trimer and cleaves readily at a site of prefer ential proteolysis to yield monomeric N and trimeric oC. These are known pr operties of full-length TSP1. Mass spectroscopy indicated that C is N-glyco sylated, and all 10 cysteine residues of C are in disulfides. By equilibriu m ultracentrifugation, C is a monomer in physiological salt solution. Circu lar dichroism, intrinsic fluorescence, and differential scanning calorimetr y experiments suggest that the stability of C is determined by the disulfid es. The two tryptophans of C are in a polar, exposed environment as assesse d by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-c oil oligomerization domain. The results indicate that the recombinant C fol ds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.