Physical Characterization of the Procollagen Module of Human Thrombospondin 1 Expressed in Insect Cells

Abstract

Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal polyhistidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography. NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C is N-glycosylated, and all 10 cysteine residues of C are in disulfides. By equilibrium ultracentrifugation, C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed 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-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.

Regulation of Integrin Function by CD47 Ligands

“…FN33 is a recombinant region of fibronectin containing its ␣ 5 ␤ 1 integrin binding site but not its ␣ 4 ␤ 1 binding sites (25). NoC1 is a recombinant trimeric portion of TSP1 (residues 1-356 of the mature protein) (26). NoC2 is the corresponding recombinant portion of TSP2 (residues 1-359 of the mature protein).…”

Thrombospondin 2 Inhibits Microvascular Endothelial Cell Proliferation by a Caspase-independent Mechanism

“…A fragment of murine TSP2 encompassing the procollagen domain and type I repeats (PC/I, amino acids 320 -549 with the initiator methionine as 1) was prepared by cloning the corresponding cDNA into pAcGP67.coco (Misenheimer et al, 2000; a gift from D. Mosher) in frame with the upstream signal sequence and downstream polyhistidine tag. A fragment of murine TSP2 encompassing the C-terminal half of TSP2 (amino acids 377-1172) was also prepared by cloning into pAcGP67.coco.…”

Thrombospondins: from structure to therapeutics

“…The disulfide bonds stabilize the protein, as demonstrated by circular dichroism in the presence and absence of dithiothreitol. Lastly, the module is most likely monomeric in solution, as ultracentrifugation experiments did not reveal any oligomerization and far UV circular dichroism experiments did not show any changes in the structure of the VWC module when in a trimeric verus monomeric environment [17].…”

“…No clear melting transitions were evident by differential scanning calorimetry [17]. The disulfide bonds stabilize the protein, as demonstrated by circular dichroism in the presence and absence of dithiothreitol.…”

“…VWC modules do not have high sequence conservation, with the exception of CXXCXC (starting at the second cysteine) and CCXXC (starting at the eighth cysteine) motifs [8]. Although the atomic structure of this module has not been solved from THBS, insights can be gained from low resolution biophysical studies of the VWC module of THBS-1 [17] and from the structure of this module from procollagen IIA, solved by NMR [8] (Fig. 2C).…”