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 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.
Misenheimer
T M
TM
Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA. tmmiesenh@facstaff.wisc.edu
Huwiler
K G
KG
Annis
D S
DS
Mosher
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D011347:Procollagen / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D019700:Thrombospondin 1 / Q000737:chemistry*; D014357:Trypsin / Q000494:pharmacology
D011347:Procollagen; D011994:Recombinant Proteins; D019700:Thrombospondin 1; D014357:Trypsin