Biophysical characterization, including disulfide bond assignments, of the anti-angiogenic type 1 domains of human thrombospondin-1.
Thrombospondin-1 (TSP1), a modular secreted glycoprotein, possesses anti-angiogenic activity both in vitro and in vivo. This activity has been localized to the thrombospondin type 1 repeats/domains (TSR). A TSP1 monomer contains three TSRs, each with a hydrophobic cluster with three conserved tryptophans (WxxWxxW), a basic cluster with two conserved arginines (RxR), and six conserved cysteines. Using the baculovirus system, we expressed TSRs of human TSP1 as either the three domains in tandem (P123) or the third domain alone (P3) and demonstrated that both P123 and P3 at nanomolar concentrations inhibit either basic fibroblast-growth-factor or sphingosine-1-phosphate induced endothelial cell migration. Far-UV circular dichroism (CD) indicated that P123 and P3 have a common global fold that is very similar to properdin, a protein with six TSRs. Near-UV CD and fluorescence quenching studies indicated the conserved tryptophans are in a structured, partially solvent-accessible, positively charged environment. N-terminal sequence and mass spectrometry analysis of trypsin-digested TSRs indicated that the RFK linker sequence between P1 and P2 is readily proteolyzed and the conserved arginines are solvent accessible. By a combination of proteolysis and mass spectrometry, the recombinant TSRs were determined to be fully disulfide bonded with a connectivity of 1-5, 2-6, and 3-4 (cysteines are numbered sequentially from N- to C-terminus). TSRs are found in numerous extracellular proteins. These TSRs share the hydrophobic and basic clusters of the TSP TSRs but some have quite different placement of cysteine residues. We propose a sorting of TSRs into six groups that reconciles our results with information about other TSRs.
Huwiler
Kristin G
KG
Department of Medicine, University of Wisconsin, 1300 University Avenue, MSC 4459, Madison, Wisconsin 53706, USA.
Vestling
Martha M
MM
Annis
Douglas S
DS
Mosher
Deane F
DF
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry*; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019700:Thrombospondin 1 / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
D020533:Angiogenesis Inhibitors; D004220:Disulfides; D010446:Peptide Fragments; D019700:Thrombospondin 1; C016952:N-acetyltryptophanamide; D014364:Tryptophan; D001120:Arginine; D014357:Trypsin; D003545:Cysteine