Biophysical Characterization, Including Disulfide Bond Assignments, of the Anti-Angiogenic Type 1 Domains of Human Thrombospondin-1

Abstract

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.

Identification and Characterization of aβ1,3-Glucosyltransferase That Synthesizes the Glc-β1,3-Fuc Disaccharide on Thrombospondin Type 1 Repeats

“…The positive ellipticity of the far-UV CD spectrum of native TSR4 is in agreement with a structure that largely consists of ␤-strands and turns and contains clustered aromatic amino acids. In fact, the spectra are very similar to that found for properdin, a protein isolated from human plasma that consists of six TSRs (35) and for recombinant TSRs from TSP-1 (34).…”

“…We examined the secondary structure of TSR4 by spectroscopic techniques that have previously been used to ascertain the correct folding of recombinant TSRs from human TSP-1 (34). At 25°C the far-UV CD spectrum of TSR4 displayed positive ellipticity with maxima at 212 and 231 nm (Fig.…”

Thrombospondins: from structure to therapeutics

“…1). TSRs are found in over 40 human proteins, both as tandem repeats and alone [19,20]. Six TSRs comprise the entire structure of properdin, and thus an alternative name for this repeat is 'properdin repeat'.…”

“…TSRs are conserved over evolution, and 14 Drosophila and 27 Caenorhabditis elegans proteins contain TSRs. The TSRs are characteristically extracellular (either in extracellular proteins or extracellular portions of transmembrane proteins), about 60 amino acids long, and contain around 12 conserved residues comprising six cysteines (for which there are two pairing strategies), two conserved arginine residues, two conserved glycine residues, and two to three tryptophan residues separated by two to four amino acids [19,20] (Fig. 2D).…”

“…Using database analyses, Huwiler et al 2002 surveyed the disulfide arrangement of TSRs found in diverse proteins, suggesting that there are two major groups of TSRs [9,20]. Group 1 TSRs, including those found in THBS-1 and THBS-2, have a disulfide pattern where the top disulfide (using the C layer) is created by a cysteine in strand B and a cysteine in strand C. However, group 2 TSRs, including those found in F-spondin, have a disulfide pattern where the top disulfide is created by a cysteine in strand A and a cysteine in strand C. This indicates that the N-terminus of group 2 TSRs is stabilized via the disulfide bond, rather than via the first jar handle, as seen in the THBS TSRs.…”