Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity.
Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH(2)-terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.
Bein
K
K
Angiogenesis Research Center, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Simons
M
M
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D017364:Collagenases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments / Q000378:metabolism; D011233:Precipitin Tests; D011414:Properdin / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D012441:Saccharomyces cerevisiae; D013917:Thrombin / Q000494:pharmacology; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019699:Thrombospondins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D000099262:Thrombospondin 2
D017364:Collagenases; D004792:Enzyme Precursors; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D011414:Properdin; D013917:Thrombin; D019700:Thrombospondin 1; D019699:Thrombospondins; D000099262:Thrombospondin 2; C120487:pro-matrix metalloproteinase 9