Expression and characterization of novel thrombospondin 1 type I repeat fusion proteins.
Thrombospondin (TSP)1 is a trimeric extracellular matrix protein that is held together by two cysteine residues. It is one of five TSP proteins that have been described to date with almost a universal heparin binding capability (TSP5 being the exception). The existence of two conformationally distinct structures in the TSP family (trimers and pentamers) prompted us to investigate the contribution of TSP1 trimeric structure to its inhibitory role in angiogenesis. We expressed full-length recombinant human TSP1, its type I repeats, and murine TSP3 in a human embryonic kidney cell line and evaluated their effect on human dermal microvascular endothelial cell (HMVEC) proliferation and sprouting into tube-like structures in vitro. Additionally, two chimaeric molecules were constructed so that the type I repeats of TSP1 were expressed as either dimers (TSP1-Ig chimaera) or pentamers (TSP1-TSP3 chimaera). Dimeric and pentameric type I constructs are novel structures. We found that, similarly to full-length TSP1, intact trimeric type I repeats were inhibitory to HMVEC angiogenesis in vitro. However, dimeric and pentameric type I repeats of TSP1 only partially inhibited HMVEC proliferation and sprouting in vitro. TSP3, which is lacking type I repeats, had no inhibitory activity, confirming that type I repeats elicit the anti-angiogenic activity of TSP1.
Qabar
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AN
Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, WA 98431, USA. aziz.qabar@apg.amedd.army.mil
Bullock
J
J
Matej
L
L
Polverini
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D020405:Dermis / Q000166:cytology; D019281:Dimerization; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D018919:Neovascularization, Physiologic; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics / Q000502:physiology*; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019699:Thrombospondins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D007141:Immunoglobulin Fc Fragments; D008222:Lymphokines; D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1; D019699:Thrombospondins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C101863:thrombospondin 3; D006493:Heparin; D003094:Collagen