An amyloid-like C-terminal domain of thrombospondin-1 displays CD47 agonist activity requiring both VVM motifs.
Two VVM-containing peptides in the C-terminal domain (CBD) of thrombospondin-1 function as CD47 agonists. A recombinant form of the CBD (rCBD) has been expressed that contains both VVM sites and exhibits CD47-dependent binding of C32 melanoma cells when coated at concentrations 100x lower than the peptide 4N1K (kRFYVVMWKk). Circular dichroism and thioflavin T binding of a recombinant form of the C-terminal domain (rCBD) of thrombospondin-1 indicated a species highly enriched in beta-sheet secondary structure, with spectra similar to those of amyloid proteins. Reduction of the CD signal with progressively higher concentrations of guanidine hydrochloride was correlated with a loss of cell-binding activity. Melanoma cell spreading on vitronectin was strongly stimulated by immobilized rCBD co-coated at concentrations more than 50x lower than 4N1K, and the effect was blocked by treatment with pertussis toxin, consistent with the known mediation of CD47 signaling by trimeric G(i). Mutations of either or both VV sequences of rCBD (1037-38 and 1123-24 of TSP1) to GG had a modest effect on cell binding, a component of which was inhibited by heparin. However, all three mutants dramatically reduced the signaling-dependent stimulation of cell spreading, indicating that the VVM motifs of rCBD are structurally linked in CD47 activation.
McDonald
J F
JF
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.
Dimitry
J M
JM
Frazier
W A
WA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D015703:Antigens, CD / Q000378:metabolism*; D052160:Benzothiazoles; D051928:CD47 Antigen; D002352:Carrier Proteins / Q000819:agonists / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects*; D002942:Circular Dichroism; D019204:GTP-Binding Proteins / Q000378:metabolism; D019791:Guanidine / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000503:physiopathology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013844:Thiazoles / Q000378:metabolism; D019700:Thrombospondin 1 / Q000235:genetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019096:Vitronectin / Q000494:pharmacology
D000682:Amyloid; D015703:Antigens, CD; D052160:Benzothiazoles; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D010446:Peptide Fragments; D013844:Thiazoles; D019700:Thrombospondin 1; D019096:Vitronectin; C009462:thioflavin T; D006493:Heparin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019791:Guanidine