Decorin inhibits cell attachment to thrombospondin-1 by binding to a KKTR-dependent cell adhesive site present within the N-terminal domain of thrombospondin-1.
Skin decorin (DCN) is an antiadhesive dermatan sulfate-rich proteoglycan that interacts with thrombospondin-1 (TSP) and inhibits fibroblast adhesion to TSP [Winnemöller et al., 1992]. Molecular mechanisms by which DCN interacts with TSP and inhibits cell adhesion to TSP are unknown. In the present study, we showed that skin DCN and bone DCN (chondroitin sulfate-rich proteoglycan) were quantitatively identical with respect to their ability to interact with TSP. Using a series of fusion proteins corresponding to the different structural domains of TSP, binding of [125I]DCN to TSP was found to be dependent of the N-terminal domain and, to a lesser extent, of the type 1 repeats and the C-terminal domain of TSP. In addition, heparan sulfate drastically inhibited [125I]DCN binding to solid-phase adsorbed TSP (80% inhibition), suggesting that DCN could bind to the N-terminal domain of TSP through interaction with heparin-binding sequences. To address this question, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), KKTR (residues 80-83) and RLRIAKGGVNDN (residues 178-189), were synthesized and tested for their ability to interact with DCN. [125I]DCN interacted only with peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS containing the heparin-binding consensus sequence KKTR. These peptides contained glycosaminoglycan-dependent and -independent binding sites because [125I]DCN binding to VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS was partially reduced upon removal of the glycosaminoglycan chain (65% and 46% inhibition, respectively). [125I]DCN poorly bound to subpeptide MKKTRG and did not bind at all to subpeptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS, suggesting that heparin-binding sequence MKKTRG constituted a DCN binding site when flanked with peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS. The sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS constitutes a cell adhesive active site in the N-terminal domain of TSP [Clezardin et al., 1997], and DCN inhibited the attachment of fibroblastic and osteoblastic cells to peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS by about 50 and 80%, respectively. Although fibroblastic cells also attached to type 3 repeats and the C-terminal domain of TSP, DCN only inhibited cell attachment to the C-terminal domain. Overall, these data indicate that modulation by steric exclusion of cell adhesion to a KKTR-dependent cell adhesive site present within the N-terminal domain of TSP could explain the antiadhesive properties of DCN.
Merle
B
B
INSERM Research Unit 403, Hôpital Edouard Herriot, Lyon, France. b-merle@cismsum.univ-lyon1-fr
Malaval
L
L
Lawler
J
J
Delmas
P
P
Clezardin
P
P
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000737:chemistry; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006497:Heparan Sulfate / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012867:Skin / Q000737:chemistry; D019700:Thrombospondin 1 / Q000378:metabolism*
D058575:Decorin; D016326:Extracellular Matrix Proteins; D006497:Heparan Sulfate; D010455:Peptides; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D019700:Thrombospondin 1; C546878:DCN protein, human; C546879:Dcn protein, mouse