title: Identification of cell adhesive active sites in the N-terminal domain of thrombospondin-1.
abstract: Using a series of fusion proteins that span almost all of the thrombospondin-1 (TSP-1) molecule, we observed in this study that Chinese hamster ovary (CHO) K1 cells strongly attached to the N-terminus but not to the other domains of TSP-1 (e.g. the C-terminus, and type 1, type 2 and type 3 repeats). In addition, attachment to the N-terminus of CHO S745 cells defective in cell-surface glycosaminoglycans (GAGs) was decreased by 47% compared with that observed with CHO K1 cells, indicating the presence of GAG-dependent cell adhesive sites. With the aim of identifying these cell adhesive sites, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), MKKTRG (residues 79-84) and TRDLASIARLRIAKGVNDNF (residues 170-189), were synthesized and tested for their ability to support CHO cell attachment. Using both centrifugation and cell-attachment assays, MKKTRG-containing peptides promoted CHO K1 cell adhesion, while ARKGSGRR-containing peptides and peptide TRDLASIARLRIAKGVNDNF did not. CHO S745 cell attachment to MKKTRG-containing peptides was partially decreased. A 36% decrease in CHO K1 cell attachment to the N-terminus was also observed when the heparin-binding consensus sequence KKTR was mutated to QNTR. In addition, peptide MKKTRG partially inhibited (25% inhibition) CHO K1 cell attachment to the N-terminus. However, peptide MKKTRG was not sufficient to fully promote cell attachment to the N-terminus of TSP-1. Peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS also supported CHO K1 cell attachment in a GAG-dependent and -independent manner respectively. Moreover, CHO K1 cell attachment to MKKTRG was found to be markedly enhanced when flanked with the sequences VDAVRTEKGFLLLASLRQ and TLLALERKDHS. Peptide VDAVRTEKGFLLLASLRQMKKTRG nearly abolished (98% inhibition) CHO K1 cell attachment to the N-terminus, while peptides MKKTRG, MKKTRGTLLALERKDHS and VDAVRTEKGFLLLASLRQ had only a moderate inhibitory effect (25, 27 and 53% inhibition respectively). These data indicate that the sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS (residues 60-94) constitutes a GAG-dependent cell adhesive site in the N-terminus of TSP-1. Moreover, a GAG-independent site, encompassing residues 189-200 (FQGVLQNVRFVF), has been identified. These two adhesive sites supported the attachment of a wide variety of cells (human breast carcinoma, melanoma and osteosarcoma cells), and a high degree of sequence homology was found between TSP-1 and TSP-2 between residues 60 and 94 (48% identity) and 189-200 (67% identity), further suggesting the functional importance of these two cell adhesive sites in the N-terminus of TSP-1.
authors.affiliation: INSERM Research Unit 403, Pavillon F, Hopital Edouard Herriot, Lyon, France.
authors.forename: P
authors.initials: P
authors.lastname: Clezardin
authors.forename: J
authors.initials: J
authors.lastname: Lawler
authors.forename: J
authors.initials: J
authors.lastname: Amiral
authors.forename: G
authors.initials: G
authors.lastname: Quentin
authors.forename: P
authors.initials: P
authors.lastname: Delmas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry; D016466:CHO Cells / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology*; D002846:Chromatography, Affinity; D006224:Cricetinae; D006025:Glycosaminoglycans / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019699:Thrombospondins
chemical_list: D006025:Glycosaminoglycans; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins