Identification of cell adhesive active sites in the N-terminal domain of thrombospondin-1

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.