IDENTIFICATION OF CELL ADHESIVE ACTIVE-SITES IN THE N-TERMINAL DOMAINOF THROMBOSPONDIN-1

Using a series of fusion proteins that span almost all of the thrombos pondin-1 (TSP-1) molecule, we observed in this study that Chinese hams ter ovary (CHO) K1 cells strongly attached to the N-terminus but not t o 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 dec reased by 47% compared with that observed with CHO K1 cells, indicatin g the presence of GAG-dependent cell adhesive sites. With the aim of i dentifying these cell adhesive sites, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), MKKTR G (residues 79-84) and TRDLASIARLRIAKGVNDNF (residues 170-189), were s ynthesized and tested for their ability to support CHO cell attachment . Using both centrifugation and cell-attachment assays, MKKTRG-contain ing peptides promoted CHO K1 cell adhesion, while ARKGSGRR-containing peptides and peptide TRDLASIARLRIAKGVNDNF did not. CHO S745 cell attac hment to MKKTRG-containing peptides was partially decreased. A 36 % de crease in CHO K1 cell attachment to the N-terminus was also observed w hen the heparin-binding consensus sequence KKTR was mutated to QNTR. I n addition, peptide MKKTRG partially inhibited (25 % inhibition) CHO K 1 cell attachment to the N-terminus. However, peptide MKKTRG was not s ufficient to fully promote cell attachment to the N-terminus of TSP-1. Peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS also supported CHO K1 cel l attachment in a GAG-dependent and -independent manner respectively. Moreover, CHO K1 cell attachment to MKKTRG was found to be markedly en hanced when flanked with the sequences VDAVRTEKGFLLLASLRQ and TLLALERK DHS. Peptide VDAVRTEKGFLLLASLRQMKKTRG nearly abolished (98% inhibition ) CHO K1 cell attachment to the N-terminus, while peptides MKKTRG, MKK TRGTLLALERKDHS and VDAVRTEKGFLLLASLRQ had only a moderate inhibitory e ffect (25, 27 and 53 % inhibition respectively). These data indicate t hat the sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS (residues 60-94) constitutes a GAG-dependent cell adhesive site in the N-terminus of TS P-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, me lanoma and osteosarcoma cells), and a high degree of sequence homology was found between TSP-1 and TSP-2 between residues 60 and 94 (48 % id entity) and 189-200 (67 % identity), further suggesting the functional importance of these two cell adhesive sites in the N-terminus of TSP- 1.