MELANOMA CELL-ADHESION AND SPREADING ACTIVITIES OF A SYNTHETIC 124-RESIDUE TRIPLE-HELICAL MINI-COLLAGEN

A great variety of cells, such as melanoma cells, fibroblasts, platele ts, keratinocytes, and epithelial cells, adhere to and migrate on spec ific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascerta ined, as no general synthetic methodology exists to allow for the stud y of peptides incorporating biologically active sequences in triple-he lical conformation. We have thus developed a novel, generally applicab le solid-phase branching methodology for the synthesis of aligned, tri ple-helical collagen-model polypeptides (i.e. ''mini-collagens''). Thr ee nascent peptide chains are carboxyl-terminally linked through one N (alpha)-amino and two N(epsilon)-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-hel ical polypeptide (THP) of 124 amino acids, incorporating residues 1263 -1277 of alpha1(IV) collagen, was synthesized. Highly metastatic mouse melanoma cells showed a profound preference for adhesion to this THP as compared with a single-stranded peptide (SSP) incorporating the sam e type IV collagen sequence or a branched peptide containing eight rep eats of Gly-Pro-Hyp (designated GPP). Specifically, 50% cell adhesion occurred at a THP concentration of 1.12 muM, while comparable levels of adhesion required [SSP] = 170 muM or [GPP] > 100 muM. Melanoma cel ls also spread on the THP to a greater extent than on the SSP or GPP. These results are the first direct demonstrations of the significance of triple helicity for cell adhesion to and spreading on a specific c ollagen sequence and support earlier conclusions of conformational dep endency for cell adhesion to and migration on types I and IV collagen. In addition, the melanoma cell THP activities support the concept tha t tumor cell adhesion and spreading on type IV collagen involves multi ple, distinct domains in triple-helical conformation. The triple-helic al peptide synthetic protocol developed here will allow eventually for the study of both structure and biological activity of specific, glyc osylated collagen sequences in homotrimeric and heterotrimeric forms.