Glycosylation of Mini-collagens Modulates Melanoma Cell Adhesion, Spreading, and Metalloproteinase Production

Introduction: Three-dimensional interactions between receptors and their ligands can modulate cell behavior. We have previously identified a region from type IV collagen, α1(IV)1263–1277, which promotes human melanoma cell adhesion and spreading in a conformationally-dependent manner [1]. These studies were expanded (a) using an alternative triple-helical construct and (b) incorporating a glycosylated hydroxylysine in position 1265. Melanoma cells were plated on the glycosylated and non-glycosylated triplehelical α1(IV)1263–1277 “mini-collagens” and subsequently tested for the promotion of adhesion and spreading, and regulation of proteolytic enzymes. The linear version of α1(IV)1263–1277 is known to bind CD44 chondroitinsulfate proteoglycan [2]. We have performed similar studies on the triplehelical version of α1(IV)1263–1277 to determine if ligand three-dimensional structure modulates receptor recognition. The studies utilize sub-popluations of M14 melanoma cells that are either enriched or deficient in cell surface CD44. The α1(IV)1263–1277 sequence was originally synthesized as a branched triple-helical peptide (THP) as described previously [1]. This construct utilizes a C-terminal branch and N-terminal 6 Gly-Pro-Hyp repeats to ensure a stable triple-helix. More recently we have described the synthesis of “peptide-amphiphiles” (PA) that incorporate an N-terminal lipid moiety that works in conjunction with Nand C-terminal Gly-Pro-Hyp repeats to form a stable triple-helix [3]. Both constructs can be use to create a triplehelix that is stable at physiological temperatures, but the later mini-collagen allows for greater flexibility in controlling Tm and enhanced ability to work on solid surfaces. The construction of tumor cell receptor ligands with their native three-dimensional structure and post-translational modifications permits a more realistic evaluation of the in vivo mechanisms of tumor cell interaction with and invasion of the basement membrane.