hydroxylysine and Melanoma

Invasion of the basement membrane is believed to be a critical step in the metastatic process. Melanoma cells have been shown previously to bind distinct triple-helical regions within basement membrane (type IV) collagen. Additionally, tumor cell binding sites within type IV collagen contain glycosylated hydroxylysine residues. In the present study, we have utilized triple-helical models of the type IV collagen alpha1(IV)1263-1277 sequence to (a) determine the melanoma cell receptor for this ligand and (b) analyze the results of single-site glycosylation on melanoma cell recognition. Receptor identification was achieved by a combination of methods, including (a) cell adhesion and spreading assays using triple-helical alpha1(IV)1263-1277 and an Asp(1266)Abu variant, (b) inhibition of cell adhesion and spreading assays, and (c) triple-helical alpha1(IV)1263-1277 affinity chromatography with whole cell lysates and glycosaminoglycans. Triple-helical alpha1(IV)1263-1277 was bound by melanoma cell CD44/chondroitin sulfate proteoglycan receptors and not by the collagen-binding integrins or melanoma-associated proteoglycan. Melanoma cell adhesion to and spreading on the triple-helical alpha1(IV)1263-1277 sequence was then compared for glycosylated (replacement of Lys(1265) with Hyl(O-beta-d-galactopyranosyl)) versus non-glycosylated ligand. Glycosylation was found to strongly modulate both activities, as adhesion and spreading were dramatically decreased due to the presence of galactose. CD44/chondroitin sulfate proteoglycan did not bind to glycosylated alpha1(IV)1263-1277. Overall, this study (a) is the first demonstration of the prophylactic effects of glycosylation on tumor cell interaction with the basement membrane, (b) provides a rare example of an apparent unfavorable interaction between carbohydrates, and (c) suggests that sugars may mask "cryptic sites" accessible to tumor cells with cell surface or secreted glycosidase activities.

Topics: Amino Acids; Aspartic Acid; Binding Sites; Carbazoles; Cell Adhesion; Chondroitin Sulfate Proteoglycans; Chromatography, High Pressure Liquid; Circular Dichroism; Collagen Type IV; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Glycosaminoglycans; Glycosylation; Humans; Hyaluronan Receptors; Hydroxylysine; Ligands; Lysine; Melanoma; Peptide Biosynthesis; Precipitin Tests; Protein Binding; Sodium Chloride; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Tumor Cells, Cultured