Abstract
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.
Highlights
Affinity chromatography studies with a single-stranded IV-H1 peptide resulted in the isolation of melanoma cell CD44 receptors, in the chondroitin sulfate proteoglycan (CSPG) form [15, 16]
Prior studies had shown that CD44/CSPG from melanoma cells binds directly to single-stranded ␣1(IV)1263–1277 [15, 16] and that CD44/CSPG binds to type IV collagen [60]
Replacement of the one negatively charged residue in ␣1(IV)1263–1277 had no effect on melanoma cell binding, indicating that melanoma cell interaction with this triple-helical ligand is not integrin-mediated
Summary
General—All standard peptide synthesis chemicals were peptide synthesis grade or better and purchased from FisherBiotech. Peptide-amphiphiles dissolved in PBS were diluted in 70% ethanol and added to the 96-well plate and allowed to adsorb overnight at room temperature with mixing. Inhibition of Cell Adhesion Assays—Cells were labeled with 5- or 6-carboxyfluorescein diacetate, and Pro-BindTM 96-well plates were coated with peptides or proteins as in the adhesion assay. The cells were added to the wells to evaluate the adhesion to coated peptides or proteins in the continued presence of mAbs or GAGs. Cells were allowed to adhere for 30 min at 37 °C, and cell adhesion was quantified as in the adhesion assay. A 4-fold molar excess of fluoroscein isothiocyanate dissolved in the same buffer was added to each GAG and incubated overnight at 4 °C with mixing. The fluorescence of 200 l from each fraction was measured at excitation ϭ 485 nm and emission ϭ 538 nm
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