Melanoma cell CD44 interaction with the alpha 1(IV)1263-1277 region from basement membrane collagen is modulated by ligand glycosylation.
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.
Lauer-Fields
Janelle L
JL
Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton 33431-0991, USA.
Malkar
Navdeep B
NB
Richet
Gérard
G
Drauz
Karlheinz
K
Fields
Gregg B
GB
D000596:Amino Acids / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002227:Carbazoles / Q000737:chemistry; D002448:Cell Adhesion; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D024141:Collagen Type IV / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006025:Glycosaminoglycans / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D006901:Hydroxylysine / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008545:Melanoma / Q000378:metabolism*; D010452:Peptide Biosynthesis; D011233:Precipitin Tests; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D002227:Carbazoles; D011508:Chondroitin Sulfate Proteoglycans; D024141:Collagen Type IV; D006025:Glycosaminoglycans; D018960:Hyaluronan Receptors; D008024:Ligands; D006901:Hydroxylysine; D001224:Aspartic Acid; D012965:Sodium Chloride; D008239:Lysine