Presence of a laminin-binding chondroitin sulfate proteoglycan at the cellsurface of a human melanoma cell Mel-85

Working with Mel-85 (a human melanoma cell line), we have been able to dete ct a laminin-binding molecule with an apparent molecular mass of 100/110 kD a (Mel-85-LBM). Reduction with beta-mercaptoethanol decreases its molecular mass but does not affect its ability to bind laminin. This laminin interac tion seems to be very specific since Mel-85-LBM binds laminin, but not fibr onectin, vitronectin or type I collagen in affinity chromatography experime nts. The molecule has a negative net charge at physiological pH and binds l aminin in a divalent cation dependent way. Mel-85-LBM was metabolically rad iolabeled with sodium [S-35]-sulfate and chemical beta-elimination of purif ied Mel-85-LBM releases chondroitin sulfate chains. Mel-85-LBM is also sens itive to chondroitinase ABC digestion. These findings show that this molecu le is a chondroitin sulfate proteoglycan. The location of this proteoglycan at the cell surface is evidenced by experiments using a polyclonal antiser um raised against purified Mel-85LBM, that specifically reacts with just on e molecule by western blotting among Mel-85 total cell extract as well as p roduces a positive signal by flow cytometry and a fluorescence profile of M el-85 cells adhered on laminin.