Characterization of a 95 kilodalton membrane glycoprotein associated with multi‐drug resistance

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Abstract
Over‐expression of a 95‐kDa membrane protein (P‐95) has been reported previously in the multi‐drug‐resistant (MDR) breast cancer cell line MCF‐7/AdrVp and the MDR small cell lung cancer line NC1‐H1688. We have now developed anti‐sera against gel‐purified P‐95 protein from each of these cell lines. Western blotting with each serum demonstrates a broad band at 95‐kDa with detergent‐solubilized membrane proteins from MCF‐7/AdrVp and NC1‐H1688 cells, which is barely detectable in membrane proteins from drug‐sensitive parental MCF‐7 cells. Each anti‐serum cross‐reacts with a 190‐kDa membrane protein (P‐190) in NC1‐H1688 but not MCF‐7/AdrVp cells. Immunoblotting and silver staining of NC1‐H 1688 membrane proteins separated by two‐dimensional gel electrophoresis demonstrates that P‐95 and P‐190 run as broad streaks with low iso‐electric points. Incubation of NC1‐H 1686 cells with tunicamycin or cleavage of carbohydrate residues of NCI‐H 1688 or MCF‐7/AdrVp membrane proteins with PNGase F leads to the appearance of a sharp 35‐kDa band reactive with anti‐P‐95 antisera. This 35‐kDa protein has been isolated by two‐dimensional gel electrophoresis. Neuraminidase digestion converts P‐95 to a broad 65‐kDa immunoreactive band, indicating the presence of terminal sialic acid residues. In conclusion, P‐95 is an N‐linked sialoglycoprotein with a 35‐kDa polypeptide core.