Galaptin-mediated Adhesion of Human Ovarian Carcinoma A121 Cells and Detection of Cellular Galaptin-binding Glycoproteins1

Previously, we have shown that galaptin, an endogenous ß-galactosidebinding lectin, is present in extracellular matrix where it may participate in the adhesion of AI21 human ovarian carcinoma cells to extracellular matrix via interaction with specific cell surface carbohydrate receptors. We now report that A121 cells adhere to polystyrene plates coated with polymerized human splenic galaptin. The carbohydrate-mediated speci ficity of this adhesive interaction was demonstrated by inhibition with lactose. Additionally, treatment of A121 cells with neuraminidase in creased cellular adherence by 30%, while ß-galactosidase treatment of cells decreased adherence by 65%. These findings prompted us to isolate and identify the cell surface galaptin receptor. I25l-labeled polymerized galaptin. In a Western blot of A121 cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, bound to a unique cellular protein having a molecular mass of 110 kDa. This receptor was enriched by affinity chromatography using polymerized galaptinSepharose. Treatment of this material with tV-glycanase ablated its galaptin-binding activity. In related studies, A121 cells metabolica!!} la beled with [•<H]glucosaminedemonstrated a radiolabeled polymerized galaptin-binding protein with an identical molecular mass of 110 kDa. These studies confirmed the glycoprotein nature of this putative endoge nous cellular galaptin receptor. Further studies with antibodies directed against two lysosomal associated membrane proteins, lamp-1 and lamp-2, demonstrated specific reactivity in Western blots with the 110kDa glycoprotein. Additionally, I25l-polymerized galaptin recognized a 110-kDa protein in Western blots of material immunoprecipitated from A121 cell lysates by lamp-1 and lamp-2 antibodies. Finally, indirect immunofluorescence using antibodies directed against lamps detected cell surface antigenicity. Therefore, lamp-1 and/or lamp-2 appear to be the putative cell surface receptors involved in the adhesion of ovarian carci noma cells to extracellular matrix mediated by galaptin.