Isolation of tumor cell surface binding sites for concanavalin A and wheat germ agglutinin.

Abstract

Novikoff ascites tumor cells were found to be agglutinable by both concanavalin A and wheat germ agglutinin (WGA). A sialoglycopeptide-containing fraction, released by papain digestion of Novikoff tumor cell suspensions, inhibits the agglutination reactions, indicating that receptor sites for these plant lectins are components of this glycopeptide fraction. The purpose of this research was to develop methods for separation of the glycopeptide fraction into its component molecules and to characterize these components regarding their ability to inhibit agglutination of Novikoff tumor cells by concanavalin A and WGA. The sialoglycopeptide-containing fraction was digested with Pronase to minimize variation in the peptide chain length of the component glycopeptides. The Pronase-digested glycopeptide fraction was submitted to gel filtration on Sephadex G-50 and subsequently to ion-exchange chromatography on DEAE-cellulose. Gel filtration resolved the glycopeptides into two molecular weight classes, a low-molecular-weight fraction (M.W. 2000 to 3300) and a higher-molecular-weight fraction (M.W. >3300). This latter fraction consists of molecules of widely different molecular weights, some of the material being excluded from Sephadex G-100. Ion-exchange chromatography of the low-molecular-weight material resolved four sialoglycopeptide fractions which inhibited agglutination of Novikoff tumor cells by concanavalin A but which were inactive in inhibiting agglutination by WGA. Ion-exchange chromatography of the higher-molecular-weight material resulted in the isolation of a sialoglycopeptide fraction which possessed high specificity as an inhibitor of agglutination by WGA.