PREPARATION OF A STABLE SUBUNIT OF JAPANESE ELDERBERRY (SAMBUCUS-SIEBOLDIANA) BARK LECTIN AND ITS APPLICATION FOR THE STUDY OF CELL-SURFACE CARBOHYDRATES BY FLOW-CYTOMETRY

A stable subunit of Sambucus sieboldiana bark lectin (MSSA) was prepared by selective reduction of disulfide bridges between the subunits and alkylation with 4-vinylpyridine. Amino acid analysis of MSSA revealed that 1.4 cysteine residues per subunit were selectively modified. MSSA failed to agglutinate rabbit erythrocytes and precipitate fetuin. However, MSSA retained the ability to bind to fetuin, as detected by ELISA. Neu5Ac-alpha-2-6lactose inhibited the binding to fetuin of both SSA and MSSA. Flow cytometric analysis showed that human histocytic lymphoma U937 cells were clearly stained with FITC-labeled MSSA (FITC-MSSA) without any detectable agglutination and that this staining was almost completely inhibited by the addition of Neu5Ac-alpha-2-6lactose (2 mM). Treatment of U937 cells with native FITC-SSA at the sub-agglutinating concentration (0.3-mu-g/ml) showed much poorer fluorescence intensity than that of MSSA, suggesting that MSSA is an invaluable tool for the detection of cell surface glycoconjugates containing NeuAc-alpha-2-6Gal/GalNAc sequences by flow cytometry.