© 2006 The Japanese Biochemical Society.
Regular Paper |
Interaction Study between Synthetic Glycoconjugate Ligands and Endocytic Receptors Using Flow Cytometry
1 NeTech Inc. KSP West 300-H, Sakado 3-2-1, Takatsu, Kawasaki, Kanagawa 213-0012; 2 Research Institute, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513; and 3 Faculty of System Design, Tokyo Metropolitan University, 6-6 Asahigaoka, Hino, Tokyo 191-0065
* To whom correspondence should be addressed. Tel: +81-42-995-1211, Fax: +81-42-991-1611, E-mail: ishihara{at}ndmc.ac.jp
Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17
-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.