Specific Binding of Glucose-derivatized Polymers to the Asialoglycoprotein Receptor of Mouse Primary Hepatocytes

• Published in: The Journal of biological chemistry 2001-09, Vol.276 (38), p.35312-35319
• Main Authors: Kim, Sang-Heon, Goto, Mitsuaki, Akaike, Toshihiro
• Format: Article
• Language: English
• Subjects: Animals; Asialoglycoprotein Receptor; Carbohydrate Sequence; Cell Adhesion; Flow Cytometry; Glucose - chemistry; Hepatocytes - metabolism; Lectins - metabolism; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred ICR; Polymers; Protein Binding; Receptors, Cell Surface - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M009749200

In this study, we designed a novel amphiphilic poly-(p-N-vinylbenzyl-d-glucuronamide) (PV6Gna) modified at the 6-OH position of glucose for hepatocyte recognition to address the mechanism of the interaction between mouse primary hepatocytes and the PV6Gna. PV6Gna bound to lectins specific for glucose but not galactose as did other glucose-derivatized polymers. However, hepatocyte adhesion onto the PV6Gna surface was inhibited in the presence of galactose and its analogues but not in the presence of glucose and its analogues. We also showed that hepatocyte adhesion to the PV6Gna surface was inhibited dose dependently by asialofetuin (ASF). Interactions between soluble PV6Gna and hepatocytes were inhibited by GalNAc, ASF, and EGTA in flow cytometry analysis using fluorescein isothiocyanate-conjugated PV6Gna. Hepatocyte adhesion to the PV6Gna surface was inhibited more effectively by GalNAc than by methyl β-d-galactose. In flow cytometry analysis and cell adhesion assay, ASF competed for the inhibition of interaction between PV6Gna and hepatocytes 0.5–4 × 105-fold more effectively than did GalNAc. These results demonstrate involvement of asialoglycoprotein receptors (ASGPRs) in the interaction between PV6Gna and hepatocytes. Furthermore, to clarify the mechanism of the interaction between glycopolymers modified at the 6-OH position of glucose and the hepatocyte, we prepared a gel particle containing 6-O-methacryloyl-d-glucose (PMglc) synthesized by an enzymatic method. ASGPRs could be detected using Western blot analysis following precipitation with PMglc in hepatocyte cell lysate. The precipitation of ASGPRs was inhibited in the presence of galactose, ASF, PV6Gna, and EGTA. The precipitation was inhibited more effectively by GalNAc than by methyl β-d-galactose. ASGPRs were rarely precipitated by PMglc in the cell lysate that had been treated with ASF-conjugated Sepharose. Taken together, we suggest that mouse primary hepatocytes adhere to the PV6Gna surface mediated by ASGPRs, which specifically interacted with the glycopolymers modified at the C-6 position of glucose.