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

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.

Carbohydrate-mediated interactions play an important role in biological processes such as receptor-mediated endocytosis, opsonization, apoptosis, and metastasis and have been applied to cell recognition studies as well as designs for biomedical materials (1)(2)(3)(4)(5). Multivalent glycopolymer ligands have been designed for clustering of L-selectin leading to the activation of the leukocyte cell surface and the inhibition of L-selectin-mediated leukocyte rolling (6,7). Interaction between the carbohydrate and the carbohydrate-binding proteins (CBPs) 1 is achieved through hydrogen bonding of the hydroxyl group of the carbohydrate to the polar amino acid side chain of the protein, including the packing of a hydrophobic sugar face against the aromatic amino acid side chain of the protein (8). CBPs interact with a particular carbohydrate by recognizing subtle differences in carbohydrate structure (9,10). Thus, the carbohydrate specificity to the cells varies according to the kind of CBPs distributed on the cell. Asialoglycoprotein receptors (ASGPRs) are lectins for receptor-mediated endocytosis found at the hepatocyte cell surface that are bound to galactose/ GalNAc-terminated ligands in a calcium-dependent manner (1,(11)(12)(13)(14). Because ASGPRs are widely used as a model for the study of receptor-mediated endocytosis, galactose-derivatized materials have been a source applied to a cell-targeted drug delivery system for hepatocytes and as an artificial adhesion matrix for liver tissue engineering (15)(16)(17)(18).
Recently we developed a novel amphiphilic glucose-derivatized PV6Gna as an artificial adhesion matrix for hepatocyte cultures and investigated the effects of the substituted position of the hydroxyl group of glucose on recognition of mouse hepatocytes (19). Mouse primary hepatocytes were specifically recognized by PV6Gna due to the modification of the C-6 position of glucose but not the C-1 and C-3 positions. The adhesion of hepatocytes to the PV6Gna surface was dependent on Ca 2ϩ and independent of temperature unlike integrin-dependent adhesion. These results indicated that hepatocytes might be interacting with PV6Gna mediated by a CBP such as a glucose transporter as well as an ASGPR on the cell membrane surface.
Mammalian hepatocytes were mainly recognized by glycopolymers that act as natural and artificial ligands having terminal galactose/GalNAc moieties, although chicken hepatocytes were recognized by terminal GlcNAc moieties (20 -22). Accordingly there are many reports that address the behavior of hepatocyte adhesion to artificial polymer surfaces having termi-nal galactose moieties (23)(24)(25). We have also developed poly-(Np-vinylbenzyl-4-O-␤-D-galactopyranosyl-D-gluconamide) (PVLA) having terminal galactose moieties as an artificial matrix for hepatocyte cultures (26). Previous studies examined hepatic functions and gene regulation of hepatocytes cultured on a PVLA surface compared with different extracellular matrixes (27,28).
To our knowledge, PV6Gna is the first polymer having glucose moieties capable of binding to mammalian hepatocytes. In this study, we evaluated the selectivity of PV6Gna against lectins and further found that the mechanism of binding of PV6Gna to hepatocytes was caused by recognition between ASGPRs and glucose moieties.

EXPERIMENTAL PROCEDURES
Materials-Glycopolymers including PV6Gna were synthesized and conjugated with fluorescein isothiocyanate (FITC) according to a method described previously (19). 13 C NMR spectra were recorded on a Varian UNITY plus 400 spectrometer using tetramethylsilane as external standard. Polystyrene (PS) plates or dishes were obtained from Becton Dickinson. Biotin-labeled lectins and peroxidase-conjugated streptavidin were purchased from EY Laboratories and Vector Laboratories, respectively. Monosaccharides and glycosides were purchased from Wako Pure Chemical (Osaka, Japan) except for methyl ␣-D-glucoside, methyl ␤-D-glucoside, and D-glucuronic acid, which were obtained from Nacalai Tesque (Kyoto, Japan). Collagenase and trypsin inhibitor were purchased from Wako Pure Chemical and Nacalai Tesque, respectively. Williams' medium E, Dulbecco's modified Eagle's medium, and fetal bovine serum were purchased from Life Technologies, Inc. Asialofetuin (ASF) and fetuin were purchased from Sigma. The DC Protein Assay kit was purchased from Bio-Rad. Proleather was kindly given by Amano Pharmaceutical Co. Ltd. (Nagoya, Japan). ASF-conjugated Sepharose was prepared by incubating CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) with 2.5 mg/ml ASF in coupling buffer (0.1 M NaHCO 3 , 0.5 M NaCl, pH 8.0) at 4°C overnight as described by the manufacturer. Mouse monoclonal anti-ASGPR was a generous gift from Daiichi Pure Chemical (Tokyo, Japan). ICR mice were obtained from Japan SLC, Inc.
Direct Lectin-Enzyme Assay-A polystyrene enzyme-linked immunosorbent assay plate (IWAKI, Tokyo, Japan) was coated with 0.1 mg/ml of PVLA, PVMEA, PVMA, PVG, and PV6Gna for 4 h and blocked with 1 wt% BSA dissolved in PBS (PBS/BSA) for 2 h at room temperature. The plate was incubated with between 0.1 and 25 M biotinlabeled Canavalia ensiformis lectin (ConA), Lens culinaris lectin (LcH), Allomyrina dichotoma lectin (Allo A), and Arachis hypogaea lectin (PNA) in the buffer recommended by the manufacturer for 1 h at room temperature and then washed five times with 0.1 wt% Tween 20 dissolved in PBS (PBS-T). To the plate was added 1 ϫ 10 Ϫ4 -fold peroxidase-conjugated streptavidin in 1 wt% PBS/BSA. The plate was incubated for 1 h at room temperature and washed five times with PBS-T. An orthophenylenediamine solution (0.4 mg/ml orthophenylenediamine and 0.4l/ml H 2 O 2 in citrate-phosphate buffer, pH 5.0) was used as a substrate of peroxidase. Absorbance at 492 nm was measured by a microplate reader (MTP-32, Corona Electric Co., Ibaraki, Japan).
Hepatocyte Isolation and Inhibition Hepatocyte Adhesion Assay-Hepatocytes were isolated from the livers of male ICR mice (5-7 weeks old) by a modification of the in situ collagenase perfusion method that includes perfusion with EGTA before collagenase treatment as described previously (29,30). The perfusion buffer, pH 7.2, contained 137 mM NaCl, 5.4 mM KCl, 0.5 mM NaH 2 PO 4 , 0.42 mM Na 2 HPO 4 , 10 mM HEPES, 0.5 mM EGTA, 4.2 mM NaHCO 3 , and 5 mM glucose. The collagenase buffer, pH 7.5, contained 137 mM NaCl, 5.4 mM KCl, 5 mM CaCl 2 , 0.5 mM NaH 2 PO 4 , 0.42 mM Na 2 HPO 4 , 10 mM HEPES, 0.15 g/liter collagenase, 0.05 g/liter trypsin inhibitor, 4.2 mM NaHCO 3 , and 0.016 mM phenol red. Briefly, the liver was perfused by the perfusion buffer and collagenase buffer through a needle aligned along the inferior vena cava. The collagenase-perfused liver was dissected, suspended in Hanks' solution (30 ml), and filtered through a cheesecloth and a 100-m nylon membrane to remove connective tissue debris and cell clumps. Hepatocytes were purified by four centrifugations (42 ϫ g, 2 min) at 4°C and a density gradient centrifugation (42 ϫ g, 10 min) using 45% Percoll solution. Cell viability measured by trypan blue exclusion was more than 90%. The isolated hepatocytes (1 ϫ 10 5 cells/ ml) were preincubated in the presence of carbohydrate inhibitors in Hanks' salt solution for 30 min on ice and allowed to adhere for 30 min at 4°C onto the polystyrene plate, which had been coated with 0.1 mg/ml PV6Gna followed by blocking with 0.5 wt% PBS/BSA to prevent nonspecific adhesion. Nonadherent hepatocytes were removed from the dish by washing with PBS three times, and protein was extracted from the adhered hepatocytes with a 0.25 wt% NaOH solution. The percentage of cell adhesion was determined from the amount of protein measured by the improved Lowry method (DC Protein Assay kit). All values were normalized to the percentage of adhesion of the maximum, which was taken as 100%.
Preparation of Erythrocytes and Cell Line Culture-Blood (0.1 ml) was collected from the heart of the ICR mouse, diluted with 0.04% EDTA (0.5 ml) in PBS solution, and washed with PBS solution (0.5 ml) by three centrifugations (1000 rpm, 5 min). Hepa 1-6 and NIH 3T3 fibroblast were incubated in tissue culture flasks containing complete Dulbecco's modified Eagle's medium with 10% fetal bovine serum under a humidified atmosphere of 5% CO 2 and 95% air at 37°C. Human umbilical vein endothelial cells were incubated in Endothelial Cell Growth Medium BulletKit-2 (EGM-2 BulletKit, Clonetics) under the same conditions described above. The media was renewed twice weekly. Cells at an 80% confluent state were rinsed and detached in 0.05% trypsin, 0.02% EDTA in PBS for 5-7 min at 37°C. The detached cells were washed with the fresh media three times by centrifugation (100 ϫ g, 5 min) and suspended in the fresh media until used for experiments.
Flow Cytometry Analysis-Freshly isolated hepatocytes (5 ϫ 10 5 cell/ml) were incubated with 100 g/ml FITC-conjugated PV6Gna in Williams' medium E containing 0.1 wt% BSA and 1 g/ml propidium iodide (PI) for 1.5 h at 4°C. All cells were washed with PBS, 2.5 mM CaCl 2 three times before the analysis of 5000 hepatocytes using a FACScan TM instrument (EPICS-XL, Beckman Coulter). The hepatocyte region was selected by adjusting the forward scatter and the side scatter. The surface-labeled hepatocytes with FITC-conjugated PV6Gna were selected from the region of PI-unlabeled hepatocytes to remove the cytoplasm-labeled damaged hepatocytes.
Preparation of PMglc and ASGPR Precipitation Assay-PMglc was prepared as a new gel particle containing 6-O-methacryloyl glucose synthesized by a commercially available protease that catalyzes transesterification of glucose with vinyl methacrylate at the C-6 position of glucose as described previously (31). Briefly, 0.6 g of D-glucose was incubated with 10 mM vinyl methacrylate in the presence of 1 g of Proleather in 10 ml of pyridine at 45°C for 48 h. The product was separated by silica gel column chromatography and characterized by 13 C NMR and HPLC analysis. The product was gelated in the presence of ammonium peroxodisulfate as an initiator in H 2 O for 30 min at 55°C and sonicated to make a fine particle. PMglc was used following removal of large particles by filtering with a 4-m membrane filter. For ASGPR precipitation with PMglc, freshly isolated hepatocytes were solubilized in lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 wt% Nonidet P-40, 20 mM n-octyl-␤-D-glucopyranoside, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 2 g/ml aprotinin) for 1 h at 4°C. Lysates were then clarified by centrifugation at 15,000 rpm for 30 min at 4°C before being added to PMglc in the lysis buffer containing 2 mM CaCl 2 for 4 h at 4°C. In the inhibitory test, the inhibitors were added in the lysates in advance. Pellets were washed four times in washing buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 wt% Nonidet P-40, 1 mM CaCl 2 ) and resuspended in SDS-polyacrylamide gel electrophoresis sample buffer containing 2 wt% sodium dodecyl sulfate, 5% (v/v) 2-mercaptoethanol. Proteins were eluted from PMglc by boiling the samples for 5 min and separated by SDS-polyacrylamide gel electrophoresis using a 12% resolving gel followed by Western blot analysis with mouse monoclonal anti-ASGPR using standard techniques. For detection, horseradish peroxidase-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch Laboratories) and the ECL method (Amersham Pharmacia Biotech) were used.

RESULTS AND DISCUSSION
Lectins Binding to PV6Gna-A direct lectin-enzyme assay was carried out to determine the type of the sugar moiety of PV6Gna compared with other glycopolymers shown in Fig. 1 that have glucose or galactose moieties. Dose-dependent binding of lectins to the glycopolymers is shown in Fig. 2. ConA and LcH showed a greater selectivity in binding to PVMA, PVG, and PV6Gna substituted at C-1, C-3, and C-6 of glucose, respectively, than to PVLA and PVMEA, which have galactose moieties. Allo A bound only to PVLA, whereas PNA bound to both PVLA and PVMEA, and neither lectin bound to the glycopolymers having glucose moieties such as PV6Gna. ConA and LcH as lectins have specificity to ␣-D-mannose or ␣-D-glucose, whereas Allo A and PNA are lectins specific to ␤-D-galactose (8,32,33). In particular, ConA bound to PVMA with a higher affinity than to PV6Gna and PVG of the glucose-derivatized polymers as shown in Fig (34). In addition, the carbohydrate in PV6Gna was characterized by 13 C NMR spectra in Me 2 SO-d 6 and compared with authentic carbohydrates such as glucuronic acid and galactose as depicted in Fig. 3. Although the PV6Gna was readily soluble in both Me 2 SO and water, NMR analysis was carried out in Me 2 SO-d 6 only because the signals were observed to broaden in the latter solvent. The galactose existed as only the ␣-anomeric form, which is represented by a peak at 92.7 ppm in Me 2 SO. The C-2-C-4 signals of the galactose appeared at 68.9 -69.5 ppm (Fig. 3a), whereas the C-2-C-5 signals of the glucuronic acid appeared at 71.6 -76.1 ppm (Fig. 3b). Specifically, the peak at 68.9 ppm represents C-3 of ␣-D-galactose according to the 13 C chemical shift for carbohydrates (35). The C-2-C-5 signals of carbohydrate in PV6Gna appeared at 71.5-76.3 ppm and paralleled those of glucuronic acid (Fig. 3c). Together the results suggest that PV6Gna is a polymer that has glucose moieties and binds to ConA and LcH mediated by the hydrophilic glucose moieties exposed on the PS surface.
Glycopolymer Selectivity of Hepatocytes-To investigate specific interactions between the synthetic glycopolymers and hepatocytes, a cell adhesion assay was carried out on a polystyrene dish coated with a particular glycopolymer. Hepatocytes exclusively adhered onto the polystyrene surface that had been coated with 0.1 mg/ml PVLA, PVMEA, and PV6Gna but not PVMA or PVG as shown in Fig. 4a. Mammalian primary hepatocytes are specifically recognized by the glycoproteins that act as natural and artificial ligands having terminal galactose/GalNAc moieties. As expected, hepatocytes interacted with PVLA and PVMEA having terminal galactose moieties. On the other hand, hepatocytes interacted only with PV6Gna among the glycopolymers that have glucose moieties. A photomicrograph of hepatocytes adhered to the PV6Gna surface is shown in Fig. 4b indicating that it did not result from contamination by nonparenchymal cells, such as Kupffer cells and sinusoidal endothelial cells, of our hepatocyte preparation. The results indicate that hepatocytes specifically recognize PV6Gna due to the substitution of the C-6 position of glucose but not at the C-1 and C-3 positions.
Inhibition of Hepatocyte Adhesion to PV6Gna Surface Exclu-sively by Galactose-type Carbohydrates-We investigated the selectivity of the carbohydrate against the CBP used in the adhesion.
, and GalNAc but not in the presence of 20 mM of glucose and its analogues. It is very interesting that the galactose-type carbohydrates exclusively inhibited hepatocyte adhesion to the PV6Gna that has glucose moieties. Our previous study showed that hepatocyte adhesion onto the PV6Gna surface occurred in a temperature-independent and Ca 2ϩ -dependent manner (19). Taken together the results indicate a relationship of the C-type lectin such as ASGPR with hepatocyte adhesion to the PV6Gna surface.
The Relationship of ASGPR to the Interaction of Hepatocytes and PV6Gna-We examined the inhibitory effect that is dependent on the concentration of those galactose-type carbohydrates on the hepatocyte adhesion onto the PV6Gna surface. Hepatocyte adhesion to the PV6Gna surface was almost completely inhibited by 1 mM GalNAc, 5 mM methyl ␤-gal, 10 mM lactose, and 20 mM melibiose, respectively, as shown in Fig. 5. Numerous studies have determined the difference of the affinity for carbohydrates of C-type lectins by using an inhibition binding assay and crystallographic analysis (36 -40). For example, mannose-binding proteins have specificity for mannose, glucose, fucose, or GlcNAc, all of which have the equatorial hydroxyl group at the C-4 position, whereas mammalian hepatic lectins have specificity for galactose/GalNAc, which has the axial hydroxyl group of C-4. Despite 85% identity in the sequences of both the hepatic and macrophage receptors, they have distinct sugar binding properties. The hepatic receptor binds GalNAc with a greater affinity than Gal, whereas the macrophage receptor binds Gal and GalNAc with almost equal affinity (38). Specifically, the rabbit and rat ASGPRs bound to GalNAc with a higher affinity than Gal. A terminal ␤-D-galactopyranosyl residue bound to the ASGPRs with a higher affinity than a ␣-D-galactopyranosyl residue and galactose when assaying the ability of the carbohydrates to inhibit 125 I-labeled asialoorosomucoid binding to the liver plasma membrane or the isolated lectin (36). In competition for binding to the carbohydrate recognition domain of rat hepatic lectin-1, GalNAc showed a greater binding affinity due to the effects of the acyl group of GalNAc binding to the carbohydrate recognition domain than did Gal (39). We show in Fig. 5 that GalNAc inhibited hepatocyte adhesion to PV6Gna with between ϳ5and 20-fold higher affinity than the other carbohydrates, indicating that GalNAc has the most inhibitory effect on hepatocyte adhesion to the PV6Gna surface, and ␤-D-galactopyranosyl showed more of an increase of the inhibitory effect on the adhesion than did ␣-D-galactopyranosyl. Interestingly this means that hepatocytes may adhere to the PV6Gna surface through ASGPR on the cell surface membrane. To verify the possibility that ASGPR is mediating the interactions between hepatocytes and PV6Gna, an inhibition cell adhesion assay was carried out on the PV6Gna surface using soluble ASF, which had been used as a natural ligand for ASGPR, as competitive inhibitors against the ASGPR-PV6Gna interaction. Hepatocyte adhesion to the PV6Gna surface was drastically reduced in the presence of soluble PVLA, PV6Gna, and ASF but not fetuin and PVMA (data not shown). Hepatocyte adhesion to PV6Gna was almost completely inhibited by 0.02 M ASF (normalized with FIG. 1. Molecular structure of glycopolymers. molecular weight). The ASF that was used is a desialylated glycoprotein from calf serum fetuin (molecular mass, 48 kDa) that has an average 13.6 sialic acid residues. Hepatocyte adhesion to the PV6Gna surface was inhibited by 1 mM GalNAc as shown in Fig. 5. The relative inhibitory effect of the carbohydrates was determined from the concentration of carbohydrate required for 50% inhibition of PV6Gna binding ([I] 50 ) to hepatocyte. The results indicate that hepatocyte adhesion to PV6Gna surface was inhibited at least 4 ϫ 10 5 -fold more efficiently by ASF ([I] 50 ϭ 1.4 ϫ 10 Ϫ6 mM) than by GalNAc ([I] 50 ϭ 0.6 mM) as shown in Fig. 5. We also showed that the soluble PVLA inhibited hepatocyte adhesion to the PV6Gna surface with a higher affinity than galactose-type monosaccharides or disaccharides (data not shown). It has already been reported that rat hepatocytes bound to PVLA with a higher affinity than to lactose. 2 In addition, we examined recognition of PV6Gna to other cell types in which ASGPRs are not expressed. Hepa 1-6 cells, erythrocytes, NIH 3T3 fibroblasts, and human umbilical vein endothelial cells rarely adhered to the PV6Gna surface compared with the collagen surface or BSA surface as shown in Fig. 6. Hepa 1-6 is a mice hepatoma cell line in which ASGPRs are not detected by reverse transcription-polymerase chain reaction and Western blot analysis (data not shown). Erythrocytes probably attached to the PS surface through a nonspecific hydrophobic interaction. The cell lines could adhere to the PS surface coated with collagen by an integrin-mediated interac-tion. The results also show that the cell lines were apt to adhere to the BSA surface through a nonspecific hydrophobic interaction compared with adherence to PVLA and PV6Gna surfaces. We observed that the hydrophobicity of the BSA surface was higher than the hydrophobicities of the PVLA and PV6Gna surfaces (data not shown). Taken together these results indicate that hepatocyte adhesion to the PV6Gna surface is mediated by ASGPRs on the cell membrane but not by nonspecific interaction.
Flow cytometry analysis as a different assay was carried out using FITC-conjugated PV6Gna to investigate ASGPR-mediated interactions between the hepatocyte cell surface and PV6Gna (Fig. 7). As shown in Fig. 7, a, b, and c, the surfacelabeled hepatocyte fraction (Region 1) was determined from the PI-unlabeled fraction (Region 1 ϩ Region 3), which was estimated as undamaged cells. Hepatocytes were labeled to 25% of the PI-unlabeled fraction in the presence of 0.1 mg/ml FITC-PV6Gna (Fig. 7b). FITC-PV6Gna binding to hepatocytes was increased with an increase of the concentration of FITC-PV6Gna that was added as represented by a hyperbolic plot in Fig. 7d. The FITC-PV6Gna binding to hepatocytes was notably decreased by the addition of EGTA, GalNAc, and ASF but not fetuin (Fig. 7, c and e). We observed that 15-20% of hepatocytes were damaged (PI-labeled fraction, Region 2 ϩ Region 4) during incubation. The FITC-labeled hepatocyte fraction (Region 2) in PI-labeled hepatocytes was also increased with an increase of the concentration of FITC-PV6Gna and inhibited by EGTA (Fig. 7c), ASF, or GalNAc (data not shown). Increasing doses of ASF or GalNAc as a competitive inhibitor produced a 2  dose-dependent decrease of the FITC-PV6Gna binding to hepatocytes as shown in Fig. 7e. Although PV6Gna binding to hepatocytes is almost completely inhibited in the presence of 10 M ASF, it is inhibited no more than 80% in the presence of 100 mM GalNAc. The soluble FITC-PV6Gna binding to hepatocytes was inhibited ϳ5 ϫ 10 4 -fold more effectively by ASF ([I] 50 ϭ 4 ϫ 10 Ϫ4 mM) than by GalNAc ([I] 50 ϭ 20 mM). These results are compatible with the multivalent effect on the inhibition of the interaction between soluble PV6Gna and hepatocytes. We also observed that FITC-PV6Gna or FITC-PVLA binding to hepatocytes was increased with an increase of Ca 2ϩ in the reaction mixture (data not shown). The optimum concentration of Ca 2ϩ for the binding of FITC-conjugated PV6Gna and PVLA to hepatocytes was ϳ0.3 and 0.1 mM, respectively. This result is in agreement with the previous observation of the Ca 2ϩ requirement of hepatocytes to adhere to PV6Gna and PVLA surfaces (19). Our data did not result from nonparenchymal cell contamination because the hepatocyte region was created by adjusting forward scatter and side scatter. These results indicate that soluble PV6Gna also binds to the hepatocyte cell surface mediated by ASGPR in a Ca 2ϩ -dependent manner. Taken together we suggest that PV6Gna specifically interacts with hepatocytes mediated by ASGPR on the cell surface.
Detection of ASGPR with Gel Particles Containing 6-O-Methacryloyl-D-glucose-Mammalian hepatocytes are rich in S-type and C-type lectins, which recognize galactose, glucose, or mannose. Therefore, we could not rule out that the interactions of the hepatocytes and the PV6Gna might be mediated by unknown C-type lectins capable of recognizing terminal galactose residues. To confirm the ability of ASGPRs and the poly-mers modified at the hydroxyl group of C-6 of glucose to associate and to further demonstrate that ASGPRs are specifically recognized by PV6Gna due to the modification of the hydroxyl group position of C-6 of glucose without the effect of the modifying group such as an amide bond, PMglc, which has an ester bond as a modifying group at the 6-OH of glucose, was prepared as described under "Experimental Procedures." We carried out the ASGPR precipitation assay with the hepatocyte cell lysate using PMglc. The precipitated ASGPR was determined by detection of mouse hepatic lectin-1 by Western blot with mono- Hepatocyte adhesion assay was carried out as described previously (19). Briefly, hepatocytes (5 ϫ 10 4 cells/cm 2 ) were allowed to adhere for 30 min at 37°C onto the polystyrene dish (Falcon 1008) that had been coated with a 0.1 mg/ml solution of PVLA, PVMEA, PVMA, PVG, or PV6Gna followed by blocking with 0.5 wt% BSA. The percentage of cell adhesion was determined as described under "Experimental Procedures." b, photomicrograph of hepatocytes adhered to the PV6Gna surface. Scale bar ϭ 100.2 m.
clonal anti-human ASGPR as shown in Fig. 8c. It has been previously shown that the monoclonal anti-human ASGPR was produced by immunizing BALB/c mice with ASGPR purified from human liver and was reactive with mouse hepatic lectin-1 (40). Mouse hepatic lectin-1 was expressed as a band at 42 kDa consistent with previous reports (41,42). We observed that the antibody was not reactive with the lysates of nonparenchymal cells as well as Hepa 1-6 cells in which ASGPR is not expressed as described above (data not shown). Dose dependence experiments showed that the increase of the precipitated ASGPR was dependent on the amounts of the PMglc added for the precipitation (data not shown). In contrast, ASGPR was rarely precipitated with the PMglc in the presence of 1 mg/ml ASF, 2 mM EGTA, or 0.1 mg/ml PV6Gna, but the precipitation was unaffected by 2 mg/ml fetuin. In addition, the precipitation was inhibited by 20 mM galactose but not glucose and mannose as monosaccharide inhibitors (data not shown). In addition, increasing doses of ASF in the reaction mixture provided a dosedependent inhibition in the precipitation of ASGPR with PMglc (data not shown). We carried out ASGPR precipitation with PMglc in the extract that had been treated with ASF-conjugated Sepharose to remove active ASGPR from the hepatocyte lysate. As shown in Fig. 9a, increasing amounts of ASF-Sepharose caused a dose-dependent decrease of ASGPR in the extract. The precipitated ASGPR was drastically reduced to less than 10% of control when the ASF-Sepharose-treated extract was precipitated with PMglc. These results that the precipitation of ASGPRs with PMglc was inhibited by ASF are enough to provide the evidence for the involvement of ASGPR in the interaction between hepatocytes and the glucose-derivatized polymers. Furthermore, we examined the inhibitory effect of GalNAc and methyl ␤-gal on the precipitation of ASGPR with PMglc. GalNAc inhibited ASGPR precipitation by PMglc more effectively than did methyl ␤-gal as shown in Fig. 9b. Because hepatic ASGPR had a greater affinity for GalNAc than for ␤-galactoside, this result demonstrates that the precipitated ASGPRs are hepatic ASGPRs. Using the technique of 13 C NMR analysis of carbohydrates (43), PMglc showed a downfield shift of the peak at C-6 from the results of NMR analysis (data not shown). These shifts resulted from the transesterification of glucose with vinyl methacrylate at the C-6 position of glucose. However, the yield of the glucose monoester from HPLC analysis was 36% of the total product containing glucose di-(52%) and triester (12%), which might act as cross-linkers for gelation of the glucose monoester shown in Fig. 8a. We thought that the reduction of regioselectivity may have been caused by a scale-up of the enzyme reaction as compared with a previous report. Despite the low content of the glucose monoester in the PMglc, hepatocytes recognized PMglc in the same manner as PV6Gna (Fig. 8b). Specifically, ASGPRs bound to PMglc in the hepatocyte cell lysate. The results show direct binding of AS-GPRs to the PMglc by a Ca 2ϩ -dependent and galactose-specific manner, indicating that the ASGPRs on hepatocytes are specifically recognized by glycopolymers modified at the C-6 position of glucose as well as by PV6Gna. Numerous studies have been reported about the regioselective formation of polymerizable sugar esters at 6-OH with various alkylating agents using commercially available protease and lipase (31, 44 -46). Our data provide new information for the design of biomimetic glycopolymers for medical applications such as liver tissue engineering and cell-targeted drug delivery.
CBPs interact with a particular carbohydrate by recognizing subtle differences in the carbohydrate structure. Interaction of galactose/GalNAc and ASGPR occurs through hydrogen bonds and nonpolar interactions between the carbohydrate recognition domain, Ca 2ϩ , and the carbohydrate. It has been reported that ASGPRs essentially require the equatorial hydroxyl group of C-3 and the axial hydroxyl group of C-4 of galactose that bind to the receptors (47,48). Nevertheless, why do ASGPRs on the mouse primary hepatocyte bind to the polymer modified at the 6-OH position of glucose? We hypothesize two combined effects to answer this question. The first combined effect is the effect of the selectivity of the substituted position of the hydroxyl group of glucose. There are many reports that demonstrate affinity for the individual hydroxyl group of galactose bound to ASGPRs. For example, substitution of another monosaccharide in the hydroxyl group of C-6 of galactose rarely affected the affinity for ASGPRs (36). Our previous study showed that hepatocytes could not interact with a glycopolymer substituted at the hydroxyl group of C-1 or C-3 of glucose. Thus, the hydroxyl group of C-6 of the carbohydrates may contribute less to recognition for ASGPRs than the hydroxyl groups of C-1, C-3, and C-4 of the carbohydrates. Alternatively, PV6Gna can bind to ASGPRs probably because the combination of 1-OH in the ␣-anomeric form and 2-OH mimics the equatorial-axial combination of galactose. In the PVMA, there is no such combination available because OH at C-1 of glucose was modified with a vinylbenzyl group. PVG may not be a good ligand because of the linkage of the benzyl group, which is too close to this pair of OH groups. The second combined effect is the polymeric effect of the PV6Gna engaging ASGPR in multivalent binding on the hepatocyte cell surface. Multivalent synthetic glycoprotein inhibits L-selectin-mediated leukocyte rolling more effectively than the corresponding monomer (7). Several reports have demonstrated a multivalent effect of ligands for an optimum recognition to the structure of ASGPRs (49,50). Hepatocyte adhesion onto the PV6Gna surface was inhibited by 2 and 16 M of soluble PVLA and PV6Gna, respectively (data not shown), although the concentrations of PV6Gna and PVLA were normalized (calculated by the molecular weight of the corresponding monomer of the polymers). The results show that on the hepatocyte adhesion, soluble PVLA (500-fold) and PV6Gna (60-fold) have a greater binding affinity than GalNAc. In addition, the hepatocyte adhesion was not affected in the presence of sodium glucuronate and isomaltose as monosaccharide or disaccharide modified at C-6 of glucose, respectively, as well as glucose. In addition, we excluded the effect of the modifying groups on the interactions of ASGPRs and the polymers modified at C-6 of glucose using a different modifying group with an amide bond or ester bond. We suggest the reason that PV6Gna can bind to ASGPR despite the loss of hydrogen bonding at C-4 of glucose might be due to the contribution differences of hydroxyl groups of the sugar and a polymeric effect of sugar ligands during the formation of hydrogen bonds.
It is well known that mammalian primary hepatocytes are FIG. 9. Inhibitory effect of ASF-Sepharose and carbohydrates on ASGPR precipitation with PMglc. a, inhibitory effect of ASF-Sepharose. ASGPR was precipitated by PMglc (25 l of a 50% suspension) from the lysate (2 mg/ml protein) that had been treated with ASF-Sepharose (40, 80, 120, and 240 l of a 50% suspension, respectively). b, inhibitory effect of GalNAc and methyl ␤-gal. ASGPR was precipitated by PMglc from the hepatocyte cell lysate (2 mg/ml protein) in the presence of between 0.01 and 10 mM GalNAc or methyl ␤-gal. MHL-1, mouse hepatic lectin-1. specifically recognized through ASGPRs by glycopolymers that act as natural and artificial ligands having terminal galactose/ GalNAc moieties. However, our data showed that the mouse primary hepatocytes specifically interacted with the PV6Gna due to the modification at the C-6 position of glucose but not at the C-1 and C-3 positions. Furthermore, we first found that ASGPRs could bind to the glucose-derivatized polymer substituted at the hydroxyl group at the C-6 position of glucose although it has been reported that the binding of ASGPR to galactose was not affected by the modification of the hydroxyl group at the C-6 position of galactose. ASGPRs have been identified in peritoneal macrophages as well as in mammalian liver. Subsequent studies demonstrated characteristic differences between the macrophage galactose receptor and the hepatic lectin in carbohydrate specificity as well as in the number and complexity of polypeptide subunits. However, Kupffer cell lectin had a greater affinity to GalNAc than to galactoside with a multivalent effect, indicating that the carbohydrate specificity of Kupffer cell lectin was similar to that of hepatic ASGPR (51,52). Kupffer cell lectin could be recognized by 6-O-phospho-D-glucose and glucosyl-BSA. In this study, we report that hepatic ASGPRs recognize glucose-derivatized polymers depending on the position of the substituted hydroxyl group. Future studies should examine whether glucose-derivatized polymers can recognize the galactose receptor of macrophages including Kupffer cells.
Carbohydrate selectivity for C-type lectins such as ASGPRs and mannose-binding proteins have been demonstrated by binding competition assay and crystallographic analysis for the carbohydrate recognition domain using monosaccharides or disaccharides as ligands. The results showed that ASGPR could not recognize glucose and its derivatives. However, most C-type lectins including ASGPR were recognized by multicarbohydrate ligands in a living body. Although PV6Gna is an artificial glycopolymer that cannot be found in a living body, our study provides new insights into carbohydrate recognition of CBPs especially for the design of biomimetic materials based on interactions between carbohydrates and CBPs.