Specific adhesion of primary hepatocytes to a novel glucose-carrying polymer

A new amphiphilic glycopolymer, poly-[N-p-vinylbenzyl-d-glucuronamide] (PV6Gna), was synthesized and characterized. Glucose moieties in the polymer were confirmed to be exposed into outer surface of polystyrene (PS) by direct lectin-enzyme assay. Hepatocytes were specifically interacted with PV6Gna substituted at C-6 of glucose but not poly-[N-p-vinylbenzyl-O--d-glucopyranosyl-[14]-d-gluconamide] (PVMA) and poly-[3-O-p-vinylbenzyl-d-glucose (PVG) substituted at C-1 and C-3 of glucose, respectively, although the glycopolymers were interacted with Con A as lectin for -d-glucose and -d-mannose. The adhesion of hepatocytes was dependent on Ca²⁺ and independent on temperature for the PV6Gna surface unlike integrin-dependent adhesion. Morphologies of hepatocytes on the PV6Gna surface were significantly different from ones on collagen type-I and affected by the coating concentration of PV6Gna onto PS dish and epidermal growth factor (EGF).     

Hepatocyte adhesion on a poly[N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-glucoamide]-coated poly(L-lactic acid) surface

A surface of poly(l-lactic acid) (PLLA) was modified by coating with poly[N-p-vinylbenzyl-4-O-beta-d-galactopyranosyl-d-glucoamide] (PVLA), which was employed to improve the hepatocyte adhesion owing to its amphiphilic property and the presence of a hepatocyte recognition motif. We characterized the surface properties through water contact angle, electron spectroscopy for chemical analysis (ESCA), and scanning probe microscopy (SPM). The effect of PVLA coating on the efficiency of hepatocyte adhesion was evaluated by protein assay and optical microscopy. The surface morphology was under the influence of the concentration of PVLA coating solution and it played a critical role in hepatocyte adhesion. It was confirmed that galactose moieties in PVLA, which can bind to the asialoglycoprotein receptor (ASGPR) on hepatocytes, have a more dominant effect on hepatocyte adhesion than enhanced hydrophilicity. We suggest that the PVLA-PLLA system will be a useful method to improve hepatocyte cell seeding and adhesion onto scaffold matrices.     

Endocytosis via galactose receptors in vivo : Ligand size directs uptake by hepatocytes and/or liver macrophages

We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.     

Carcinoma autoantigens T and Tn and their cleavage products interact with Gal/GalNAc-specific receptors on rat Kupffer cells and hepatocytes

We studied interactions of isolated Thomsen-Friedenreich (T)- and Tn-specific glycoproteins with the Gal/GalNAc-specific receptors on rat Kupffer cells and compared them to those with rat hepatocytes. Immunoreactive T and Tn are specific pancarcinoma epitopes. Electron microscopy of gold-labelled T and Tn antigens revealed their specific binding to Kupffer cells, followed by their uptake via the coated pit/vesicle pathway of receptor-mediated endocytosis. Preincubation of Kupffer cells with GalNAc and GalNAc-BSA, but not GlcNAc or GlcNAc-BSA specifically inhibited binding of the T and Tn glycoproteins. Desialylated, isologous erythrocytes (T RBC) are known to bind to the Gal/GalNAc receptors of rat Kupffer cells and hepatocytes. This attachment was specifically inhibited by T and Tn in a concentration-dependent manner: 50% T RBC-Kupffer cell contacts were inhibited at 8.5.10(-6) mM T and 8.5.10(-5) mM Tn antigen concentrations, respectively. The corresponding figures for hepatocytes were 6.10(-6) mM T and 1.2.10(-6) mM Tn antigen. Amino-terminal cleavage products of the T glycoprotein, possessing clusters terminating in non-reducing Gal/GalNAc, inhibited T RBC binding to Kupffer cells and hepatocytes usually at 10(-2) to 10(-5) mM concentrations, whereas GalNAc, galactose and galactose glycosides inhibited at millimolar concentrations. Galactose-unrelated carbohydrates were inactive at concentrations greater than or equal to 50 mM.     

Immobilization of galactose ligands on acrylic acid graft-copolymerized poly(ethylene terephthalate) film and its application to hepatocyte culture

Surface modification of argon-plasma-pretreated poly(ethylene terephthalate) (PET) films via UV-induced graft copolymerization with acrylic acid (AAc) was carried out. Galactosylated surfaces were then obtained by coupling a galactose derivative (1-O-(6'-aminohexyl)-D-galactopyranoside) to the AAc graft chains with the aid of a water-soluble carbodiimide (WSC) and N-hydroxysulfosuccinimide (sulfo-NHS). The modified PET films were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and water contact-angle measurements. The galactosylated PET films were used as substrates for hepatocyte culture. The effects of surface carboxyl group concentration on the extent of galactose ligand immobilization, the extent of hepatocyte attachment, and the surface morphology were investigated. The amount of the galactose ligands immobilized on the PET surface increased with the AAc polymer graft concentration. AFM images revealed that the surface roughness of the PET film increased after graft copolymerization with AAc, but did not change appreciably with the subsequent immobilization of the galactose ligands. At the surface carboxyl group concentration of about 0.56 micromol/cm(2) or galactose ligand concentration of about 0.51 micromol/cm(2), the hepatocyte culture on the galactosylated surface exhibited the optimum concentration and physiological functions and formed aggregates or spheroids after just 1 day of culture. The albumin and urea synthesis functions of these hepatocytes were comparable to or higher than those of the hepatocytes cultured on the collagen-modified PET substrates.     

Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor

The ligand-binding activity of the galactose/N-acetylgalactosamine-specific receptor (Gal/GalNAc receptor) present on the surface of hepatocytes can be modulated under a number of conditions in the intact cell. The carboxylic acid ionophores monensin and nigericin inhibit endocytosis by the Gal/GalNAc receptor in a concentration-dependent manner. Monensin at a concentration of 100 microM reduces the number of binding sites for asialo-orosomucoid and a tri-branched glycopeptide (F2) 5-10-fold; however, the number of Gal/GalNAc receptor subunits detected at the cell surface by a competitive radioimmunoassay and by immunoprecipitation of surface labeled receptor is not significantly altered. Replacement of NaCl in the medium with either N-methylglucamine or sorbitol to isotonicity also inhibits binding and endocytosis. The monensin, nigericin, N-methylglucamine, and sorbitol treatments have in common the ability to alkalinize the cytosol of the hepatocyte. None of these agents has any effect on binding by the isolated Gal/GalNAc receptor nor is the intracellular pH shift of such a magnitude that it would alter binding by the isolated Gal/GalNAc receptor. This has led us to conclude that the ligand-binding properties of the Gal/GalNAc receptor at the cell surface can be modulated in a transmembrane fashion by events other than those involving pH or Ca2+ regulation at the ligand-binding site itself. Such transmembrane modulation of ligand binding by the Gal/GalNAc receptor may provide a rapid and efficient mechanism for mediating ligand release and immediate return of the receptor to the cell surface.     

Cell-type-specific adhesion onto polymer surfaces from mixed cell populations

The targeted adhesion of a specific cell type from a mixed cell suspension via the surface presentation of a cell-specific ligand is demonstrated. This generic strategy is illustrated by the covalent attachment of a galactose derivative to a polylysine backbone via the amine functionality. Following adsorption of the resultant material to a polymer surface, hepatocyte adhesion is increased via the interaction between galactose and asialoglycoprotein receptors in a concentration-dependent manner. The selective nature of the material is demonstrated by the approximate doubling in the adhesion of hepatocytes relative to a nontargeted cell type (hepatic stellate cells), and an inability of the modified polymer surface to attract additional numbers of the nontargeted cells. This strategy provides a mechanism for controlling the ratios of cell types adhering to scaffold supports, thus enabling the rapid creation of defined coculture systems from heterogeneous cell suspensions.     

Two different cell adhesion molecules--cell-CAM 105 and a calcium-dependent protein--occur on the surface of rat hepatocytes

We have recently identified a 105000 D plasma membrane glycoprotein, denoted cell-CAM 105 (CAM, cell adhesion molecule), that is involved in intercellular adhesion of reaggregating rat hepatocytes (Ocklind &; Öbrink, J biol chem 257 (1982) 6788 [11]). In this communication we identify another cell surface protein that is also involved in hepatocyte cell-cell adhesion. This protein has an apparent molecular weight (MW) of 70000 and can be released from the surface membrane by chelation of calcium with EGTA. Results are presented indicating that it is identical with a previously discovered protein, CDP-1 (CDP, calcium-dependent protein) (Öbrink, Lindström &; Svennung, FEBS lett 70 (1976) 28 [28]). Antisera produced against either cell-CAM 105 or CDP-1 inhibit hepatocyte aggregation, but not attachment to collagen. Cell-CAM 105 and CDP-1 are present on the cell surface as separate components, as judged by the fact that both EGTA treatment and trypsin treatment of hepatocytes selectively make the cells insensitive to blocking of aggregation by antibodies against CDP-1 but not by antibodies against cell-CAM 105. However, although much less efficiently, the antibodies against CDP-1 can recognize a 105000 D protein which is also bound by the antibodies against cell-CAM 105, and under certain conditions the antibodies against cell-CAM 105 seem to recognize a 70000 D protein. CDP-1 may thus be derived from cell-CAM 105, or the two proteins might have a common precursor.     

Selective biorecognition and preservation of cell function on carbohydrate-immobilized phosphorylcholine polymers

To obtain synthetic materials capable of selectively recognizing proteins and cells, and preserving their functions, biomembrane mimetic polymers having a phospholipid polar group and carbohydrate side chains were designed. Poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-2-lactobionamidoethyl methacrylate (LAMA)] (PMBL) was synthesized and coated on substrates by solvent evaporation. Selective binding of galactose-recognized lectin, RCA120, to a PMBL surface was investigated by measurement of surface plasmon resonance. The binding of RCA120 to the PMBL surface was confirmed by a remarkable change in resonance angle. The apparent affinity constant of RCA120 to PMBL3.0 (3.0 mol % LAMA unit in the feed) per LAMA unit was 2.77 x 10(5) M(-1). When a glucose-recognized lectin, concanavalin A, was in contact with PMBL, no change in the resonance angle was observed, and any nonspecific fouling of protein on PMBL was effectively reduced. Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on polymer surfaces. On poly(BMA) (PBMA), many adherent cells were observed and were well-spread with monolayer adhesion, but cell adhesion was reduced on poly(MPC-co-BMA) (PMB). HepG2 adhesion was observed on PMBL because the cell has ASGPRs; the number of cells adhering to the PMBL polymer surfaces increased with an increase in the density of galactose residues on the surface. In contrast, adhesion of NIH-3T3 cells to PMBL was reduced in a manner similar to that on PMB because the NIH-3T3 cells did not have ASGPRs. Cell adhesion to the PMBL surface was well-regulated by ligand-receptor interactions. Furthermore, some of the cells adhering to the PMBL surface had a spheroid form, and similarly shaped spheroids were scattered on the surface. Although poly(BMA-co-LAMA) (PBL) has galactose residues, the adherent cells were spread in a manner similar to those on PBMA. The MPC units in PMBL contribute to make a spheroid formation of HepG2 cells. The amount of albumin secreted from a cell was compared with the chemical structure of the substrate. The spheroid shaped cells cultured on the PMBL surface secreted much more albumin than did the spreading cells that adhered to the PBMA. In conclusion, the biomembrane mimetic carbohydrate-immobilized phosphorylcholine polymers produced a suitable surface for biorecognition and preservation of cell function.     

Studies on the liver sequestration of lymphocytes bearing membrane-associated galactose-terminal glycoconjugates: reversal with agents that effectively compete for the asialoglycoprotein receptor

The removal of "effete" glycoproteins from the circulation represents a proposed physiologic role for the hepatocyte asialoglycoprotein receptor. Our experiments support the hypothesis that this receptor may also be directly involved in the removal from the circulation of cells bearing asialoglycoconjugates. We report that the enhanced liver localization of neuraminidase-treated lymphocytes can be competitively inhibited by the coinjection of asialofetuin (ASF). Fetuin itself was without effect. Competitive inhibition of the liver receptor allowed normal localization to lymphoid tissues of the enzyme-treated lymphocytes, a condition which persisted as long as free ASF was present in the circulation. Our studies support the concept that cell surface carbohydrates play an important role in the tissue distribution of circulating lymphocytes. The process of thymocyte maturation, bone marrow transplantation, and the adoptive immunotherapy with continuous T-cell lines represent conditions where recirculation potential may be influenced by the presence of galactose terminal glycoconjugates.     

Cell surface component(s) involved in rat hepatocyte intercellular adhesion

Aggregation of rat hepatocytes was effectively inhibited by monovalent antibodies (Fab fragments) directed against hepatocyte plasma membranes, but monovalent antibodies against some distinct, known hepatocyte surface antigens had no effect. Surface antigens, which neutralized the Fab inhibiting effect on cell aggregation, could be solubilized from plasma membranes by limited proteolytic digestion. Thus, hepatocyte intercellular adhesion seems to involve specific cell surface components, which may be proteins or protein derivatives.     

Rat hepatocytes bind to synthetic galactoside surfaces via a patch of asialoglycoprotein receptors

The binding of rat hepatocytes to flat polyacrylamide surfaces containing galactose is sugar-specific, requires Ca+2, and occurs only above a critical concentration of sugar in the substratum [Weigel et al., 1979, J. Biol. Chem., 254, 10,830). Binding is completely inhibited by asialo-orosomucoid but not by orosomucoid or asialo- agalacto-orosomucoid, suggesting that cell binding is mediated by asialoglycoprotein receptors. Asialo-orosomucoid was labeled with fluorescein isothiocyanate and used as a direct fluorescent probe to monitor the distribution of cell surface asialoglycoprotein receptors before and after hepatocyte binding to galactoside or control substrata. Cells bound at 37 degrees C were de-adhered at 4 degrees C using the Ca+2 chelator EGTA. The released cells were then stained with fluorescein-asialo-orosomucoid, fixed, washed, and examined by fluorescence microscopy. On freshly isolated cells before binding, the distribution of asialoglycoprotein receptors appears diffuse and nonclustered. However, more than half of the cells released intact from a galactoside surface had a single large (4 micrometer2) fluorescent patch. The receptor patch cannot be detected on cells while they are bound to a galactoside surface but rather only on released cells, indicating that the cell-substratum junction is the site of the receptor patch. No asialoglycoprotein receptor patches (less than or equal to 1%) were observed on cells that were incubated on, but did not bind to, an underivatized polyacrylamide surface or to a surface with a galactose concentration below the critical concentration for binding. Furthermore, no receptor patches were present on cells that had bound to and were subsequently released from substrata that did not contain galactose, including glass, tissue culture plastic, nontissue culture plastic, and collagen. The distribution of asialoglycoprotein receptors is preserved at 4 degrees C because at 37 degrees C the patches disappear with a half-life of approximately 2.6 min. The results directly demonstrate that a large cluster of asialoglycoprotein receptors mediates the binding of rat hepatocytes to a galactoside surface.     

Regulation of asialoglycoprotein receptor expression in the proliferative state of hepatocytes

It is necessary to proliferate hepatocytes and to increase the number of hepatocytes for development of bioartificial liver (BAL) and reconstitutive therapy. But usually the cell has a precarious balance between proliferation and differentiation: as the cell proliferation increases, functional differentiation decreases. Therefore, it is desirable for the hepatocytes to be functional by differentiation as a material for such clinical use not to be proliferative. In this study, we investigated the background of hepatocyte proliferation for the springboard of control between proliferation and differentiation of hepatocytes, and we focused attention to the asialoglycoprotein receptors (ASGP-R) of the hepatocytes. Partially hepatectomized (PH) rats were used as a model animal. When the isolated hepatocytes were plated onto the artificial extracellular matrix of poly-(N-p-vinylbenzyl-O-beta-d-galactopyranosyl-d-gluconamide) (PVLA) having galactose residues as cell-specific ligand, the rate of adhesion was decreased along with liver regeneration. Interestingly, the release of the ASGP-R from hepatocytes in serum after PH in vivo and reduction of ASGP-R of the hepatocytes in the proliferative state occurred due to cell growth in vitro. It is suggested that the ASGP-R on the hepatocyte surface during the differentiation was released in the proliferative state.     

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy--d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10^–8 M and 10^–9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.Abbreviations Z benzyloxycarbonyl - EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride - AH 6-aminohexyl - ASOR aslaloorosomucoid - DMF N-dimethylformamide - DMSO dimethylsulfoxide - Lac lactosyl     

Studies on the inhibition of glycogenolysis by D-galactosamine in rat liver hepatocytes.

Effect of galactosamine on glycogenolysis was studied in isolated hepatocytes. It was found that addition of galactosamine strongly inhibited glycogenolysis in normal hepatocytes. Galactosamine-inhibited glycogenolysis was not stimulated by epinephrine or glucagon. This inhibition was specific as no such inhibition was observed with galactose, 2-deoxy-glucose or glucosamine. The glucagon-stimulated cyclic AMP formation in galactosamine-treated hepatocytes was the same as in normal cells; Glc-1-P and Glc-6-P did not accumulate nor was lactate formation enhanced. The glucose production by hepatocytes from regenerating liver was only slightly inhibited by galactosamine and glucagon addition stimulated glycogenolysis in the presence of the amino sugar.     

Endocytosis of bovine lactoperoxidase by two carbohydrate-specific receptors in rat liver

A perfused rat liver took up bovine lactoperoxidase (LPO) by a Ca2+-dependent, saturable process. This endocytosis was accomplished by both hepatocytes and Kupffer or other nonparenchymal cells (NPCs). The mediating receptors were the Gal/GalNAc lectin of hepatocytes and the Man/GlcNAc lectin of NPCs. Blocking either one of these receptors caused a large shift in distribution of accumulated LPO into the cell type whose receptor was left unblocked, but the extent of uptake was unaffected and the rate was only moderately reduced. Effective inhibition of overall uptake into the perfused organ required the presence of competitors for both receptors. Conversely, LPO was an effective competitor of other ligands (asialoorosomucoid or mannan) for either of the two receptors. The major clearance capacity for LPO was associated with hepatocytes which in suspension took it up by a process completely inhibitable by asialofetuin (ASF) and at a rate more than three times greater than for ASF. A faster cycling time for Gal/GalNAc receptors when bound to LPO is suggested. The glycoprotein selectively lost its affinity for Man/GlcNAc receptors when digested by endoglycosidase H (endo H), suggesting that LPO contains mannose-rich oligosaccharides.     

Correlation between cell-surface hydrophobicity of Candida albicans and adhesion to buccal epithelial cells

Adhesion of four isolates of Candida albicans to buccal epithelial cells was determined after growth of the yeasts in defined medium containing 50 mM glucose or 500 mM galactose as the carbon source. With each isolate, adhesion of galactose-grown yeasts was significantly higher than that of glucose-grown organisms. Yeast cell-surface hydrophobicity was assessed by two methods, a modified hydrocarbon adhesion assay and a more sensitive polystyrene microsphere assay. All four isolates were significantly more hydrophobic after growth on 500 mM galactose than after growth on 50 mM glucose. Overall, a strong positive correlation between adhesion and surface hydrophobicity was observed (r = 0.965). These results are discussed in relation to the role of yeast-surface hydrophobicity in pathogenesis.     

Involvement of leukocyte function-associated antigen-1 (LFA-1) in the invasion of hepatocyte cultures by lymphoma and T-cell hybridoma cells

We studied the interaction of MB6A lymphoma and TAM2D2 T cell hybridoma cells with hepatocyte cultures as an in vitro model for in vivo liver invasion by these tumor cells. A monoclonal antibody against leukocyte function-associated antigen-1 (LFA-1) inhibited adhesion of the tumor cells to the surface of hepatocytes and consequently strongly reduced invasion. This effect was specific since control antibodies, directed against Thy.1 and against T200, of the same isotype, similar affinity, and comparable binding to these cells, did not inhibit adhesion. This suggests that LFA-1 is involved in the formation of liver metastases by lymphoma cells. TAM2D2 T cell hybridoma cells were agglutinated by anti-LFA-1, but not by control antibodies. Reduction of adhesion was not due to this agglutination since monovalent Fab fragments inhibited adhesion as well, inhibition was also seen under conditions where agglutination was minimal, and anti-LFA-1 similarly affected adhesion of MB6A lymphoma cells that were not agglutinated. The two cell types differed in LFA-1 surface density. TAM2D2 cells exhibited 400,000 surface LFA-1 molecules, 10 times more than MB6A cells. Nevertheless, the level of adhesion and the extent of inhibition by the anti-LFA-1 antibody were only slightly larger for the TAM2D2 cells.     

Effects of O-linked glycosylation on the cell surface expression and stability of decay-accelerating factor, a glycophospholipid-anchored membrane protein

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane glycoprotein that protects mammalian host cells from inadvertant complement lysis. The effects of inhibiting mucin-type O-glycosylation on the cell surface expression of DAF were studied by introducing an expression vector for human DAF into wild-type Chinese hamster ovary and ldlD cells. The ldlD cells express reversible defects in the addition of galactose and N-acetylgalactosamine (GalNAc) to oligosaccharide chains on glycoproteins and glycolipids. Mucin-type O-glycosylation of proteins is inhibited in ldlD cells and can be selectively corrected by the addition of GalNAc to the culture medium. The attachment of a phosphatidylinositol phospholipase C-sensitive glycolipid anchor to DAF and its efficient sorting to the cell surface in ldlD cells were independent of galactose and GalNAc additions to glycolipids and proteins. Attachment of galactose and GalNAc to DAF's glycolipid anchor were apparently not required for its normal function. However, in the absence of O-glycosylation DAF was proteolytically cleaved soon after reaching the cell surface, and a large fragment of DAF was released into the culture medium. This rapid proteolysis/release resulted in the expression of very low steady state levels of O-glycosylation-deficient DAF as measured by immunoblotting. These results, in conjunction with those obtained from studies of three other membrane glycoproteins expressed in ldlD cells, suggest that O-linked sugars on membrane glycoproteins may frequently play a role in determining the level of cell surface expression of these proteins.     

Substructural specificity and polyvalent carbohydrate recognition by the Entamoeba histolytica and rat hepatic N- acetylgalactosamine/galactose lectins

Both the Entamoeba histolytica lectin, a virulence factor for the causative agent of amebiasis, and the mammalian hepatic lectin bind to N-acetylgalactosamine (GalNAc) and galactose (Gal) nonreducing termini on oligosaccharides, with preference for GalNAc. Polyvalent GalNAc- derivatized neoglycoproteins have >1000-fold enhanced binding affinity for both lectins (Adler,P., Wood,S.J., Lee,Y.C., Lee,R.T., Petri,W.A.,Jr. and Schnaar,R.L.,1995, J. Biol. Chem ., 270, 5164-5171). Substructural specificity studies revealed that the 3-OH and 4-OH groups of GalNAc were required for binding to both lectins, whereas only the E.histolytica lectin required the 6-OH group. Whereas GalNAc binds with 4-fold lower affinity to the E.histolytica lectin than to the mammalian hepatic lectin, galactosamine and N-benzoyl galactosamine bind with higher affinity to the E. histolytica lectin. Therefore, a synthetic scheme for converting polyamine carriers to poly-N-acyl galactosamine derivatives (linked through the galactosamine primary amino group) was developed to test whether such ligands would bind the E.histolytica lectin with high specificity and high affinity. Contrary to expectations, polyvalent derivatives including GalN6lys5, GalN4desmosine, GalN4StarburstTMdendrimer, and GalN8StarburstTMdendrimer demonstrated highly enhanced binding to the mammalian hepatic lectin but little or no enhancement of binding to the E.histolytica lectin. We propose that the mammalian hepatic lectin binds with greatest affinity to GalNAc "miniclusters," which mimic branched termini of N-linked oligosaccharides, whereas the E.histolytica lectin binds most effectively to "maxiclusters," which may mimic more widely spaced GalNAc residues on intestinal mucins.