N-acetylglucosamine and adenosine derivatized surfaces for cell culture: 3T3 fibroblast and chicken hepatocyte response

3T3 fibroblasts and primary chicken hepatocytes were cultured on derivatized polystyrene surfaces to examine the effect of cell-specific ligands on cellular morphology and growth. Surfaces were prepared by derivatizing chloromethylated polystyrene with N-acetylglucosamine (GlcNAc; recognized by the chicken asialoglycoprotein receptor) and adenosine (not recognized by adult hepatocytes). These surfaces were compared with tissue culture polystyrene (TCPS), acid-cleaned glass, and the unmodified chloromethylated polystyrene. The spreading, cytoskeletal structure and growth of the fibroblasts following attachment to these surfaces were examined. The extent of attachment, total protein levels, and DNA contents for surfaces-attached chicken hepatocytes were also measured. Fibroblast spreading was greatest on polymer surfaces derivatized with GlcNAc, whereas cytoskeletal structure and growth rate were independent of surface chemistry. Although chicken hepatocytes attached most efficiently to the GlcNAc derivatized polymer, the total protein and DNA levels of the surface-attached cells were not affected. In anticipation of the application of these polymers for cell culture and hybrid artificial organ design, the GlcNAc-derivatized polystryrene was fabricated into porous microcarriers. Fibroblasts grew avidly on the microcarriers, whereas chicken hepactocytes adhered well to the formed large aggregates arounds the microcarriers.     

Tumor cell haptotaxis on immobilized N-acetylglucosamine gradients

Polyacrylamide surfaces covalently derivatized with quantifiable gradients of glycosides superimposed on a uniform adhesive background of coimmobilized Arg-Gly-Asp-containing adhesion peptide were synthesized. Substrate-directed cell redistribution (haptotaxis) was measured by seeding derivatized surfaces uniformly with B16F10 murine melanoma cells. After 4-32 hr, cells on gradients of N-acetylglucosamine (GlcNAc) redistributed markedly; higher cell densities were found at gel positions having a higher immobilized GlcNAc density. In contrast, cells seeded on otherwise identical gels having a uniform concentration of immobilized GlcNAc, or on gels having gradients of glucose or galactose, did not redistribute. Soluble inhibitors containing nonreducing terminal GlcNAc (but not those with terminal GalNAc or Gal) blocked redistribution on immobilized GlcNAc gradients. Redistribution was not affected by the presence or absence of serum in the medium. An affinity-purified antibody against beta-1,4-galactosyltransferase, a GlcNAc-binding protein reported to be expressed on B16F10 cell surfaces, attenuated GlcNAc-directed redistribution. When cells were seeded on surfaces derivatized with various uniform densities of immobilized GlcNAc coimmobilized with an invariant density of immobilized Arg-Gly-Asp-peptide, neither cell attachment nor proliferation rate were enhanced on the gels having a higher GlcNAc density. These data indicate that the redistribution on immobilized GlcNAc gradients was due to cell motility. Although gels derivatized with Arg-Gly-Asp-peptide alone supported strong B16F10 cell adhesion, surfaces derivatized with uniform high concentrations of GlcNAc did not. We conclude that cell recognition of substratum gradients that support, at best, weak adhesion (GlcNAc) on an otherwise uniform strongly adhesive background (Arg-Gly-Asp-peptide) may be sufficient to direct cell migration.     

Intact cell adhesion to glycan microarrays

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.     

Multiple carbohydrate receptors on lymphocytes revealed by adhesion to immobilized polysaccharides

Phosphomannan polysaccharides and fucoidan, a polymer of fucose 4-sulfate, have been demonstrated to inhibit adhesion of lymphocytes to tissue sections that contain high endothelial venules (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). We have investigated the potential cell surface carbohydrate receptors involved by quantitating adhesion of rat cervical lymph node lymphocytes to purified polysaccharides immobilized on otherwise inert polyacrylamide gels. One-sixth of the lymphocytes adhered specifically to surfaces derivatized with PPME (a phosphomannan polysaccharide prepared from Hansenula holstii yeast), whereas up to half of the cells adhered to surfaces derivatized with fucoidan. Several lines of evidence demonstrated that two distinct receptors were involved. Adhesion to PPME-derivatized gels was labile at 37 degrees C (decreasing to background levels within 120 min) whereas adhesion to fucoidan-derivatized gels was stable. Soluble PPME and other phosphomannans blocked adhesion only to PPME-derivatized gels; fucoidan and a structurally related fucan blocked adhesion to fucoidan-derivatized gels. Other highly charged anionic polysaccharides, such as heparin, did not block adhesion to either polysaccharide-derivatized gel. Adhesion to PPME-derivatized gels was dependent on divalent cations, whereas that to fucoidan-derivatized gels was not. The PPME-adherent lymphocytes were shown to be a subpopulation of the fucoidan-adhesive lymphocytes which contained both saccharide receptors. These data reveal that at least two distinct carbohydrate receptors can be found on peripheral lymphocytes.     

Specific adhesion of rat hepatocytes to beta-galactosides linked to polyacrylamide gels

Rat hepatocytes, isolated by a collagenase perfusion technique, specifically bind to polyacrylamide gel containing covalently immobilized 6-aminohexyl beta-D-galactopyranosyl groups. Less than 5% of these cells bind to polyacrylamide or to gels with the following covalently linked ligands: 6-aminohexanol, or the 6-aminohexyl D-pyranosides of alpha-mannose, beta-glucose, beta-2-acetamido-2-deoxyglucose, beta-cellobiose, beta-maltose, or beta-melibiose. Cell binding to beta-D-galactoside gels occurs after a lag period at 37 degrees and 65 to 100% (depending on the cell preparation) of the cells adhere. The duration of the lag period is inversely related to the beta-D-galactoside content of the gel but preincubation of the cells at 37 degrees reduces the lag period. Cell-gel binding is a threshold phenomenon. Adhesion of cells to gels does not occur when the glycoside concentration is less than about 900 nmol per cm2 x 0.25 mm thick gel piece. Above this critical concentration, cell-gel binding occurs and becomes maximal when the concentration is increased by only 20%. If these in vitro results apply to cellular interactions in vivo, they suggest that slight changes in the levels of cell surface or extracellular matrix carbohydrates may profoundly influence the behavior of neighboring cells.     

The sugar-specific adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii is a sensorium that continuously monitors nutrient levels in the environment

Our earlier studies on cell adhesion to immobilized carbohydrates are extended here to a marine bacterium, Vibrio furnissii. Apparently one lectin mediates the binding of these cells to glycosides of N-acetylglucosamine, mannose, and glucose covalently linked to Agarose beads. Kinetic studies show that protein synthesis is required for initiating and for maintaining adhesion to the glycosides. Furthermore, a pro- mutant binds to GlcNAc-beads at Pro concentrations insufficient to support cell growth. Expression of the functional lectin therefore predominates under conditions of limiting protein synthesis. Thus, cells adhere to the sugars in an environment compatible with protein synthesis, and deadhere when depleted of any required nutrient, presumably to migrate to a more favorable locale. The adhesion-deadhesion apparatus thereby permits constant monitoring of the surrounding environment, comprising a "nutrient sensorium".     

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.     

Binding characteristics of N-acetylglucosamine-specific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin

Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent attachment of an Arg-Gly-Asp sequence peptide to derivatizable polyacrylamide surfaces: support of fibroblast adhesion and long-term growth

A synthetic nonapeptide (Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser), which includes the adhesive Arg-Gly-Asp (RGD) sequence, was covalently immobilized on chemically well-defined polyacrylamide gel surfaces utilizing N-succinimidyl active esters. The amount of peptide immobilized varied linearly with the concentration added to the gels. Immobilization was approximately 80% efficient (based on peptide added), resulting in up to 17.5 nmol peptide/cm2 gel surface. Balb/c 3T3 mouse fibroblast cells adhered readily to peptide-derivatized surfaces, even in the absence of serum. Furthermore, surfaces derivatized with 2 nmol peptide/cm2 gel supported long-term fibroblast growth at a rate and to an extent comparable to that on tissue culture plastic. Surfaces derivatized with a control nonapeptide having no RGD sequence were nonsupportive of cell attachment or growth. The immobilization technology used to derivatize the gel surfaces with adhesive nonapeptide can be modified to allow coderivatization with proteins, glycoproteins, glycosides, or other amine-containing compounds to test their effects on long-term cell behaviors.     

Immobilized glycoconjugates for cell recognition studies

Specific cell-cell recognition and adhesion may involve cell surface glycoconjugates on one cell binding the complementary carbohydrate receptors on an apposing cell surface. Such interactions have been modeled by immobilizing simple synthetic glycosides, glycoproteins, glycosaminoglycans, and glycolipids on otherwise inert plastic surfaces and incubating them with intact cells. Using this approach, the ability of several cell types to recognize specific carbohydrates has been demonstrated. This carbohydrate-directed cell adhesion may depend on cell surface carbohydrate receptors which mediate both the initial specific adhesion and complex postrecognition cellular responses. While the relationship of the cell adhesion demonstrated here to cell-cell recognition in vivo has yet to be determined, this well-controlled biochemical approach may reveal new information on the way in which cells analyze and respond to their immediate external environment.     

Ion-Affinity Electrophoresis. Preparation and Properties of Phosphorylated Polyacrylamede Gels

Strong, pliable polyacrylamide gels containing covalently bound phosphate groups have been formed by radical-initiated copolymerization of acrylamide and a phosphorylated, N-substituted derivative of acrylamide. Under conditions of disc electrophoresis, the gels provide enhanced resolution in the separation of hemoglobin A from S, and separate two forms of yeast glucose 6-phosphate dehydrogenase and rabbit muscle glyceralde-hyde 3-phosphate dehydrogenase. Preliminary evidence suggests that the improved resolution is due to ionic adsorption of the proteins to immobi-lized phosphate groups, implying that the gels may function as supports for electrophoretically powered separations involving ion-exchange or affinity chromatography.     

Direct implication of surface mannosyl residues in cell adhesion of Dictyostelium discoideum

In the cell adhesion of aggregation-competent Dictyostelium cells, the requirement for the carbohydrate moiety of the glycoprotein appeared to be indirect in that it acts to protect the protein moiety from proteolytic degradation; however, the effect was limited to the tunicamycin (TM)-sensitive carbohydrate moiety (Hirano, T., et al. (1983) J. Biochem. 93, 1249-1257). In the present study, we showed that the EDTA-stable adhesion of aggregation-competent Dictyostelium cells was abolished by the treatment of intact cells with jack bean alpha-mannosidase, whereas neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, or alpha-L-fucosidase had no effect. The EDTA-stable cohesiveness of TM-treated cells in the presence of leupeptin (TM/LP cells) was also abolished by the treatment of the cells with alpha-mannosidase. The effect of alpha-mannosidase was not prevented in the presence of LP. The N-glycoside-deficient contact site A (an adhesion-mediating glycoprotein) was obtained from TM/LP cells and was shown to have a molecular weight of 70,000. This protein (p 70) was shown to still have carbohydrates as detected by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and subsequent staining of the gel with periodic acid-silver stain. Moreover, p 70 reacted with anti-gp 68, which has a specificity against alpha-mannosyl residues of carbohydrate chains. However, p 70 treated with alpha-mannosidase showed decreased reactivity with anti-gp 68. The monovalent antibody fragment of anti-contact site A or anti-p 70 inhibited EDTA-stable cell adhesion of both control and TM/LP cells. These results indicated that TM-resistant mannosyl residues of contact site A are directly involved in EDTA-stable adhesion of aggregation-competent cells. This is the first report of the direct involvement of the carbohydrate moiety in cell adhesion of aggregation-competent Dictyostelium cells. A schematic model is presented of the role of the carbohydrate moiety in EDTA-stable cell adhesion, including the direct effect of carbohydrates.     

Hepatocyte adhesion to carbohydrate-derivatized surfaces. II. Regulation of cytoskeletal organization and cell morphology

Rat hepatic lectins mediate adhesion of isolated rat hepatocytes to synthetic surfaces derivatized with galactosides. Initial weak adhesion is followed by rapid adhesion strengthening. After hepatocytes contact galactose-derivatized gels, the hepatic lectins move rapidly into an inaccessible patch at the adhesive surface (Weisz, O. A., and R. L. Schnaar. 1991. J. Cell Biol. 115:485-493). Hepatic lectin patching, which occurs both at 37 degrees C and 4 degrees C, is not responsible for adhesion strengthening, which does not occur at 4 degrees C. Of various cytoskeletal and metabolic perturbants tested, only a combination of hyperosmotic medium, colchicine, and cytochalasin caused a marked (72%) reduction of adhesion strengthening (without reducing weak cell adhesion). Clathrin and actin were readily detected in the adhesive patch by immunofluorescence microscopy. Rat hepatocytes also adhered avidly to surfaces derivatized with asialofetuin, a high-affinity ligand for the rat hepatic lectins. However, hepatic lectin molecules did not migrate into a patch on the asialofetuin-derivatized surface, suggesting that hepatic lectin-asialofetuin binding may have resulted in the rapid formation of a ring of essentially irreversibly adherent receptors that prevented diffusion of additional lectin molecules into the contact site. The cells were unable to increase their adhesive contact area by flattening onto the derivatized surface. Treatment of cells with cytochalasin, however, did result in an increase in the size of the contact area. Cells adhering to surfaces derivatized with an adhesion-promoting peptide (containing an arg-gly-asp sequence) had larger contact areas than those adhering to galactoside-derivatized surfaces. A model is proposed in which carbohydrate-mediated adhesion causes specific reorganization of cytoskeletal components, leading to strengthened adhesion and a characteristic spherical cell morphology.     

Carbohydrate-specific cell adhesion is mediated by immobilized glycolipids

We describe a technique for examining the ability of one important class of cell surface complex carbohydrates, glycosphingolipids, to mediate carbohydrate-specific cell recognition and adhesion. Analogs of natural glycosphingolipids were synthesized, consisting of 1-glycosyl derivatives of 3-deoxyceramide (N-palmitoyl-2-aminostearol) radiolabeled in the fatty acid portion. Methods were developed to efficiently adsorb both these synthetic glycolipids and natural glycosphingolipids (including gangliosides) from aqueous ethanol solution onto plastic wells. The glycolipids remained firmly attached to the surface in aqueous solutions, but could be recovered using detergents or organic solvents. The ability of the adsorbed glycolipids to elicit specific adhesion of intact hepatocytes was tested using specific adhesion of intact hepatocytes was tested using a cell adhesion assay based on that of McClay, D. R., Wessel, G. M., and Marchase, R. B. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4975-4979. When otherwise nonadhesive plastic surfaces were adsorbed with N-acetylglucosaminyl 3-deoxyceramide, they supported adhesion of 80-95% of the chicken hepatocytes added to the well. No adhesion above background levels (10-25%) was observed to surfaces adsorbed with other synthetic glycolipids including glucosyl, galactosyl, mannosyl, or lactosyl 3-deoxyceramide, 3-deoxyceramide, or to the naturally occurring glycosphingolipids, lactosyl ceramide or ganglioside GM1. Chicken hepatocyte adhesion to surfaces adsorbed with N-acetylglucosaminyl 3-deoxyceramide was inhibited by soluble N-acetylglucosamine (IC50 = 3 m M), but not by other soluble sugars. Rat hepatocytes adhered preferentially to surfaces adsorbed with lactosyl 3-deoxyceramide, but not to surfaces adsorbed with the N-acetylglucosaminyl derivative. These studies demonstrate the ability of adsorbed glucolipids to mediate carbohydrate- and cell-specific adhesion from intact cells. Using these techniques, the ability of naturally occurring complex glycosphingolipids to elicit specific cellular responses from a variety of cell types can be examined.     

Comparative study of chicken ovalbumin subfractions having different carbohydrate chain from each other by high performance anion exchange chromatography

1. Ovalbumin was fractionated to six fractions according to their phosphate content by high performance anion exchange chromatography. 2. This method was applied to analyze the phosphate content of ovalbumin subfractions having different carbohydrate chain from each other which were prepared by concanavalin A/Sepharose chromatography from oviduct slices incubated with [2-3H]mannose. 3. Most biosynthetic intermediates bearing a carbohydrate chain of Man8 or 9 GlcNAc2 was not phosphorylated while other fractions bearing differently processed carbohydrate chains such as Man5 or 6 GlcNAc2 or hybrid type carbohydrate chain was phosphorylated at their peptide portion.     

Preparation of 6-aminohexyl d-aldopyranosides

6-Aminohexyl glycosides covalently linked to solid matrices are effective reagents for the isolation of proteins that bind to carbohydrates [Schnaar and Lee, Biochemistry, 14 (1975) 1535–1541], and for the study of interactions between intact cells and immobilized carbohydrates [Weigel et al., J. Biol. Chem., 253 (1978) 330–333]. The preparation of the 6-aminohexyl glycosides of the following D-pyranoses is now described: β-glucose, β-galactose, 2-acetamido-2-deoxy-β-glucose, α-mannose, β-maltose, β-melibiose, β-lactose, and β-cellobiose. These glycosides were prepared by glycosylation of 6-(trifluoroacetamido)hexanol with the appropriate acetylated glycosyl halide in 1:1 (v/v) benzene-nitromethane, with mercuric cyanide as the catalyst. Deacylation of the glycosides was achieved in two steps: use of sodium methoxide for O-deacetylation, and of an anion-exchange resin for N-de(trifluoroacetyl)ation.     

Carbohydrate recognition systems in avian sinusoidal liver cells

We studied the carbohydrate recognition systems on liver sinusoidal cells of adult chicken and 20-day-old embryos. We localized and quantified the binding sites for glycoproteins exposing terminal N-acetylglucosamine (GlcNAc), mannose and galactose (Gal) residues. Sinusoidal liver cells from animals of both ages express on their cell surfaces binding sites for GlcNAc, mannose and galactose residues, while hepatocytes bind glycoproteins with GlcNAc resiudes. The gold particles distribution on Kuffer cells depend on the binding sites and the age considered. Binding sites for GlcNAc and Gal residues are generally present as clusters of gold granules, while mannose-specific binding sites are always as single gold granules. Ligand-gold complexes bound on endothelial cells are always present on the coated regions of the cell surface. The number of GlcNAc and Gal-specific receptors expressed on the cell surface of Kupffer cells undergoes modifications between embryonal and adult life.     

Embryonal carcinoma cell adhesion: the role of surface galactosyltransferase and its 90K lactosaminoglycan substrate

Embryonal carcinoma (EC) cells possess a complex cell surface glycoconjugate called lactosaminoglycan, whose core structure is composed of repeating N-acetyllactosamine (Gal leads to GlcNAc) disaccharides. Recent studies suggest that the cell surface receptor for lactosaminoglycan is galactosyltransferase, which binds terminal GlcNAc residues on various side chains, thus anchoring the glycoconjugate to the cell surface (Shur, B. D. (1982). J. Biol. Chem. 257, 6871-6878.). The results described in this paper suggest that multivalent lactosaminoglycans mediate EC cell adhesions by binding to their surface galactosyltransferase receptors. In the presence of UDPgalactose, but not other sugar nucleotides, EC cell adhesion is reduced and preformed cell adhesions are dissociated. UDPgalactose interferes with EC cell adhesion by forcing the galactosyltransferase reaction to completion, thus dissociating the enzyme from its galactosylated substrate (i.e., lactosaminoglycan), and thereby dissociating EC cells from one another. Lactosaminoglycans purified from EC cell cultures rapidly agglutinate EC cells, and EC cells preferentially adhere to substrates irreversibly derivatized with protein- and lipid-free lactosaminoglycan side chains. Under identical conditions, EC cells do not adhere to either hyaluronate- or chondroitin sulfate-derivatized substrates, relative to underivatized control surfaces. EC cell adhesion to other cells and to lactosaminoglycan-derivatized surfaces can be inhibited by reagents that selectively interfere with surface galactosyltransferase activity. First, alpha-lactalbumin specifically reduces the galactosyltransferase's affinity for its lactosaminoglycan substrate and simultaneously inhibits adhesion. Similar levels of bovine serum albumin have no effect. Second, selective inhibition of surface galactosyltransferase with UDP-dialdehyde also inhibits adhesion, while similar levels of AMP-dialdehyde do not. Results show that 1 mM Ca2+ protects the surface galactosyltransferase activity from proteolysis, which suggests the galactosyltransferase is one of the Ca2+-dependent EC cell adhesion molecules. SDS-PAGE fluorography and gel chromatography analyses have determined that the principal lactosaminoglycan substrate for EC surface galactosyltransferase has an apparent molecular weight of 90K. Taken together, these results suggest that lactosaminoglycans participate in EC cell adhesion by binding to their surface galactosyltransferase receptors.     

Cell attachment and long-term growth on derivatizable polyacrylamide surfaces

Important cellular characteristics, including selective adhesion, growth rate, motility, and differentiation, are controlled, in part, by signals received at the cell surface. The molecular mechanisms for the cell surface control of these cell behaviors are largely unknown. In order to probe the role of specific extracellular molecules in controlling cell function, we report the development of synthetic surfaces which generally support the long-term growth of cells yet can be readily derivatized with a wide variety of molecules of biological interest. Polyacrylamide gels containing a gradient of active ester groups were prepared and then the esters were displaced with ligands to generate a gradient of carboxylic acid, tertiary amine, or hydroxyl groups. When untransformed mouse fibroblasts (BALB/3T3) were seeded on the various surfaces, they attached and grew only on those derivatized with carboxylic acids or hydroxyl groups within narrow concentration ranges. Cell growth rate, density, and morphology on polyacrylamide gels containing the optimal concentration of carboxylic acid groups (approximately 30 mumol/ml) were comparable to those on tissue culture plastic, whereas growth on hydroxyl group-derivatized gels was less extensive. In contrast, short-term (90-min) adhesion to hydroxyl group-derivatized gels was greater than that to carboxylic acid-derivatized gels. Both short-term adhesion and long-term growth required serum. Growth-supportive polyacrylamide gels were readily derivatized with molecules of biological interest. The techniques reported here are applicable to other types of cell in culture since the nature and concentration of substratum functional groups can be easily varied and tested for support of long-term cell growth.     

Coated pits and asialoglycoprotein receptors redistribute to the substratum during hepatocyte adhesion to galactoside surfaces

Rat hepatocytes bind in a sugar-specific and concentration-dependent manner to flat polyacrylamide matrices containing covalently attached galactosyl (Gal) groups. Previous studies (Weigel, P.H., J. Cell Biol. 87, 855, 1980) concluded that binding was likely mediated by the asialoglycoprotein receptor. Here we confirm that adhesion is mediated by this receptor, since cell binding is inhibited by antireceptor antibody and a threshold binding response is also observed when hepatocytes adhere to surfaces coated with asialoorosomucoid, a ligand for this receptor. Cells that had bound to a Gal surface and were then sheared from the surface left a membrane patch behind on the substratum. The cytoplasmic side of these plasma membrane patches was visualized on the substratum by indirect immunofluorescence using antireceptor antibody or anticlathrin antibody. The density of punctate coated pits, visualized with the latter antibody, was enriched in a circular membrane region of about 4 microns 2 area that mediated cell binding. This zone also contained concentrated receptors, although the staining pattern with antireceptor antibody was more uniform and less punctate. The results show that both asialoglycoprotein receptors and coated pits are redistributed at the substratum interface on hepatocytes bound to Gal surfaces.