Specific cell adhesion to immobilized glycoproteins demonstrated using new reagents for protein and glycoprotein immobilization
D D Pless, Y C Lee, S Roseman, R L Schnaar
Journal of Biological Chemistry
Cell surface complex carbohydrates have been implicated in the control of cell-cell interactions. We have modeled such interactions using intact cells and "cell surface analogs," carbohydrates immobilized on otherwise inert synthetic surfaces. Our previous studies demonstrated the ability of very simple monosaccharide ligands immobilized on polyacrylamide gels to elicit specific adhesion from intact hepatocytes (Schnaar, R. L., Weigel, P. H., Kuhlenschmidt, M. S., Lee, Y. C., and Roseman, S.
... 78) J BioL Chem. 253, 7940-7951). In the current report, we describe reagents which permit the covalent immobilization of peptides and proteins (including glycopeptides and glycoproteins) on polyacrylamide gels. The immobilized glycoproteins elicit carbohydrate-specific responses fiom intact cells. T w o bifunctional reagents were synthesized having an acrylyl group at one end and an active ester at the other with aminohexanoic acid@) as spacers. These reagents were readily copolymerized with acrylamide and bisacrylamide, resulting in activated polyacrylamide gels with the desired shapes and porosities. The active ester groups were readily displaced in aqueous buffers at 4 "C by a variety of ligands having primary amino groups, releasing N-hydroxysuccinimide and forming a stable uncharged amide linkage between the ligands and the gels. Conditions for optimal immobilization of aminohexyl glycosides, glycopeptides, and a variety of proteins and glycoproteins are described. The efficiency of protein and glycoprotein immobilization on the activated gels depended on the concentration ¶ molecular size, and type of protein used, the concentration and type of active ester in the gel, the pH and type of buffer used during displacement, and on the porosity of the gel. Although immobilization of glycoproteins on the surfaces of low porosity gel slabs was low (1-2% of the input protein bound) protein staining demonstrated that most of the immobilization took place at the gel surface, as desired for cell adhesion studies. A series of glycoproteins including orosomucoid (sialic acid termini), asialo-orosomucoid (galactose termini), and asialo-agalacto-orosomucoid (N-acetylglucosamine termini) were immobilized on activated polyacrylamide gel slabs. Rat hepatocytes bound only to the asialo-orosomucoid-derivatized gels and chicken hepatocytes only to the asialo-agalacto-orosomucoidderivatized gels, consistent with their known cell surface carbohydrate-binding proteins. The techniques described in this paper can be used to immobilize a variety of peptides, proteins, glycopeptides, and glycoproteins for the study of the control of cell behavior by cell surface receptors, and for a variety of other applications. In earlier studies (1, 2) we reported that intact cells recognize and adhere to specific carbohydrates covalently linked to an otherwise inert insoluble matrix. Chicken hepatocytes bound specifically to polyacrylamide surfaces derivatized with ligands containing N-acetylglucosamine and rat hepatocytes to ligands containing galactose. Our initial studies with hepatocytes demonstrated that: (a) cell-gel adhesion was both cell and sugar specific; ( b ) adhesion was remarkably dependent on the sugar concentration on the synthetic surface, exhibiting a threshold-binding phenomenon; (c) the binding was absolutely dependent on the presence of calcium ions; (d) initial adhesion could be blocked or reversed by addition of the appropriate free sugar. Thus, "cell surface analogs" consisting of simple carbohydrate ligands immobilized on synthetic surfaces were detected by intact cells. Cell surface complex carbohydrates include glycoproteins, glycolipids, and proteoglycans having one or multiple oligosaccharide units which may consist of dozens of carbohydrate residues in highly complex branched arrays (3,4). By contrast our earlier "cell surface analogs" consisted of single carbohydrate residues or disaccharides immobilized on polyacrylamide surfaces. While these carbohydrates were recognized by certain intact cells, they comprise very simple analogs of true ceU surface complex carbohydrates. This was exemplified by inhibition studies on chicken hepatocyte adhesion to GlcNAc gels (1). While soluble N-acetylglucosamine blocked adhesion at millimolar concentrations, more complex glycoproteins having nonreducing terminal N-acetylglucosamine residues blocked adhesion at nanomolar concentrations. In the present studies our ability to detect carbohydrate-directed responses from cells is expanded by devising methods for immobilizing purified glycoproteins on synthetic surfaces for use as cell surface analogs. We have previously reported the synthesis and use of Nsuccinimidyl acrylate for the efficient immobilization of low molecular weight ligands containing primary amino groups on polyacrylamide gels (5). The derivatized gels were synthesized by copolymerizing acrylamide, bisacrylamide, and N-succinimidyl acrylate, followed by treatment of the "activated gel" with the desired ligand, a glycoside containing an amino group 2340 by guest on March 22, 2020 http://www.jbc.org/ Downloaded from