Biochemistry zyxwvu 1987, zyxwvut 26, 3949-3957 3949 Assay and Characterization of Carbohydrate Binding by the Lectin Discoidin I Immobilized on Nitrocelluloset Russell E. Kohnken and Edward A. Berger* Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01 545 Received December 2, 1986; Revised Manuscript Received February zyxw 23, 1987 ABSTRACT: Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of '251-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole of discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 OC stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity. A time course of digestion with trypsin showed that a slightly smaller fragment of discoidin I retained carbohydrate binding activity but that fragments approximately half the size of intact discoidin I did not. Chemical modification of discoidin I suggested that oxidation-sensitive, sulfhydryl, and possibly carboxylate side chains are iinportant for carbohydrate binding. Protection by N-acetylgalactosamine indicated that some of those residues may be at or near the carbohydrate binding site, suggesting approaches with which to identify that site. Discoidin 1 is a galactose binding lectin synthesized by the cellular slime mold Dictyostelium discoideum during starva- tion-induced development (Simpson et al., 1974). It is a tetrameric protein composed of four identical subunits (Simpson et al., 1974) of relative molecular weight (Mr)' 28 100 (Poole et al., 1981). Unlike a number of lectins, a function for discoidin I in vivo has been proposed. Discoidin I contains a tetrapeptide sequence homologous to the cell binding site of fibronectin (Pierschbacher et al., 1985), and a cell surface receptor on Dictyostelium cells has been iden- tified which appears to interact with that domain of discoidin I (Gabius et al., 1985). On the basis of experiments demon- strating inhibition of cell-substratum adhesion by both syn- thetic peptides identical with the fibronectin-like domain of discoidin I and specific monovalent antibodies directed against discoidin I, it has been suggested that discoidin I mediates the cell-substratum adhesion which is required for orderly cellular aggregation during development of this organism (Springer et al., 1984). It is not clear whether carbohydrate binding is required for this function, although packaging of discoidin I into multilamellar bodies and subsequent externalization do appear to involve carbohydrate binding (Barondes et al., 1985; Cooper et al., 1986). Unlike the cell binding site, the mmain of discoidin I involved in carbohydrate binding has yet to be localized. 'This work was supported by NIH Grants GM zyxwvutsr 25588, GM 30986, zyxwvutsrqp * Correspondence should be addressed to this author. and CA 12708. In order to facilitate the identification of the carbohydrate binding site of discoidin I, we have examined in detail the carbohydrate binding behavior of this lectin. On the basis of hemagglutination (Frazier et al., 1975), binding to fixed Dictyostelium cells (Bartles & Frazier, 1980), and binding of lactosyl-conjugated bovine serum albumin (Cooper et al., 1983), some characteristics of carbohydrate binding by dis- coidin I have been described. These include sugar specificity (Frazier et al., 1975), metal dependence (Alexander et al., 1983), and sensitivity to a component extracted from Dict- yostelium cells with chloroform/methanol (Bartles et al., 1979). In this paper, we describe an assay which measures binding of radioiodinated asialofetuin (ASF) to lectins im- mobilized on nitrocellulose. This assay is sensitive, simple, and quantitative and has allowed us to characterize further the carbohydrate binding behavior of discoidin I. With this assay, we have identified several reagents which appear to interact with or modify discoidin I at or near its carbohydrate binding site, thereby suggesting strategies for localization of that site. I Abbreviations: ASF, asialofetuin; RCA 60, Ricinus communis ag- glutinin, type I; RCA 120, Ricinus communis agglutinin, type 11; Con A, concanavalin A; GalNAc, N-acetylgalactosamine; SDS, sodium do- decyl sulfate; M,, relative molecular weight; SD, standard deviation about the mean; EDC, zyxwv l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; PBS/Az, 20 mM potassium phosphate, pH 7.2, 150 mM NaCI, and 1 mM NaN,; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone. 0006-2960/87/0426-3949$01.50/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1987 American Chemical Society