Proc. Natl. Acad. Sci. USA
Vol. 74, No. 11, pp. 5056-5059, November 1977 Cell Biology
Selective loss of wheat germ agglutinin binding to agglutininresistant mutants of Chinese hamster ovary cells (cell surface/Scatchard plots/positive cooperativity)
PAMELA STANLEY* AND JEREMY P. CARVER Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8
Communicated by Elkan R. Blout, August 24,1977
ABSTRACT The binding of "25I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 106-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was complex and exhibited positively cooperative binding at the high affinity sites. One of the WGA-resistant mutants (WgaRm) was apparently not altered in its WGA-binding ability compared with parental cells. However, two of the WGA-resistant lines (WgaRI and WgaR') had distinct alterations in their WGA-binding properties specific to certain regions of the binding curve. Neither appeared to be affected in either the highest or lowest affinity regions of the binding curve. Thus, lectin-resistant cell mutants altered in specific lectin-binding sites at the cell surface provide a direct approach to analysis of the complex binding parameters that characterize the interaction of WGA with the plasma membrane.
Cell surface carbohydrate structures are becoming increasingly implicated in the modulation of cellular responses. A major tool in the exploration of the nature of cell surface changes has been the family of carbohydrate-binding proteins known as lectins (1). However, the molecular bases of their interactions with the cell surface are poorly understood and, because of this, quantitative interpretation of lectin studies has not been possible. Biochemical approaches to describing lectin binding sites are severely complicated by the fact that they probably reside in carbohydrate side chains distributed among many glycoproteins or glycolipids of different structure and function. Even acrylamide gel analysis of surface-labeled membranes is ambiguous because glycoproteins can contain more than one type of carbohydrate structure (2). More important perhaps is the problem of characterizing cell surface topography and interactions between binding sites in situ. Detailed binding studies, on the other hand, have the potential of distinguishing between different classes of sites on the basis of their number, affinities, and cooperative behavior. The main problem then becomes the interpretation of the complex binding data and the establishment of correlations between binding parameters and structures
to investigate the correlations between binding parameters and structural entities at the cell surface. In this paper, we describe the detailed binding properties of three such WGA-resistant CHO cell mutants (WgaRI, WgaRli, and WgaRm). The three mutants belong to different complementation groups (4) and exhibit different degrees of resistance to WGA (3, 5). WgaRs cells lack a specific N-acetylglucosaminyltransferase activity that appears to be the biochemical lesion responsible for its complex phenotype (5-8). WgaRnI and WgaRm cells exhibit different defects in the sialylation of surface glycoproteins (unpublished data). WgaRI and WgaRn cells show different alterations in WGA binding specific to the intermediate-affinity region of the binding curve; WgaRxII cells exhibit binding essentially identical to that of parental cells. The extreme complexity of the binding curves revealed by examining a 106-fold range of WGA concentrations combined with the fact that two of the mutant cell lines show specific alterations in particular regions of the curve strongly suggests that it should be possible to use this approach both to test the validity of theoretical interpretations of the parental binding curve and to deduce correlations between specific binding parameters and particular cell surface structures.
MATERIALS AND METHODS Materials. WGA was obtained from Sigma Chemical Co. Solutions were made at 4 mg/ml in phosphate-buffered saline (Pi/NaCI), pH 7.4, filtered through a 0.22-Mm Millipore filter, and stored at 40. Concentrations were determined by using EA = 15 (9). Iodine-125 (100 mCi/ml) was obtained from the Radiochemical Centre, Amersham, England; bovine serum albumin was from Sigma Chemical Co.; N,N',N"-triacetylchitotriose (GlcNAca) was a gift from Nicole Lascelle; Sephadex G-25 (medium) was from Pharmacia Fine Chemicals, Uppsala, Sweden; other chemicals were reagent grade from British Drug Houses, England, or Fisher Scientific Co. Fetal calf serum was obtained from Flow Laboratories. The selection of WgaR mutants from parental CHO lines Pro-5 (a proline-requiring auxotroph) and Gatr2 (a glycine-, adenosine-, thymidine-requiring auxotroph) have been described in detail (5). Three distinct WgaR complementation groups have recently been delineated (4)-I(WgaRI), II(WgaRn) and III(WgaRIn). In those studies, the line Gat-2WgaRn4c was shown to complement with the line previously designated Pro-5WgaRM4B (3, 5). The latter has therefore been reclassified as Pro-5WgaRUI4B (4). The mutant cell lines used in the present
at the cell surface. In an attempt at better definition of lectin receptor specificities, we have undertaken a detailed binding study of wheat germ agglutinin (WGA) to Chinese hamster ovary (CHO) cells. Scatchard analysis of the binding data over a 106-fold range of WGA concentrations gives rise to a curve that exhibits marked positive cooperativity in the high-affinity region and that has not reached saturation even at high WGA concentrations (3). It seemed likely that WGA-resistant mutants of CHO cells known to possess specific alterations in the glycosylation of their
surface glycoproteins would provide the ideal tool with which
Abbreviations: WGA, wheat germ agglutinin; CHO cells, Chinese hamster ovary cells; Pi/NaCl, phosphate-buffered saline; GlcNAc3, N,N',N"-triacetylchitotriose; 125I-WGA, 125-Ilabeled WGA; r, amount of WGA bound per cell; A, free WGA concentration. * To whom to address reprint requests c/o Albert Einstein College of Medicine, 130 Morris Park Avenue, New York, NY 10416.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5056
Proc. Natl. Acad. Sci. USA 74 (1977).
Cell Biology: Stanley and Carver study were Pro-5WgaR13C (4,5), Pro-5WgaRil6A (a new isolate which failed to complement with Gat-2WgaRil4C and behaved recessively in hybrids and is therefore presumed to also belong to complementation group II; ref. 4), and Pro 5WgaRm4B (4, 5). All cell lines were cultured in suspension at 34? in complete a medium containing 10% fetal calf serum as described (5). Iodination of WGA. WGA was labeled to -12-15 MCi/tig by the chloramine-T method in the presence of dimethyl sulfoxide as described (3). The usual reaction mixture contained: 30Mg of WGA, 5.ul of dimethyl sulfoxide, 0.5 mCi of carrierfree 125I, and 2.5 ,g of chloramine-T in 50,Ml of Pi/NaCl. After 15 min at room temperature, the reaction was stopped by the addition of 5 ,ug of potassium metabisulfite and 10 MAl of 0.1 M potassium iodide. Unbound 125I was removed by chromatography on a 9 X 0.6 cm column of Sephadex G-25 (medium). The first peak of radioactivity was pooled, adjusted to 1 ml with Pi/NaCI/0.1% in bovine serum albumin and stored at 40. Experiments with 30 Mg of unlabeled'WGA gave -70% recovery after these procedures. Thus, it was assumed that each preparation of 125I-labeled WGA (125I-WGA) contained -20 Mg of 125I-WGA in 1 ml of Pi/NaCI/0.1% bovine serum albumin. The agglutination and cytotoxic properties and the circular dichroism properties of 125I-WGA prepared in this manner did not differ significantly from those of unlabeled WGA (3). Binding Assay. Cells from exponentially growing cultures (5 to 7 X 105 cells per ml) were washed three times with cold Pi/NaCl, counted in a Particle Data counter, and resuspended at 107 cells per ml with Pi/NaCI at room temperature. 'Each glass assay tube contained 125I-WGA of the appropriate specific activity, Pi/NaCl, and bovine serum albumin (2.5% final concentration) in a volume of 150'ul. laI-WGA of different specific activities was made up as six stock solutions covering the concentration range 0.001 to 1000 Mg/ml. Tubes of the same final WGA concentrations but containing 125I-WGA of different specific activities were included at the overlap of each logarithm of concentration as a control to show that the 125I-WGA and unlabeled WGA had identical binding properties. Tubes were prepared in ice water on the day before an assay and stored at 40 overnight. Radioactivity in each tube was counted in a Nuclear-Chicago Autogamma Counter (efficiency, 41%) before cells (107/ml) were added in a 5S0-M aliquot. The tubes were shaken once and then left for 1 hr at room temperature, by which time equilibrium had been reached even at the lowest 125I-WGA concentration. Shaking the reaction tubes during the incubation did not alter the binding results. Unbound 125I-WGA was removed by filtration of the cells through glass filters (Whatman GF/C) that had been soaked for at least 2 hr in 10% bovine serum albumin (fraction V).- Control experiments showed that
Vol. 74, No. 11, pp. 5056-5059, November 1977 Cell Biology
Selective loss of wheat germ agglutinin binding to agglutininresistant mutants of Chinese hamster ovary cells (cell surface/Scatchard plots/positive cooperativity)
PAMELA STANLEY* AND JEREMY P. CARVER Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8
Communicated by Elkan R. Blout, August 24,1977
ABSTRACT The binding of "25I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 106-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was complex and exhibited positively cooperative binding at the high affinity sites. One of the WGA-resistant mutants (WgaRm) was apparently not altered in its WGA-binding ability compared with parental cells. However, two of the WGA-resistant lines (WgaRI and WgaR') had distinct alterations in their WGA-binding properties specific to certain regions of the binding curve. Neither appeared to be affected in either the highest or lowest affinity regions of the binding curve. Thus, lectin-resistant cell mutants altered in specific lectin-binding sites at the cell surface provide a direct approach to analysis of the complex binding parameters that characterize the interaction of WGA with the plasma membrane.
Cell surface carbohydrate structures are becoming increasingly implicated in the modulation of cellular responses. A major tool in the exploration of the nature of cell surface changes has been the family of carbohydrate-binding proteins known as lectins (1). However, the molecular bases of their interactions with the cell surface are poorly understood and, because of this, quantitative interpretation of lectin studies has not been possible. Biochemical approaches to describing lectin binding sites are severely complicated by the fact that they probably reside in carbohydrate side chains distributed among many glycoproteins or glycolipids of different structure and function. Even acrylamide gel analysis of surface-labeled membranes is ambiguous because glycoproteins can contain more than one type of carbohydrate structure (2). More important perhaps is the problem of characterizing cell surface topography and interactions between binding sites in situ. Detailed binding studies, on the other hand, have the potential of distinguishing between different classes of sites on the basis of their number, affinities, and cooperative behavior. The main problem then becomes the interpretation of the complex binding data and the establishment of correlations between binding parameters and structures
to investigate the correlations between binding parameters and structural entities at the cell surface. In this paper, we describe the detailed binding properties of three such WGA-resistant CHO cell mutants (WgaRI, WgaRli, and WgaRm). The three mutants belong to different complementation groups (4) and exhibit different degrees of resistance to WGA (3, 5). WgaRs cells lack a specific N-acetylglucosaminyltransferase activity that appears to be the biochemical lesion responsible for its complex phenotype (5-8). WgaRnI and WgaRm cells exhibit different defects in the sialylation of surface glycoproteins (unpublished data). WgaRI and WgaRn cells show different alterations in WGA binding specific to the intermediate-affinity region of the binding curve; WgaRxII cells exhibit binding essentially identical to that of parental cells. The extreme complexity of the binding curves revealed by examining a 106-fold range of WGA concentrations combined with the fact that two of the mutant cell lines show specific alterations in particular regions of the curve strongly suggests that it should be possible to use this approach both to test the validity of theoretical interpretations of the parental binding curve and to deduce correlations between specific binding parameters and particular cell surface structures.
MATERIALS AND METHODS Materials. WGA was obtained from Sigma Chemical Co. Solutions were made at 4 mg/ml in phosphate-buffered saline (Pi/NaCI), pH 7.4, filtered through a 0.22-Mm Millipore filter, and stored at 40. Concentrations were determined by using EA = 15 (9). Iodine-125 (100 mCi/ml) was obtained from the Radiochemical Centre, Amersham, England; bovine serum albumin was from Sigma Chemical Co.; N,N',N"-triacetylchitotriose (GlcNAca) was a gift from Nicole Lascelle; Sephadex G-25 (medium) was from Pharmacia Fine Chemicals, Uppsala, Sweden; other chemicals were reagent grade from British Drug Houses, England, or Fisher Scientific Co. Fetal calf serum was obtained from Flow Laboratories. The selection of WgaR mutants from parental CHO lines Pro-5 (a proline-requiring auxotroph) and Gatr2 (a glycine-, adenosine-, thymidine-requiring auxotroph) have been described in detail (5). Three distinct WgaR complementation groups have recently been delineated (4)-I(WgaRI), II(WgaRn) and III(WgaRIn). In those studies, the line Gat-2WgaRn4c was shown to complement with the line previously designated Pro-5WgaRM4B (3, 5). The latter has therefore been reclassified as Pro-5WgaRUI4B (4). The mutant cell lines used in the present
at the cell surface. In an attempt at better definition of lectin receptor specificities, we have undertaken a detailed binding study of wheat germ agglutinin (WGA) to Chinese hamster ovary (CHO) cells. Scatchard analysis of the binding data over a 106-fold range of WGA concentrations gives rise to a curve that exhibits marked positive cooperativity in the high-affinity region and that has not reached saturation even at high WGA concentrations (3). It seemed likely that WGA-resistant mutants of CHO cells known to possess specific alterations in the glycosylation of their
surface glycoproteins would provide the ideal tool with which
Abbreviations: WGA, wheat germ agglutinin; CHO cells, Chinese hamster ovary cells; Pi/NaCl, phosphate-buffered saline; GlcNAc3, N,N',N"-triacetylchitotriose; 125I-WGA, 125-Ilabeled WGA; r, amount of WGA bound per cell; A, free WGA concentration. * To whom to address reprint requests c/o Albert Einstein College of Medicine, 130 Morris Park Avenue, New York, NY 10416.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5056
Proc. Natl. Acad. Sci. USA 74 (1977).
Cell Biology: Stanley and Carver study were Pro-5WgaR13C (4,5), Pro-5WgaRil6A (a new isolate which failed to complement with Gat-2WgaRil4C and behaved recessively in hybrids and is therefore presumed to also belong to complementation group II; ref. 4), and Pro 5WgaRm4B (4, 5). All cell lines were cultured in suspension at 34? in complete a medium containing 10% fetal calf serum as described (5). Iodination of WGA. WGA was labeled to -12-15 MCi/tig by the chloramine-T method in the presence of dimethyl sulfoxide as described (3). The usual reaction mixture contained: 30Mg of WGA, 5.ul of dimethyl sulfoxide, 0.5 mCi of carrierfree 125I, and 2.5 ,g of chloramine-T in 50,Ml of Pi/NaCl. After 15 min at room temperature, the reaction was stopped by the addition of 5 ,ug of potassium metabisulfite and 10 MAl of 0.1 M potassium iodide. Unbound 125I was removed by chromatography on a 9 X 0.6 cm column of Sephadex G-25 (medium). The first peak of radioactivity was pooled, adjusted to 1 ml with Pi/NaCI/0.1% in bovine serum albumin and stored at 40. Experiments with 30 Mg of unlabeled'WGA gave -70% recovery after these procedures. Thus, it was assumed that each preparation of 125I-labeled WGA (125I-WGA) contained -20 Mg of 125I-WGA in 1 ml of Pi/NaCI/0.1% bovine serum albumin. The agglutination and cytotoxic properties and the circular dichroism properties of 125I-WGA prepared in this manner did not differ significantly from those of unlabeled WGA (3). Binding Assay. Cells from exponentially growing cultures (5 to 7 X 105 cells per ml) were washed three times with cold Pi/NaCl, counted in a Particle Data counter, and resuspended at 107 cells per ml with Pi/NaCI at room temperature. 'Each glass assay tube contained 125I-WGA of the appropriate specific activity, Pi/NaCl, and bovine serum albumin (2.5% final concentration) in a volume of 150'ul. laI-WGA of different specific activities was made up as six stock solutions covering the concentration range 0.001 to 1000 Mg/ml. Tubes of the same final WGA concentrations but containing 125I-WGA of different specific activities were included at the overlap of each logarithm of concentration as a control to show that the 125I-WGA and unlabeled WGA had identical binding properties. Tubes were prepared in ice water on the day before an assay and stored at 40 overnight. Radioactivity in each tube was counted in a Nuclear-Chicago Autogamma Counter (efficiency, 41%) before cells (107/ml) were added in a 5S0-M aliquot. The tubes were shaken once and then left for 1 hr at room temperature, by which time equilibrium had been reached even at the lowest 125I-WGA concentration. Shaking the reaction tubes during the incubation did not alter the binding results. Unbound 125I-WGA was removed by filtration of the cells through glass filters (Whatman GF/C) that had been soaked for at least 2 hr in 10% bovine serum albumin (fraction V).- Control experiments showed that
