monoclonal antibodies to the glucose transporter from human ~ryt

THE JOURNAL OF BIOLOGICAL CHEMISTBY 0 1% by The American Society of Biological Chemists, fnc. Vol. 260, No. 15, Issue of July 25, pp. 8668-8675, 19%

Printed in U. S. A.

Monoclonal Antibodies to the Glucose Transporter from Human ~ r y t ~ r o c y t e s IDENTIFICATION OF THE TRANSPORTER AS A M, = 55,000 PROTEIN*

(Received for publication, January 29, 1985)

W. Jeffrey Allard and Gustav E. Lienhard From the Department of Biochemistry, Dartmouth Medical School, Hanouer, New Hampshire 037.56

Using the preparation of purifiedglucoss transporter from human erythrocytes as antigen, we have prepared and characterized six monoclonal antibodies. Three of these antibodies have been shown to be to the glucose transporter by several criteria: they immunoprecipi- tate the transport activity, the cytochalasin €3 binding activity, and 75% of the protein from the solubilized purified preparation. The remaining three antibodies were shown to recognize the same polypeptide by a Western blot procedure. All of the antibodies reacted with the deglycosylated transporter and are thus against peptide determinants; most bound to the cytoplasmic domain of the transporter. The antibodies exhibited a range of effects on cytochalasin B binding, from slight enhancement to modest inhibition to strong inhibition; for this reason they must bind to at least three different epitopes. Western blot analysis of erythrocyte membranes prepared in the presence of protease inhibitors showed that all six antibodies bound to a polypeptide of average M, = 55,000. More- over, by immunological assay this polypeptide ac- counted for 5.3% of the membrane protein, a value similar to that given by cytochalasin B binding. Thus, the proposal that the native transporter is a Mr = 100,000 polypeptide is highly unlikely. The antibodies also react with the glucose transporter in other human cell types, but not with that in rodent or avian cells.

A transport system for glucose of the facilitated diffusion type is present in virtually all animal tissues (1). It is partic- ularly abundant in the human erythrocyte, where it constitutes about 5% of the membrane protein (see Footnote 1). There are conflicting estimates of the molecular weight of the erythrocyte glucose transporter. We and others have described the purification of a glycoprotein of average Mr = 55,000 that, upon reconstitution into vesicles, catalyzes D-glucose transport with characteristics similar to those for transport in the erythrocyte (3-6). The same preparation has been shown to bind cytochalasin B, a potent competitive inhibitor of glucose

* This work was supported by Research Grant GM22996 from the National Institutes of Health. 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. Section 1734 solely to indicate this fact.

The value of 5.1% is calculated from the following data obtained in this laboratory: there are 3.2 nmol of cytochalasin B sites per mg of protein (by amino acid analysis) in erythrocytes depleted of peripheral proteins (2, 37) and l mg of protein of these membranes is derived from 2.9 mg of protein (by amino analysis) of erythrocyte membranes (4, 37). It should be noted that this value is higher than that of 2.5% that is calculated from the literature values of 250,000 sites per erythrocyte ghost (36) and 7.5 X 10“’ mg of protein per ghost (37).

transport (7, 81, with a stoichiometry of about 0.7 per polypeptide chain (4).

In contrast, Langdon and others have proposed that the native erythrocyte transporter is a protein of MI = 100,000 (9, 10,42). This proposal was originally based upon the finding that maltosyl isothiocyanate, a covalent inhibitor of the trms- port system, labeled a protein of MI = 100,000 (9). More recently, Shelton and Langdon developed t p purification procedure that yields a prepar~tion consisting largely of a protein of M, = 100,000 (10). Upon reconstitution into vesicles, this preparation has been shown to catalyze D-glucose transport. Carruthers and Melchior (43) have also reported that a preparation of the erythrocyte M,= 100,000 band 3 protein(s) will carry out glucose transport when reconstituted in a suitable manner.

To account for the discrepancy in the molecular weights, it has been suggested that the protein of M, = 55,000 is a proteolytic fragment of a transporter of M, = 1 0 0 , ~ O (9, 10, 42). In support of this position, it was found that incubation of maltosyl isothiocyanate-labeled membranes leads to the gradual conversion of the label from a M, = 100,000 to a MI = 55,000 species (9). Recently, Shelton and Langdon (10) reported that the Mr = 55,000 protein cannot be detected by gel electrophoresis in erythrocyte membranes prepared from freshly drawn blood in the presence of protease inhibitors (10). Also, when the isolation of the transporter from these membranes was attempted by a slight modification of the previously published procedures for the purification of the MI = 55,000 protein, no protein was recovered in the expected fraction (10). In contrast with these findings, two groups have prepared rabbit antisera to the preparation of MI = 55,000 transporter and these antisera react with a protein of average MI = 55,000 in erythrocyte membranes even when these membranes are isolated in the presence of protease inhibitors (11, 12). Moreover, cytochalasin B can be incorporated into the transporter by photolysis, and p h o t o ~ ~ n i t y labeling of membranes prepared from freshly drawn blood in the presence of protease inhibitors labels a polypeptide of M, = 55,000 in a D-glucose-inhibitable manner (13, 14).

The present study describes the first preparation and the definitive characterization of monoclonal antibodies to the glucose transporter from human erythrocytes. These have been used to resolve the question of the M, of native transporter in erythrocytes. In addition, we intend that this report serve as the basis for several other studies involving the use of these antibodies for the identification and/or isolation of the human glucose transporter (see “Discussion”).

EXPERIMENTAL PROCEDURES

Materials-Commercially available reagents were obtained from the following sources: polyethylene glycol 1500, Merck-Schuchardt (Munich); 2,5,10,14-tetramethylpentadecane (pristane), Aldrich; rabbit antibodies specific for mouse isotypes, Miles; Sepharose C1-6B

8668

Monoclonal Antibodies to the Glucose Transporter 8669

and protein A coupled to Sepharose C1-4B, Pharmacia; octyl glucoside, Calbiochem-Behring; [3H]cytochalasin B, D-["c]glUCOSe, and L- [3H]glucose, New England Nuclear. lZ5I-labeled rabbit anti-mouse Fab was a gift from Dr. S. C. Froehner, Dartmouth Medical School. Erythrocyte membrane lipids were prepared by extraction of membranes with chloroform/methanol(15).

Production and Purification of Monoclonal Antibodies-Female BALB/c mice were immunized with the preparation of purified transporter according to the following protocol (16). Transporter (30 pg) in a 100-pl volume of PBS2 was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously. Seven days later, an injection of 30 pg in incomplete adjuvant was given. After 1 to 5 months, the mice were injected intraperitoneally with 60 pg of transporter in PBS on days 7, 3, 2, and 1 prior to the fusion. After the final boost, a small volume of serum was obtained by tail bleeding and tested for the presence of circulating antibodies to the transporter in an ELISA. For the ELISA, samples were applied to 96-well plastic plates coated with 500 ng of purified transporter/well and anti- transporter antibodies were detected colorimetrically by subsequent applications of alkaline phosphatase conjugated to rabbit anti-mouse immunoglobulin and of p-nitrophenyl phosphate (17). The sera from all mice used as spleen cell donors were positive in the ELISA at a dilution of at least 1 to 400.

Fusions were performed with a 5:3 ratio of immune spleen cells to NS-1 myeloma cells (a gift from Dr. Froehner) in 40% polyethylene glycol 1500 (18). Hybrids were selected by culture in Dulbecco's modified Eagle medium (Gibco) containing 10% fetal bovine serum (Hyclone), 50 pg/ml gentamycin, 50 p M mercaptoethanol, 100 p M hypoxanthine, 0.04 p~ aminopterin, and 16 p~ thymidine. After 2 weeks, hybridoma supernatants were screened for anti-transporter activity by the ELISA. At the same time, positive supernatants were assayed for immunoglobulin isotype by a nitrocellulose dot technique that requires only a few microliters of culture supernatant (19). This screening was useful for the elimination of IgMs, which are inconven- ient because they do not bind to protein A and, in our experience, had low affinities for the transporter. Selected hybridomas were cloned by limiting dilution and the supernatants again were assayed in the ELISA and isotyping procedures.

Ascites fluids were produced by the intraperitoneal injection of 3- 7 X lo6 hybridoma cells in 0.5 ml of PBS into BALB/c mice that had been primed 1 to 3 weeks earlier with 0.5 ml of pristane. IgGs were purified from ascites fluid by chromatography on protein A-Sepharose C1-4B as described by Ey et al. (20). IgMs were purified by precipi- tation with 50% ammonium sulfate, followed by gel filtration of the precipitatedprotein on Sepharose Cl-6B (20). Antibody concentration was determined by absorbance at 280 nm, with the value for 1 mg/ ml taken as 1.4 (20). The purified IgM antibodies were sterilized by filtration through 0.2-pm filters and stored in PBS, 3 mM NaN3 at 5 "C. IgGs were stored in the same buffer at -70 'C.

The two hybridomas producing IgMs to the transporter (mabs M1 and M2) were kindly generated for us by Dr. M. W. Fanger of Dartmouth Medical School, according to a different immunization protocol and by fusion with the P3 myeloma line (21). The control IgMs, mabs CM1 and CM2, were gifts from Dr. Fanger. These antibodies (designated PMN 6 and PM 81 in the literature, respectively) are known to be to certain glycolipids (22); they were purified from ascites fluid in exactly the same way as mabs M1 and M2. There is evidence that the transporter is one of the erythrocyte membrane proteins that carries the AB0 group determinants (23) and during this study we obtained a hybridoma producing an anti-A mab of the IgM class upon immunization with transporter from type A erythrocytes. This antibody has been used as a positive control (see Table 111). The control mab CG1 was prepared from ascites fluid elicited with the mouse myeloma MPC 11 (American Type Culture Collec- tion).

Sealed Ghosts, Erythrocyte Membranes, and Purified Transporter- Sealed ghosts were prepared by incubation of human erythrocyte membranes in PBS at 37 "C, exactly as described by Steck and Kant (24). The fraction of sealed ghosts in the preparations, as measured by the accessibility of NADH-cytochrome c oxidoreductase (24), was 95% or greater.

Erythrocyte membranes were prepared from erythrocytes in two ways. One preparation, which is referred to as "normal," was obtained

The abbreviations used are: PBS, 150 mM NaC1, 10 mM sodium phosphate, pH 7.4; ELISA, enzyme-linked immunosorbent assay; mab, monoclonal antibody; SDS, sodium dodecyl sulfate.

from freshly drawn blood according to the procedure described by Steck and Kant (24). The other, which is referred to as "protease- inhibited" was obtained from freshly drawn blood as described by Shelton and Langdon (10); it was sent to us frozen in dry ice by these investigators and stored at -70 "C. The difference between these two preparations is that in the latter leukocytes are removed completely by passage of the cells through cellulose and also the erythrocytes are treated with protease inhibitors before osmotic lysis. These preparations of membranes were used without further treatment for the experiments described in Figs. 4 and 5.

Erythrocyte membranes depleted of peripheral cytoskeletal proteins, the purified glucose transporter, and purified transporter that had been deglycosylated with endoglycosidase F were prepared exactly as described previously (4, 23, 25). The purified preparation consists of the transporter reconstituted into membranes of erythrocyte lipids that accompany the transporter during purification; the ratio of protein to lipid is about 1:3 by weight.

Immunoprecipitation of the Purified Transporter-Protein A-Seph- arose (48 pl) was incubated with 260 pg of each monoclonal IgG in 1.4 ml of PBS for 1 h at 0 "C. The supernatant was carefully aspirated and the beads were washed once with 500 p1 of 3 mg/ml erythrocyte lipids, 62 mM octyl glucoside, 1 mM EDTA, 50 mM Tris-C1, pH 7.4. The capacity of the beads is 5 mg of IgG/ml of beads (20). The purified glucose transporter, at 60 pglml, was solubilized in 3 mg/ml erythrocyte lipids, 62 mM octyl glucoside, 1 mM EDTA, 100 mM NaC1, 50 mM Tris-C1, pH 7.4, at 0 "C. After a 10-min incubation, the mixture was centrifuged at 175,000 X g for 1 h, to remove insoluble material. Aliquots of the supernatant (0.8 ml, 50 pg of transporter) were added to the IgG-protein A-Sepharose and incubated at 0 "C with intermit- tent mixing for 1 h. The supernatants were then removed, made 1 mM in dithiothreitol, and dialyzed for 15 h against 1 mM EDTA, 100 mM NaC1,50 mM Tris-C1, pH 7.4, at 4 "C. The inclusion of additional erythrocyte lipids in this procedure served two purposes. It inhibited the denaturation of the transporter, which occurs slowly at high ratios of octyl glucoside to phospholipid (4), and it led to the reconstitution of the transporter into sealed vesicles, which form upon the removal of octyl glucoside by dialysis (26).

The simultaneous uptake of 0.1 mM D-[14c]glucose and L-[~H] glucose by the vesicles was measured at 6 "C exactly as previously described (5).

SDS-Gel Electrophoresis and Western Blots-Samples for electrophoresis were prepared by solubilization in 2% SDS (Pierce, lauryl grade), 25 mM dithiothreitol, 10% glycerol, 1 mM EDTA, 0.002% bromphenol blue, 100 mM Tris-C1, pH 6.8. Unless noted otherwise, the samples were prepared without heating. Electrophoresis was performed on slab gels of 10% acrylamide as previously described (27). For Western blotting, the proteins were transferred to nitrocellulose paper; the paper was treated sequentially with bovine serum albumin, the antibody to be tested, and Iz5I-labeled rabbit anti-mouse Fab at 150,000 dpm/ml, and then subjected to autoradiography, according to the procedure in Ref. 27. In some cases, the amount of radioactivity in a band was determined by cutting the band and a blank region of equal size from the nitrocellulose strip and measuring these in a y counter.

Cytochalasin B Binding-The binding of [3H]cytochalasin B to the transporter was measured by equilibrium dialysis, according to the method described previously (28). Binding was measured at a single concentration of cytochalasin B (3 X lo-' M ) considerably below the dissociation constant (1.3 X lo" M). Under these conditions, the value of the ratio of the bound cytochalasin B concentration to the free cytochalasin B concentration is equal, after correction for nonspecific binding to the lipid, to the ratio of the total concentration of transporter to the dissociation constant for cytochalasin B (see the derivation in Ref. 28). Thus, in the immunoprecipitation experiments, this ratio serves as a measure of the concentration of transporter. In the experiments on the effect of the antibodies on binding, pertur- bation of this ratio could be due to either a change in the concentration of transporters capable of binding cytochalasin B, a change in the value of the dissociation constant, or both. Nonspecific binding of cytochalasin B to lipids was determined by measuring the bound/ free ratios for samples held at 100 "C for 2 min. These values, which were 20% or less of the values for the unheated samples, have been subtracted from the corresponding values for the unheated samples.

RESULTS

Immunoprecipitation of Transport Actiuity, Cytochalasin B Binding Sites, and Protein-The seven monoclonal antibodies

8670 Monoclonal Antibodies to the Glucose Transporter

used in these studies, together with the isotype of each, are listed in Table I. Mabs CG1, CM1, and CM2 are control antibodies that do not react with components of the human erythrocyte membrane, whereas the other monoclonal antibodies are the ones to the glucose transporter that we have obtained.

The initial selection of the hybridomas secreting antibodies to the transporter was done by means of a solid phase ELISA. Since the preparation of transporter used in this assay is known to contain the erythrocyte nucleoside transporter (about 3 weight % of the preparation) (29) and could contain as much as 25% of other proteins by weight in all (4), it was necessary to demonstrate that the monoclonal antibodies were specific for the glucose transporter. The preparation of purified transporter was solubilized with octyl glucoside in the presence of added erythrocyte lipids and any reacting protein was absorbed with monoclonal antibody bound to protein A- Sepharose. The proteins remaining in the supernatant were reconstituted into vesicles by dialysis and two functions of the transport system, glucose transport and cytochalasin B binding, were measured.

Representative results for the assay of transport are presented in Fig. 1. In control experiments, it was found that 20 p~ cytochalasin B had no effect on the rate of uptake of L- glucose, but largely inhibited the uptake of D-glucose (data not shown). Thus, the transport activity was taken as the difference between the initial rates of uptake of D- and L- glucose. Immunoprecipitation with mabs G1, G2, and G3 removed 89, 75, and 83% of the transport activity, respectively. The same vesicle preparations that were used in the transport assay were also assayed for their capacity to bind cytochalasin B. The results presented in Table I1 show that

TABLE I Roster of monoclonal antibodies

Isotype Isotype IgG 2a

IgG 2a IgG 1 CM1 IgM

CG1 IgG 2b CM2 IgM

0.161

50 04 7 L e e - l 0.5 Time 1.0 ( m i n l 1.5 2.0

FIG. 1. Removal of transport activity by immunoprecipita- ion. Transporter was solubilized in octyl glucoside with added eryth- xyte lipid and immunoprecipitated by monoclonal antibodies G1 I, 0) or CG1 ( 0 , O ) coupled to protein A-Sepharose. Supernatants ‘ere reconstituted by dialysis and the transport activity was deter- lined by measuring the simultaneous uptake of ~-[’H]glucose (0,O) nd D-[14C]glUCOse (m, 0). See “Experimental Procedures” for details. ’he uptake curves for transporter carried through the procedure lithout the protein A-Sepharose treatment were identical with those .hown for mab CG1.

TABLE I1 Zmmunoabsorption of cytochalasin B binding sites and transporter

protein The vesicles prepared according to the procedure described in the

legend to Fig. 1 were assayed for cytochalasin B binding by equilibrium dialysis (see “Experimental Procedures”). For measurement of the immunoabsorption of protein, 40 pg of transporter was incubated in 150 pl with 150 pg (experiment 1) or 210 pg (experiment 2) of monoclonal antibody coupled to protein A-Sepharose, in a buffer of 43 m M octyl glucoside, 1.2 mM dioleoyl phosphatidylcholine, 1 mM EDTA, PBS. After a 60-min incubation at 0 “C, the protein remaining in the supernatant was measured colorimetrically (41).

Bound % of total protein [cytochalasin B]’ % immunoabsorbed

Mab free Exwriment Exwriment

[cytochalasin B] 1 2

None 2.74 CG1 2.83 9 1 G1 0.44 85 74 72 G2 0.44 85 78 77 G3 0.31 89 74 74

“Mean of duplicate determinations. The individual values were within HI% of the mean.

mabs G1, G2, and G3 absorbed 85 to 89% of the cytochalasin B binding sites, as compared with mab CG1.

Immunoabsorption was also used to determine the fraction of the total protein in the transporter preparation that was bound by the monoclonal antibodies. Mabs G1, G2, and G3 each bound about 75% of the protein at molar ratios of antibody to transporter of about 1.2 and 1.6 (Table 11).

These results show that mabs G1, G2, and G3 are to the glucose transporter and that approximately 75% of the protein in the purified preparation is transporter. Since mabs G4, M1, and M2 are isotypes that bind weakly or not at all to protein A (ZO), they could not be used in the immunoabsorption experiments. However, by means of an alternate method (see below), these have also been shown to react with the transporter.

Binding of the Monoclonal Antibodies to the Deglycosylated Transporter-The glucose transporter is a heterogeneously glycosylated glycoprotein that consists of about 15% carbohydrate by weight. We have recently found that most of the oligosaccharides can be cleaved from the protein with endoglycosidase F (23). In order to determine whether any of the monoclonal antibodies were to carbohydrate determinants, the binding of the antibodies to the endoglycosidase F-treated transporter was examined by the Western blot procedure (Fig. 2). Upon SDS-polyacrylamide gel electrophoresis, the purified deglycosylated transporter preparation exhibits a single sharp band of M, = 46,000 (Fig. 2, lane SS) (23). Each monoclonal antibody to the transporter bound to this polypeptide, whereas no binding was observed with the control antibodies.

These results also suggest that mabs G4, M1, and M2 were to the transporter. In order to be certain of their specificity, we examined whether they bound to the same polypeptide as mab G3, which, as described above, is to the transporter. The supernatants from immunoprecipitation of the purified transporter with mabs G3 and CG1 were used in the Western blot procedure with mabs G4, M1, and M2 (Fig. 3). In each case, the blot with the control supernatant shows a broad band of M, = 50,000 to 65,000 typical of the glycosylated transporter (see Fig. 4C) whereas no band is seen with the supernatant from which the transporter was absorbed.

Localization of the Antigenic Determinants-In order to determine whether the antibodies bound to the inner or outer domain of the transporter, we measured binding to sealed ghosts and to unsealed membranes. The procedure consisted


GI G2 63 G 4 CGI SS M I M 2 CMI CM2 “““fmrnvp-.

215 -

08 - 72 -

i

43 -

29-

FIG. 2. Binding of monoclonal antibodies to the deglycosylated glucose transporter. Endoglycosidase F-treated transporter was subjected to SDS-polyacrylamide gel electrophoresis on a slab gel. One lane was cut from the gel and silver-stained (designated SS) (38). The protein in the remainder of the gel was transferred to nitrocellulose and strips were treated with the designated mabs according to the Western blot procedure. The figure is a composite of results from separate experiments with the IgGs and IgMs. For simplicity, only the silver-stained slice from the IgG experiment is shown; in the experiment with the IgMs, the protein stain also coincided with the antibody binding. In the case of the IgGs, each strip contained 190 ng of transporter and was treated with mab at 20 pg/ml; for the IgMs, the amounts were either 100 (Ml) or 300 (M2) ng and 10 pg/ml, respectively. The approximate mobilities of some major erythrocyte membrane proteins, which we use as standards, are indicated by their M. value in thousands (39).

of incubating each antibody with these preparations, separat- ing the membranes by centrifugation, and assaying the supernatant for protein. The unsealed membranes were ones that had been stripped of peripheral proteins with dilute alkali. This preparation has the advantages that there is no cyto- skeleton to interfere with binding to the cytoplasmic domain and that the membranes are known to be unsealed (25). About 5% of the ghost protein and 12% of the protein in the stripped membranes is transporter ((2) and Footnote 1) and the binding was carried out with a molar ratio of transporter to antibody of 3 or greater.

Mabs G2, G3, and G4 showed significant binding only with the unsealed membranes and are thus to determinants in the cytoplasmic domain (Table 111). This is probably also the case for mab G1, but the conclusion is less certain due to the lower value for binding to the unsealed membranes. Mabs M2 and M3 exhibited no significant binding to either membrane preparation. They may have too low an affinity to bind extensively under these conditions or they may be against determinants that are sterically inaccessible when the transporter is incorporated into membranes.

The antibodies were also tested for their ability to agglutin- ate erythrocytes, as judged by the formation of visible clumps of cells upon mixing the antibodies with the erythrocytes. In agreement with the results of the assay described above, none of the antibodies to the transporter was positive. Under the same conditions, the monoclonal antibody against blood group A (Table 111) completely agglutinated type A erythrocytes.

Effects of Antibodies on Cytochulsin B Binding-The effect of each monoclonal antibody on the binding of cytochalasin B to the purified transporter was examined. This experiment was feasible because the preparation of purified transporter

G 4 M I M 2

FIG. 3. Identification of the glucose transporter as the antigen for mabs G4, M1, and M2. Purified solubilized glucose transporter (100 pglrnl) was treated at 4 ‘C with either G3 or CG1 bound to protein A-Sepharose (550 pg of mab/llO pl of protein A- Sepharose per ml) in 46 mM octyl glucoside, 1.7 mM dioleoyl phosphatidylcholine, 1 mM EDTA, 100 mM NaC1,50 mM Tris-C1, pH 7.4. After 1 h, the supernatants were withdrawn and the octyl glucoside was removed by dialysis. Samples of the two dialyzed supernatants were subjected to SDS slab gel electrophoresis and the protein was transferred to nitrocellulose. Pairs of strips, one from each blot, were treated with the designated mab at 20 pglml. In the absence of any immunoprecipitation, each strip would contain 250 ng of transporter. For each pair, the lejt and right blots are those obtained with the mab CG1 and G3 supernatants, respectively.

TABLE I11 Binding of monoclonal antibodies to sealed ghosts and unsealed

membranes A 20-pg quantity of monoclonal antibody was incubated either

alone or with sealed ghosts (500 pg of protein) or with protein- depleted erythrocyte membranes (225 pg of protein) in a 175-p1 volume of PBS for 2 h on ice. Membranes were pelleted by centrifugation at 27,000 X g for 15 min and the protein remaining in the supernatants was assayed by the Lowry method described in Ref. 41. For each membrane preparation, a blank containing membranes alone was run; these gave values of 13.6 and 1.5 pg for the ghosts and unsealed membranes, respectively. Each incubation was performed in duplicate and in each case the individual values for protein were within &5% of the average value. In order to calculate the per cent of antibody protein bound to the membranes, we first subtracted the blank value from each of the corresponding values for the mixtures with antibody plus membrane. Then, the amount of antibody protein in the supernatant was subtracted from the total amount of antibody protein, given by the corresponding value for the incubations with antibody alone. In several instances, this difference was negative, due to partial absorption of the antibody to the polyethylene centrifuge tube in the absence of additional protein. Finally, this difference was divided bv the value for the total amount of antibodv urotein.

Preparation G1 G2 G3 G4 CG1 M1 M2 CM2 Anti-A’ % antibody protein bound

Sealedghosts 31 12 26 21 13 11 (-11) (-14) 88 Unsealed mem- 51 81 74 94 (-16) 0 (-15) (-40) NDb

hrnnen

’ A mab of the IgM class against blood group type A, which was the type of the ghosts used.

Not determined.


TABLE IV Effects of monoclonal antibodies on the binding of cytochahin B to

the glucose transporter Cytochalasin B binding was measured by equilibrium dialysis, as

described under “Experimental Procedures.” The purified glucose transporter with or without added antibody was dialyzed against [3H] cytochalasin B, in PBS. The respective concentrations of transporter and antibody were 33 and 200 pg/ml in the experiments with the IgG mabs and 31 and 800 pg/ml in the experiments with the IgM mabs. Dialysis with each of the antibodies alone gave bound/free values of 0.02 or less and thus the antibodies themselves do not bind cytochalasin B.

Experiment Mab Bound [cytochalasin B]“/ % free [cytochalasin B]

1 None 2.80 (0.12) CG1

-llC

G1 3.17 (0.02) 1.36 (0.11)

G2 -57

3.61 (0.09) +14 G3 0.61 (0.06) G4

-81 1.46 (0.14)

2 -54

None CM2

2.14 (0.05) 2.53 (0.04)

-15‘

M1 M2

1.52 (0.04) 1.50 (0.10)

-40 -41

“Mean of triplicate determinations, with standard error of the mean in parentheses.

* 100 (value for control mab - value for mab)/(value for control rnab).

‘The values are probably slightly lower than the corresponding values with control mab present due to slight loss of transporter by absorption to the dialysis cell in the absence of additional protein.

consists of largely unsealed membranes; in this preparation, 80% of the transporters have their external domain accessible to neuraminidase and 80% also have their cytoplasmic domain accessible to t r y p ~ i n . ~ A molar excess of antibody to transporter (1.9 for the IgGs and 1.2 for the IgMs) was used.

The results in Table IV show that the monoclonal antibodies to the transporter exhibited a range of effects. Mab G3 almost completely inhibited the binding of cytochalasin B; mabs GI, G4, M1, and M2 partially inhibited binding; and mab G2 slightly enhanced binding. These results thus indicate that the six antibodies recognize at least three different epitopes. There is evidence that the site for cytochalasin B lies in the cytoplamsic domain (30) and thus it is not surprising that its binding is altered by antibodies to this domain.

Identification of the Glucose Transporter in Human Eryth- rocyte Membranes-The glucose transporter was identified in preparations of normal and protease-inhibited erythrocyte membranes by the Western blot procedure (Fig. 4A). With both preparations, each of the relevant antibodies reacted with a broad band of M , = 50,000-65,000 that coincided in mobility with that given by the purified transporter (Fig. 4C, UT). Moreover, the amount of antibody bound to each of the two preparations and therefore the amount of transporter in the two preparations was virtually the same. This conclusion was reached by measuring the radioactivity in the transporter band of each strip treated with an IgG. The values for the ratio of radioactivity in the band from the protease-inhibited membranes to that from the normal membranes were 0.96, 0.80, 0.92, and 0.95 for mabs G1 through 4, .respectively. A control experiment in which different amounts of membrane were used in the Western blots showed that for each monoclonal IgG the radioactivity in the transporter band was approximately proportional to the amount of membrane applied.

Because the samples for the SDS-gel electrophoresis in Fig.

J. R. Appleman and G. E. Lienhard, unpublished experiments.

4A were not heated, it could be argued that both membrane preparations contained a protease that rapidly cleaved the SDS-denatured transporter. The results in panel B of Fig. 4 indicate that this possibility is unlikely. When the samples for electrophoresis were prepared by plunging the membranes into SDS at 100 “C, the Western blots showed no significant difference between the two membrane preparations in either the pattern of antibody binding or the amount of transporter as measured by the radioactivity in the M , = 55,000 band. Heating of the purified transporter in SDS results in a broader band of slightly greater mobility; moreover, there is a reduc- tion in its intensity concomitant with the appearance of a dimer and aggregates of higher M, (Fig. 4C). These changes were evident upon comparison of the Western blots of the unheated and heated samples of membranes (Fig. 4, A and B).

It could also be argued that the monoclonal antibodies only recognize a proteolytic fragment of the transporter that constitutes a small fraction of the total transporter present in the membranes. In order to test this possibility, we performed quantitative Western blots in which the radioactivity associ- ated with the transporter band in the protease-inhibited ghosts was compared with the amounts of radioactivity asso- ciated with known amounts of purified transporter in lanes on the same blots (Fig. 5). According to this procedure, the membranes contained 5.2 f 0.4% transporter by weight. This value agrees with that of 5.1% that can be estimated from a literature value for the number of D-glucose-inhibitable cytochalasin B binding sites and the apparent M, of the deglycosylated transporter.’

Immunological Detection of the Glucose Transporter in Other Celk-We have used the Western blot procedure to search for binding of the monoclonal antibodies to the transporter in several other cell types. In each case, cells, or a membrane preparation therefrom, that had been assayed for glucose transporter through measurement of D-glucose-inhibitable cytochalasin B binding (31) were subjected to the Western blot procedure with mabs G1, G2, G3, G4, and CG1. A sufficient amount of each preparation was used so that if the transporter in it were as reactive as the human erythrocyte transporter, a band would be seen on the Western blot. The following were examined (listed as preparation; source; picomoles of transporter per mg of protein): (a) Hep G2 cells, a human liver cell line (32); Dr. J. D. Yager, Jr., Dartmouth Medical School; 9; (b) low density microsomes from rat adipocytes (31); Dr. D. W. Schroer, Dartmouth Medical School; 35; (c) cellular membranes from mouse 3T3 L1 adipocytes; Drs. S. C. Frost and M. D. Lane, Johns Hopkins University School of Medicine; 9; ( d ) cellular membranes from glucose-starved, Rous sarcoma virus-transformed chick embryo fibroblasts (33); S. A. Olson and Dr. M. J. Weber, University of Virginia School of Medi- cine; 50. The blots of the Hep G2 cells with the anti-transporter antibodies showed a single broad band of M , = 50,000- 65,000 (Fig. 6). The blots of the other three preparations exhibited no bands (data not shown). In a study to be reported in detail elsewhere, we have found that mab G3 binds to the transporter in human adipocytes.4 Thus, on the basis of this limited survey, the monoclonal antibodies bind to the glucose transporter in other human cell types but not in other species.

DISCUSSION

The results show clearly that the six monoclonal antibodies characterized in this study are ones to the glucose transporter. Through the use of these antibodies, the proposition that the

D. W. Schroer and G. E. Lienhard, unpublished results.


A. CB GI G2 G3 G4

215--W

88-jl! 72----

35"

B.

CGI MI M2 CM2 6"1 .,,, ICI I '

C. MI M2 UT HT

m

FIG. 4. Identification of the glucose transporter in human erythrocyte membranes. Normal and protease-inhibited membranes were subjected to SDS-slab gel electrophoresis. A strip was cut off and stained with Coomassie Blue (designated CB). The proteins in the remaining gel were transferred to nitrocellulose. Strips containing the proteins from normal and protease-inhibited membranes were treated in pairs with the various mabs. The figure is a composite of separate experiments done with the IgGs and IgMs. For simplicity, only one of the protein-stained strips from the IgG experiment is shown; there was no difference between the staining patterns of the normal and protease-inhibited membranes and the position of the transporter relative to the stained bands was the same for the IgM experiment as that shown for the IgG experiment. In the case of the IgG experiment, each strip contained 2.2 pg of membrane protein and was treated with 20 pg/ml mab; the values in the IgM experiment were 6 pg and 10 pg/ml, respectively. Each pair of blots is designated by the mab; the left and right strips are those for normal and protease-treated membranes, respectively. Panel A , membranes were solubilized in the SDS sample buffer at room temperature. M, values for the various membrane proteins (39) are given in thousands. Panel B, membranes were solubilized at 100 "C. The SDS sample buffer was placed in a boiling water bath for 1 min and then an equal volume of membrane suspension was added and the mixture was held in the bath for 2 min. Panel C, Coomassie Blue staining patterns given by purified glucose transporter (2.4 pg) with the sample unheated (UT) or heated (HT) as described under Panel B.

purified glucose transporter (average M, = 55,000) is a proteolytic fragment of a M , = 100,000 species in the membrane (9, 10, 42) has been demonstrated to be very unlikely. Each monoclonal antibody detected only a M , = 55,000 polypeptide in both normal and protease-inhibited membranes. The amount of this polypeptide in the two preparations was the same. This amount agreed with the amount expected from the number of transporter-specific cytochalasin B binding sites in the membrane.

I t might be argued that there is a M , = 100,000 precursor of the M , = 55,000 transporter and that, for steric reasons, the monoclonal antibodies cannot bind to this precursor. However, because the six monoclonal antibodies recognize at least three different epitopes, this case is highly improbable. Also, we note that because the M , = 55,000 species constitutes about 5% of the protein in membranes isolated from freshly drawn erythrocytes in the presence of protease inhibitors, any

proteolysis would have most likely occurred in uiuo; and if this were so, the M , = 55,000 transporter must be considered a native species.

Although the conclusion that the transporter has a M, = 55,000 has been reached previously from studies with poly- clonal sera to the human erythrocyte transporter (11,12), the present work is more definitive on two accounts. First, although it was established that these sera contained antibodies to the transporter, they may also have contained antibodies to other erythrocyte membrane proteins of M , = 50,000- 70,000. The reason is that the preparation of purified transporter used as immunogen may include as much as 25% of other proteins; it is known to include the nucleoside transporter, which constitutes about 3% of the protein in the preparation (4, 29). Second, unlike past studies, we have examined the transporter in erythrocyte membranes prepared by the procedure of Shelton and Langdon (10).


A B C D E

FIG. 5. The amount of glucose transporter polypeptide in erythrocyte membranes. A single slab gel contained the following samples; lanes A, B, C, and D, 120, 80, 40, and 0 ng (by amino acid analysis) of the purified preparation of glucose transporter, respectively, and lane E, 1.2 pg (by amino acid analysis) of the protease- inhibited erythrocyte membranes. Lanes A-D also contained 1.2 pg of rabbit erythrocyte membranes as carrier protein. The gel was subjected to the Western blot procedure with mab G3 used at 10 pg/ ml and the radioactivity in the transporter band in each lane on the nitrocellulose was measured. This experiment was repeated 5 times and the value for the percent of transporter given in the text is the average one f standard error of the mean. In this calculation it was assumed that only 75% of the purified preparation is transporter (see Table 11).

G I G 2 G 3 G 4 C G I GI G 2 G 3 G 4 C G I - . I .. -

FIG. 6. Identification of the glucose transporter .in Hep 62 cells. Hep G2 cells or human erythrocytes at 20% cytocrit in PBS were treated with protease inhibitors (2 mM diisopropyl fluorophos- phate, 0.1 mM Ep 475 (40) 5 mM EDTA, 1 pg/ml pepstatin). Aliquots were dissolved in SDS sample buffer and subjected to electrophoresis and the Western blot procedure. Each nitrocellulose strip contained 28 pg of total protein and about 15 ng of transporter. The left and right sets are those blots of the Hep G2 cell and the erythrocyte, respectively, with the designated mabs. In this experiment, the samples of Hep G2 cells and erythrocytes were run on the same slab gel and transferred to the same nitrocellulose sheet, which was subse- quently cut into strips. Thus, the electrophoretic mobility of the transporter in Hep G2 cells is the same as that of the erythroycte transporter.

Our results clearly disagree with the conclusion that these membranes are devoid of M , = 55,000 transporter (10). This conclusion was based upon the observations that the protease- inhibited membranes do not exhibit a significant amount of the M, = 55,000 transporter polypeptide upon SDS-gel electrophoresis and that the M, = 55,000 transporter cannot be purified from them (10). We have no sure explanation for the failure to detect this polypeptide. It seems possible that because the transporter runs as a broad band on electrophoresis, especially after heating of the sample (Fig. 4C), it was not detected and that because the purification procedure was modified slightly, the transporter did not elute in the expected fraction.

We also have no sure or complete explanation to.account for the evidence that the transporter is a polypeptide of M, = 100,000 (see the introduction). It seems possible that maltosyl isothiocyanate, an amino group reagent, tags the anion transporter ( M I = 100,000) as well as the glucose transporter, simply because there is about 8 times as much anion transporter as glucose transporter by weight in the erythrocyte membrane (34). The anion transporter is known to be cleaved by endogeneous proteases to a fragment of about M, = 55,000 (35). With regard to the preparations consisting largely of M, = 100,000 proteins that exhibit glucose transport activity upon reconstitution (10, 43), we note that for one of these (10) there has been no report of the specificity for sugars nor of the effect of cytochalasin B. It also seems possible that the conditions used for the isolation of this protein, which include a very high concentration of octyl glucoside (170 mM) and no thiol, lead to the irreversible dimerization of the M I = 55,000 transporter (see Ref. 23). In the case of the other report (43), the specific transport activity of the band 3 preparation is only 5 to 9% of that of the MI = 55,000 preparation. As the authors note, this result may be due to the contamination of their band 3 fraction from DEAE-chromatography with the MI = 55,000 transporter. Since the transporter is heterogeneously glycosylated, a small portion of it may behave differ- ently upon chromatography.

Several of the monoclonal antibodies described here have proven crucial for other projects. They have enabled the identification of the glucose transporter as a substrate for protein kinase C, both in membranes and in the intact cell.5 They have served to detect putative clones containing cDNA coding for the transporter in a library prepared from the mRNA of Hep G2 cells? In future, it should be possible to purify the glucose transporter from erythrocytes and other human tissues to homogeneity by immunoaffinity chromatography. This will allow rigorous protein chemistry to be carried out with these transporters. In addition, because the nucleoside transporter is a major impurity in the preparation of erythrocyte transporter, substantial purification of the former should also be achieved,

Acknowledgments-We are deeply indebted to Drs. M. W. Fanger and S. C. Froehner and their associates for assistance in the production of the monoclonal antibodies. We thank Drs. R. L. Shelton, Jr. and R. G. Langdon for their generosity in providing us with the protease-inhibited erythrocyte membranes.

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