/ . Biochem. 82, 661-669 (1977)
Membrane Receptors of Mouse Lymphocytes for Various Lectins1 Makoto IWATA, Hayao IDE, Tadao TERAO, and Toshiaki OSAWA Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, April 28, 1977
The major glycoproteins which bind various lectins (Bauhinia purpurea, Lens culinaris, Pisum sativum, Ricinus communis, and wheat germ hemagglutinins) were isolated from splenic lymphocytes of DD-mice. The surface of the cells was radioiodinated using the enzyme lactoperoxidase and the crude membranes (microsomal fraction) were isolated from the radioiodinated cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on affinity adsorbents prepared by coupling the lectins to activated Sepharose 4B. The glycoproteins specifically eluted with haptenic sugars from the affinity adsorbents were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The mitogenic lectins {P. sativum, L. culinaris) and R. communis hemagglutinin, which preferentially interact with the sugar chains of the type found in serum glycoproteins (scrum glycoprotein-type sugar chains), bind strongly membrane glycoproteins with molecular weights ranging from 80,000 to 20,000, whereas the other lectins, which can interact with sugar chains such as those of mucins (mucin-type sugar chains), bind membrane glycoproteins with molecular weights higher than 100,000. To characterize the receptor sites for mitogenic lectins on the surface of lymphocytes, the membrane glycoproteins of C3H/He-mouse splenic lymphocytes, which were specifically eluted from a column of/". jaC/v«m-Sepharose 4B, were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, and the histocompatibility-2-complex proteins (H-2D, H-2K, and la antigens) were found to be major receptor sites.
The lectins have several peculiar biological activities. These activities are assumed to stem from initial binding of the lectins to receptor sites of carbohydrate nature on the cell surface (/, 2). 1
The structure of these cell surface receptor sites has often been inferred simply from the specificities of lectins disclosed by hapten inhibition assays using simple sugars as hapten inhibitors. In previous
This investigation was supported by research grants from the Ministry of Education, Science and Culture of Japan and from the Japan Society for the Promotion of Science (the Japan-U.S. Cooperative Science Program). Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M; IgD, immunoglobulin D; a-MM, methyl a-D-mannopyranoside; la antigens, I-region associated antigens. Vol. 82, No. 3, 1977
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studies (3, 4), we showed that the results of hapten hemagglutinin (10), L. culinaris hemagglutinin (11) inhibition assays using simple sugars as inhibitors and R. communis hemagglutinin (12) were purified cannot always be correlated to the structure of according to the methods described previously. receptor sites on the cell surface. We have ac- P. sativum hemagglutinin was purified by the cordingly carried out extensive studies on the method of Entlicher et al. (13). These purified carbohydrate-binding specificities of lectins by lectins were coupled to activated Sepharose 4B by means of hemaggultination inhibition assays using the method described by Matsumoto and Osawa various glycopeptides (5) and chemically synthe- (14). Wheat germ agglutinin-Sepharose 6MB was sized oligosaccharides (6, 7) as hapten inhibitors purchased from Pharmacia Fine Chemical and the inhibition of lectin-binding to human (Uppsala, Sweden). erythrocytes and lymphocytes with various glycoPreparation of Lymphocytes—Spleens of mice proteins and glycopeptides (8). From the results were teased apart at 4°C in Hank's balanced salt of these investigations, we can classify the lectins solution (15) purchased from Nissui Co., Tokyo. into three groups. The members of the first After removal of clumps by sedimentation for 10 group (class I), which contains Agaricus bisporus, min, cells were collected by centrifugation (150 X g Arachis hypogaea, and Bauhinia purpurea hemag- for 10 min), washed twice with the same medium glutinins, bind preferentially to the O-)S-D-galac- and suspended in fetal calf serum (Grand Island topyranosyl-( 1 —>3) - 2 - acetamido - 2 - deoxy-D-galac- Biological Co., Madison, Wis., U.S.A.). Purified tose sugar sequence in sugar chains as those of lymphocytes were obtained by subjecting the cell mucins (mucin-type sugar chains), whereas the suspension to Ficoll (Pharmacia Fine Chemical)— members of the second group (class II), which Urografin (Schering, Berlin, Germany>density contains concanavalin A, Lens culinaris, Pisum gradient centrifugation as described by Kawaguchi sativum, and Ricinus communis hemagglutinins, et al. (16). The cells were washed three times with bind primarily to different but overlapping portions Hank's balanced salt solution and three times with of the sugar chains of the type found in serum 10 mM sodium phosphate buffer (pH 7.2) containglycoproteins (serum glycoprotein-type sugar ing 0.15 M NaCl. Labeling of Lymphocytes—Radioiodination of chains). On the other hand, the members of the third group (class III), which contains soy bean lymphocytes was performed by virtually the same and wheat germ agglutinins, can bind to both types method as that described by Hubbard and Cohn of sugar chains. The presence of these two types (17). To the suspension of lymphocytes (6x10* of sugar chains on the surface of lymphocytes has cells) in 1 ml of 10 mM sodium phosphate buffered saline (pH 7.2) were added 3.6 munits of lactobeen confirmed (9). Mannheim, To reveal the nature of the major receptor peroxidase (Boehringer Mannheim, 1M Germany) and 300 ftCi of Na[ r] (New England sites for these three classes of lectins on mouse splenic lymphocytes and to characterize the re- Nuclear Corp., Boston, Mass., U.S.A.). Then ceptor sites for the lectins which are mitogenic to 3.6 munits of glucose oxidase (Boehringer) and lymphocytes, we analyzed membrane glycoproteins 5 ftmol of glucose were added to the mixture. of mouse splenic lymphocytes using affinity ad- After incubation for 20 min at room temperature, sorbents prepared by coupling various lectins to 10 ml of ice-cold 10 mM sodium phosphate buffered Sepharose 4B. The results of these studies are saline (pH 7.2) containing 10 mM Nal was added to stop the reaction. The labeled cells were then described here. washed several times with ice-cold 10 mM sodium phosphate buffered saline (pH 7.2). MATERIALS AND METHODS Isolation of Crude Plasma Membranes—Crude Animals—DD mice (7 to 10 weeks old) were plasma membrane fraction was obtained according obtained from Nippon Bio-Supp. Center, Tokyo to the methods of Allan and Crumpton (18) and and C3H/He mice (7 weeks old) were obtained Kornfeld and Siemers (19) with a few modifications. s from the Institute of Medical Science, University of The lymphocytes (6 x 10 cells) were suspended in 2 ml of ice-cold 10mM Tris-HCl buffer (pH 7.2) Tokyo. Lectins and Affinity Adsorbents—B. purpurea containing 0.15 M NaCl and 1 mM EDTA. To /. Biochem.
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this cell suspension, 2 ml of 10 mM Tris-HCl buffer extensive dialysis, this fraction was concentrated (pH 7.2) containing 1 mM EDTA was added slowly and analyzed by sodium dodecyl sulfate polyacrylwhile the cell suspension was being stirred. The amide gel electrophoresis. mixture was left at 0°C for 5 min, and then 10 ml of Sodium Dodecyl Sulfate Polyacrylamide Gel 10 mM Tris-HCl buffer (pH 7.2) containing 0.18 M Electrophoresis—The eluate from the affinity NaCl and 1 mM EDTA was added to restore the column was dissolved by boiling in an equal volume NaCl concentration to 0.15 M. TO this mixture, of 2% sodium dodecyl sulfate for 5 min. 2phenylmethanesulfonyl fluoride (Sigma Chemical Mercaptoethanol was added to a final concentration Co., St. Louis, Mo., U.S.A.) in ethanol was added of 40 mM to reduce disulfide bonds. The electroto a final concentration of 1 mM. Then the cells phoresis was carried out in 5.6% polyacrylamide were homogenized in a loosely fitting glass Potter- gel at pH 7.4 according to the method of Fairbanks Elvehjem homogenizer using five to seven gentle et al. (20). In the case of immunoprecipitate, the strokes. The homogenate was centrifuged at discontinuous sodium dodecyl sulfate disc gel 300 x g for 15 min to remove the cell nuclei. The electrophoresis system was used as described by supernatant was collected and centrifuged again as Laemmli (27). After electrophoresis, the gels were above. The volume of this nucleus-free super- immersed in 10%(w/v) trichloroacetic acid for 12 h. natant was measured, and an amount of 3 M LiCl The fixed radioactivity in each slice was determined sufficient to give a final concentration of 0.1 M LiCl in an Alloka well-type autogamma counter. Mouse was added to the supernatant, which was then immunoglobulins ft and y and light chains were centrifuged for 45 min at 45,000xg in a Hitachi used as reference proteins. The following proteins, 65P ultraccntrifuge. The crude membranes thus obtained from Mann Research Laboratories obtained were suspended in 5 ml of lOmM Tris- (Orangeburg, N.Y., U.S.A.), were used as standHCl buffer (pH 7.2) containing 1 mM EDTA and ards for the estimation of molecular weights 1 mM phenylmethanesulfonyl fluoride, and then (molecular weights shown in parentheses): horse homogenized to a fine suspension by three passes heart cytochrome c (12,400), ox pancreas chymoin a Potter-Elvehjem homogenizer. These re- trypsinogen A (25,000), ovalbumin (45,000), bovine suspended crude membranes (microsomal fraction) serum albumin (67,000), and human ^-globulin were again centrifuged at 45,000 x g for 45 min. (160,000). The distance of migration of the memIsolation of Membrane Receptors for Lectins brane proteins was found by determination of —The radioactively labeled microsomal fraction radioactivity and those of the standard proteins obtained from 6x10* cells was suspended in 1 ml by staining the gels with Coomassie Brilliant Blue._ of 10 mM Tris-HCl buffer (pH 7.0) containing 1 mM Indirect Precipitation of Cell Membrane Prophenylmethanesulfonyl fluoride. Triton X-100 teins—The Triton X-100 extract of the crude mem(Wako pure Chemical Co., Tokyo) was added to a brane fraction prepared from splenic lymphocytes final concentration of 0.2%. The cell suspension (6x10* cells) of C3H/He mice was applied to a was mixed vigorously and incubated at 4°C for 1 h. column of P. sativum hemagglutinin-Sepharose 4B After centrifugation at 3,000 x g for 10 min at 4°C, (0.9x13.5 cm) equilibrated with 10 mM Tris-HCl the Triton X-100 extract was applied to an affinity buffer (pH 7.0) containing 0.2% Triton X-100. column (0.6 x 10 cm unless otherwise specified) Elution was carried out with the same buffer. equilibrated with 10mM Tris-HCl buffer (pH 7.0) After the first peak had been eluted completely, the containing 0.2% Triton X-100. Elution was car- active fraction was eluted with 0.2 M methyl a-Dried out with the same buffer and, after the first mannopyranoside solution containing 0.2% Triton peak had been completely eluted, the active fraction X-100. Fractions of 1.6 ml were collected at 4 ml/ was eluted with an appropriate haptenic sugar h at room temperature. The specifically eluted solution containing 0.2 % Triton X-100. Fractions fraction was then concentrated to one third of the of 0.8 ml were collected at 4 ml/h at room temper- volume in a collodion bag (SM 13200; Sartorius ature. Radioactivity was tested on each fraction Membrane Filter Co., Gottingen, Germany). Then in an Alloka autogamma counter. The specifically 10 fi\ of 1 M solution of phenylmethanesulfonyl eluted fractions were pooled and dialyzed against fluoride was added to inhibit enzymic breakdown 50% (v/v) ethanol to remove the detergent. After of labeled proteins, and the mixture incubated for Vol. 82, No. 3, 1977
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30 min at 4°C. To deplete a component which binds to antigen-antibody complexes (22), the following specific immunoprecipitation experiments were always preceded by three precipitations achieved by adding goat anti-human IgG serum (Miles Laboratories, Kankakee, IU., U.S.A.) and an equivalent amount of human IgG (Miles). After centrifugation at 15,000 xg for 15 min, 0.1 ml of rabbit anti-mouse immunolglobulin serum (Miles) was added and the mixture incubated at 37°C for 30 min. Precipitation was caused by adding an equivalent amount of C3H/He mouse serum to provide a carrier protein. Incubation was allowed to proceed for 30 min at 37°C and then at 4°C overnight. The precipitates were centrifuged at 15,000xg for 15 min, washed with 10ml sodium phosphate buffered saline (pH 7.2) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis by the method of Laemmli (21). The supernatants were then treated with a mouse alloantiserum specific for the H-2D antigen and rabbit anti-mouse immunoglobulin serum. The precipitates obtained were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The same sandwich technique was also used to precipitate the H-2K antigen and I-region associated antigens (la antigens) from the supernatant by using an antiserum specific to the H-2K. and la antigens and rabbit anti-mouse immunoglobulin serum. The specific alloantiserum specific for the H-2D antigen, (AxBALBG)Fi anti-BALB-K, and the alloantiserum against the H-2K antigen and the la antigens, C3HOL anti-C3H, were kindly provided by Dr. S.G. Nathenson (Albert Einstein College of Medicine, Bronx, N.Y., U.S.A.). RESULTS AND DISCUSSION The solubilized crude membrane fraction prepared from radioiodinated DD-mouse splenic lymphocytes was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis by the method of Fairbanks et al. (20) using a gel concentration of 5.6%. As shown in Fig. 1, many protein peaks of various molecular weights were seen, which shows the complex nature of membrane glycoproteins of lymphocytes. To characterize the membrane receptor sites for B. purpurea hemagglutinin, which belongs to
Relative
mobility
Fig. 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of crude membranes (microsomal fraction) of radioiodinated mouse splenic lymphocytes. The microsomal fraction was extracted with 0.2% Triton X-100 at 4°C for 1 h and subjected to electrophoresis in 5.6% polyacrylamide gel at pH 7.4 in the presence of 1 % sodium dodecyl sulfate according to the method of Fairbanks et al. (20). p, r, and L refer to positions of standard mouse immunoglobulin chains. The direction of protein migration is from left to right. class I and binds preferentially to mucin-type sugar chains (5, 8), the solubilized crude membrane fraction was subjected to affinity chromatography on a column of Sepharose 4B coupled with B. purpurea hemagglutinin (Fig. 2). When the column was washed with 10 mM Tris-HCl buffer (pH 7.0) containing 0.2% Triton X-100, approximately 90% of the radioactivity was recovered without retardation, and thereafter, a slow release of the remaining radioactivity was observed continuously. The strongly adsorbed membrane glycoproteins were finally eluted with 0.2% Triton X-100 solution containing 0.2 M lactose, which is a specific inhibitor against B. purpurea hemagglutinin. Total recovery of the radioactivity usually reached more than 95 %. The fractions which were specifically eluted with lactose (strongly-bound fractions), and those which were shown by a double-headed arrow in Fig. 2 (loosely-bound fractions), were then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the apparent molecular weights of radioactive glycoproteins separated were estimated by comparison with the migration of the standard J. Biochem.
MEMBRANE RECEPTORS FOR LECTINS
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0.2M Lactose
E a
10
20 30 40 Fraction number
50
60
70
Fig. 2. Affinity chromatography of solubilized crude membranes of radioiodinated mouse splenic lymphocytes on a B. purpurea-Sephaiose 4B column (0.6 X 15 cm). Experimental details are described in the text. a
tr
proteins. As shown in Fig. 3a, the strongly-bound fractions mainly consist of glycoproteins having molecular weights greater than 100,000 whereas the loosely-bound fractions are mainly composed of glycoproteins of smaller molecular weights (Fig. 3b). A peak with an approximate molecular weight of 210,000 is most marked in the stronglybound fractions. These results and the fact that this lectin strongly binds to the O - /3 - galactopyranosyl-{l—>3)-2-acetamido-2-deoxy-D-galactose sugar sequence in mucin-type sugar chains (5; Kaifu, R. and Osawa. T., unpublished observation) suggest that the glycoproteins with high molecular weights (> 100,000) bear mucin-type sugar chains in their molecules. In contrast, the loosely-bound fractions shows the strongest radioactivity in the peak having approximate molecular weight of 55,000. Then the solubilized crude membrane fraction of radioiodinated DD-mouse splenic lymphocytes was subjected to affinity chromatography on columns of Sepharose 4B coupled with mitogenic lectins, P. sativum and L. culinaris hemagglutinins, both of which belong to class II and bind preferentially to the sugar sequences containing amannopyranosyl residues in serum glycoproteintype sugar chains (6). A typical example of these chromatographies is shown in Fig. 4. In these affinity chromatographies, approximately 8 % of the Vol. 82, No. 3, 1977
Relative
mobility
Fig. 3. Sodium dodecyl sufate polyacrylamide gel electrophoresis of radioiodinated membrane glycoproteins eluted from B. purpurea-Scpheuose 4B. Experimental details are in the text, (a) Strongly-bound fractions, (b) loosely-bound fractions. The numbers in the figure are the estimated molecular weights of the peaks indicated by arrows. The other symbols are used as described in the legend to Fig. 1.
Triton X-100-extracted radioactivity remained bound to the affinity adsorbents, even after extensive washing with 10 mM Tris-HCl buffer (pH 7.0) containing 0.2% Triton X-100. Total recovery of the radioactivity after elution of strongly bound glycoproteins with 0.2 M methyl a-D-mannopyranoside (a-MM) solution varied between 96 and 98%. When the lymphocyte membrane glycoproteins specifically eluted from a column of P. sativumSepharose 4B were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, radioactive peaks corresponding to approximate molecular weights of 80,000, 52,000, 46,000, 31,000, 24,000, and 15,000 were reproducibly observed as
666
M. IWATA, H. IDE, T. TERAO, and T. OSAWA
1.0 E Q.
>
K
E a.
0.2Ma-MM
X
A
0.5
oct
| o
TJ
a a.
o o •o a a.
i
10 20 30 Fraction number
40 0.5
1.0
Fig. 4. Affinity chromatography of solubilized crude Relative mobility membranes of radioiodinated mouse splenic lympho- Fig. 6. Sodium dodecyl sulfate polyacrylamide gel cytes on a P. sativum-SephSiiosc 4B column. Experi- electrophoresis of radioiodinated membrane glycopromental details are described in the text. teins specifically eluted with 0.2 M a-MM from L. culinara-Sepharose 4B. The same symbols are used as described in the legend to Fig. 3. Experimental conditions are described in the text. E
a
o o
•5 o 0.5 Relative
1.0
mobility
Fig. 5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glycoproteins specifically eluted with 0.2 M a-MM from P. sativwn-Sepharose 4B. The same symbols are used as described in the legend to Fig. 3. Experimental conditions are described in the text. shown in Fig. 5. Within the experimental error of the molecular weight estimation, almost the same set of radioactive peaks was seen in sodium dodecyl sulfate polyacrylamide gel electrophoresis of the membrane glycoproteins that were specifically eluted from a column of Sepharose 4B coupled with L. culinaris hemagglutinin (Fig. 6). Furthermore,
the glycoproteins of the same range of molecular weights (20,000-80,000) as that observed in the cases of P. sativum and L. culinaris hemagglutinins had also been observed by Nilsson and Waxdal (23) in the membrane glycoproteins of mouse lymphocytes specifically eluted from the affinity matrix prepared by attaching concanavalin A to Affi-gel 10. However, relative intensities of these radioactive peaks were different among these mitogenic lectins used for the preparation of affinity adsorbents, possibly reflecting a subtle difference in carbohydrate-binding specificities of these mannosebinding mitogenic lectins. R. communis hemagglutinin also belongs to class II, and it has strong affinity to the O-/3-Dgalactopyranosyl-(l—>4)-2-acetamido-2-deoxy-Dglucose sugar sequence in serum glycoprotein-type sugar chains (5, 7, 8). The solubilized crude membrane fraction of radiolabeled DD-mouse splenic lymphocytes was applied to a column of R. com7rtun£y-Sepharose 4B and the bound radioactivity (approximately 5 % of the radioactivity of the solubilized membrane fraction) was eluted with 0.2 M lactose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the specifically eluted fractions (Fig. 7) exhibited many radioactive glycoproteins of apparent molecular weights ranging
/ . Biochem.
MEMBRANE RECEPTORS FOR LECT1NS
667
E a.
o o a a. 0.5 0.5
1.0
Relative
1.0 mobility
Fig. 8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glycoproFig. 7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glyco- teins specifically eluted with 0.5 M N-acetyl-D-glucosaproteins specifically eluted with 0.1 M lactose from mine from wheat germ agglutinin-Sepharose 4B. The R. communis-Sepharose 4B. The same symbols are used same symbols are used as described in the legend to as described in the legend to Fig. 3. Experimental Fig. 3. Experimental conditions are described in the text. conditions are described in the text. Relative
m o b i l i ty
from 140,000 to 24,000. However, strong radioactivity was always found in the peaks of the molecular weights between 80,000 and 41,000. The peaks in this region of molecular weights were also found to be strongly radioactive in the experiments which employed other class II lectins (P. sativum and L. culinaris hemagglutinins) having a strong affinity to mannose-containing sugar sequences in serum glycoprotein-type sugar chains, as shown in Figs. 5 and 6. These results are consistent with our previous data (5, 8) that R. communis hemagglutinin belongs to class II. However, the large molecular weight peaks with relatively weak radioactivity in Fig. 7 suggest that this lectin has rather broad carbohydrate-binding specificity and can weakly bind also to /3-D-galactopyranosyl residues in mucin-type sugar chains. Finally, the solubilized crude membrane fractions were subjected to affinity chromatography on a column of wheat germ agglutinin-Sepharose 6MB. Wheat germ agglutinin belongs to class III in our classification and can bind to both serum glycoprotein-type and mucin-type sugar chains (Saito, M., Toyoshima, S., and Osawa, T., unpublished observation). The glycoproteins that Vol. 82, No. 3, 1977
bound to the affinity adsorbent were specifically eluted with 0.5 M N-acetyl-D-glucosamine solution containing 0.2% Triton X-100. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, the specifically eluted glycoproteins exhibited many radioactive peaks with almost equal intensity of radioactivity over the range of molecular weights from 140,000 to 30,000 (Fig. 8). These results are in good agreement with the assumption that this lectin belongs to class III and has broad carbohydrate-binding specificity. Although we cannot exclude the presence of membrane glycoproteins which could not be eluted from the affinity adsorbents with haptenic sugars, mitogenic lectins invariably bind the glycoproteins of apparent molecular weights ranging from 80,000 to 20,000. Since the heavy and light chains of immunoglobulins and the his.tocompatibility-2complex proteins are found in this molecular weight range (24-28), immunoprecipitation with specific antisera was carried out to characterize these glycoproteins. The specifically eluted membrane glycoproteins of C3H/He-mouse splenic lymphocytes from P. sativum-Sepharose 4B affinity adsorbent were first treated with rabbit anti-mouse immunoglobulin serum. Precipitation was effected by
668
M. IWATA, H. IDE, T. TERAO, and T. OSAWA
adding an equivalent amount of C3H/He mouse serum to provide a carrier protein. When the precipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, three peaks were observed as shown in Fig. 9. On the basis of the approximate molecular weights estimated and the migration of mouse immunoglobulin (i and r and light chains, the component of molecular weight 73,000 was tentatively identified as the ft chain from cell surface IgM, and the component of molecular weight 65,000 as the d chain from cell surface IgD. These estimated molecular weights agrees well with the values reported by other investigators {24,25). However, the lack of specific antisera precludes the final characterization of these immunoglobulins. The protein peak with •a molecular weight of around 24,000 is possibly 1he light chain of the cell surface immunoglobulins. These immunoglobulins might have come from B-cells in mouse splenic lymphocytes. The supernatant, after precipitation of the cell-sulface imrmunoglobulin as above, was further treated with a n alloantiserum specific for the H-2D antigen or an alloantiserum specific for the H-2K and la antigens. Precipitation was brought about by the sandwich technique, by adding an equivalent amount of rabbit anti-mouse immunoglobulin serum. When the precipitates obtained using the
anti-H-2D serum were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, only one peak, with an approximate molecular weight of 45,000, was observed (Fig. 10a). The precipitates with the alloantiserum specific for the H-2K and la antigens were likewise analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 10b). Two distinct peaks, with molecular weights around 46,000 and 31,000, were always seen, and in some experiments, an additional peak with a molecular weight 24,000 was observed. From these results and from the estimated molecular weights of the peaks, the glycoproteins with approximate molecular weights of 45,000 and 46,000 could be the H-2D and H-2K antigens and the proteins with approximate molecular weights of 31,000 and 24,000 are possibly la antigens.
10 20 30 40 Sigmin t numb* r
50
60
Fig. 9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glycoprogeins eluted with a-MM from P. jo/iVum-Sepharose 4B after immunoprecipitation by adding rabbit anti-mouse immunoglobulin serum and an equivalent amount of C3H/He mouse serum. The same symbols are used as described in the legend to Fig. 3. The experimental conditions are described in the text.
20 Sigmtnl
30
40
50
60
number
Fig. 10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the specifically eluted radioiodinated membrane glycoprotein from P. MZ/vum-Sepharose 4B which remained after immunoprecipitation with rabbit anti-mouse immunoglobulin serum, (a) Immunoprecipitation of H-2D antigen with an alloantiserum specific for the H-2D antigen, (b) Immunoprecipitation of H-2K and la antigens with an alloantiserum specific for the H-2K and la antigens. /. Biochem.
MEMBRANE RECEPTORS FOR LECTINS These estimated molecular weights of H-2 and la antigens correspond closely to those reported by other investigators (25-28). Since the glycoproteins with molecular weights which correspond to those of H-2 complex proteins are invariably seen in the specifically eluted fraction from the column of Sepharose 4B coupled with T-cell mitogens [P. sativum and L. culinaris hemagglutinins in this paper and concanavalin A (23)], and since the presence of la antigens on T-cells has been indicated by analyzing the pokeweed mitogenbinding glycoproteins from the membrane of purified T-cells (29), these H-2-complex proteins may play an important role in the mitogenic triggering of T-cells, serving as major cell surface receptor sites for mitogenic Iectins. A final conclusion must await further investigations of cell surface receptor sites of several other T-cell mitogens, and the molecular architecture of H-2 and la antigens. We wish to thank Dr. S.G. Nathenson for the generous gift of antisera directed against H-2-complex proteins and Drs. S. Migita and T. Kishimoto for mouse IgG and IgM. REFERENCES 1. Lis, H. & Sharon, N. (1973) Annu. Rev. Biochem. 42, 541-574 2. Nicolson, G.L. (1974) Int. Rev. Cytol. 39, 89-120 3. Toyoshima, S., Fukuda, M., & Osawa, T. (1972) Biochemistry 11, 4000-4005 4. Osawa, T., Terao, T., Kawaguchi, T., Fukuda, M., Toyoshima, S., & Irimura, T. (1974) Co/log. Int. C.N.R.S. 221, 765-776 5. Irimura, T., Kawaguchi, T., Terao, T., & Osawa, T. (1975) Carbohyd. Res. 39, 317-327 6. Kaifu, R., Osawa, T., & Jeanloz, R.W. (1975) Carbohyd. Res. 40, 111-117 7. Kaifu, R. & Osawa, T. (1976) Carbohyd. Res. 52, 179-185 8. Kawaguchi, T. & Osawa, T. (1976) Biochemistry 15, 4581^586
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669 9. Saito, M., Toyoshima, S., & Osawa, T. (1977) / . Biochem. 81, 1203-1208 10. Irimura, T. & Osawa, T. (1972) Arch. Biochem. Biophys. 151, 475-482 11. Toyoshima, S., Osawa, T., & Tonomura, A. (1970) Biochim. Biophys. Ada 11\, 514-521 12. Tomita, M., Kurokawa, T., Onozaki, K., Ichiki, N., Osawa, T., & Ukita, T. (1972) Experimemia 28, 84-85 13. Entlicher, G., Kostin, J.V., & Kocourek, J. (1970) Biochim. Biophys. Acta 111, 272-281 14. Matsumoto, I. & Osawa, T. (1972) Biochem. Biophys. Res. Commun. 46, 1810-1815 15. Hanks, J.H. & Wallace, R.E. (1949) Proc. Soc. Exp. Biol. Med. 71, 196-200 16. Kawaguchi, T., Matsumoto, I., & Osawa, T. (1974) /. Biol. Chem. 249, 2786-2792 17. Hubbard, A.C. & Cohn, Z.A. (1972) /. Cell Biol. 55, 39(MO5 18. Allan, D. & Crumpton, M.J. (1970) Biochem. J. 120, 133-143 19. Kornfeld, R. & Siemens, C. (1974) / . Biol. Chem. 249, 1295-1301 20. Fairbanks, G., Steck, T.L., & Wallach, D.F.H. (1971) Biochemistry 10, 2606-2616 21. Laemmli, U.K. (1970) Nature (London) 227,680-683 22. Goding, J.W., White, E., & Marchalonis, J.J. (1975) Nature (London) 257, 230-231 23. Nilsson, S.F. & Waxdal, M.J. (1976) Biochemistry 15, 2698-2705 24. Melchers, U., Vitetta, E.S., McWilliams, M., Lumm, N.E., Phillips-Quagliata, J.M., & Uhr, J.W. (1974) /. Exp. Med. 140, 1427-1431 25. Finkelman, F.D., Shevach, E.M., Vitetta, E.S., Green, I., & Paul, W.E. (1975) /. Exp. Med. 141, 26. Schwartz, B.D. & Nathenson, S.G. (1971) / . Immunol. 107, 1363-1367 27. Cullen, S.E., David, C.S., Schereffler, D.C., & Nathenson, S.G. (1974) Proc. Natl. Acad. Sci. U.S. 71, 648-652 28. Rask, L., Lindblom, J.B., & Peterson, P.A. (1974) Nature (London) 249, 833-836 29. Yokoyama, K., Terao, T., & Osawa, T. (1977) Biochem. J. 165, 431^37
Membrane Receptors of Mouse Lymphocytes for Various Lectins1 Makoto IWATA, Hayao IDE, Tadao TERAO, and Toshiaki OSAWA Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, April 28, 1977
The major glycoproteins which bind various lectins (Bauhinia purpurea, Lens culinaris, Pisum sativum, Ricinus communis, and wheat germ hemagglutinins) were isolated from splenic lymphocytes of DD-mice. The surface of the cells was radioiodinated using the enzyme lactoperoxidase and the crude membranes (microsomal fraction) were isolated from the radioiodinated cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on affinity adsorbents prepared by coupling the lectins to activated Sepharose 4B. The glycoproteins specifically eluted with haptenic sugars from the affinity adsorbents were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The mitogenic lectins {P. sativum, L. culinaris) and R. communis hemagglutinin, which preferentially interact with the sugar chains of the type found in serum glycoproteins (scrum glycoprotein-type sugar chains), bind strongly membrane glycoproteins with molecular weights ranging from 80,000 to 20,000, whereas the other lectins, which can interact with sugar chains such as those of mucins (mucin-type sugar chains), bind membrane glycoproteins with molecular weights higher than 100,000. To characterize the receptor sites for mitogenic lectins on the surface of lymphocytes, the membrane glycoproteins of C3H/He-mouse splenic lymphocytes, which were specifically eluted from a column of/". jaC/v«m-Sepharose 4B, were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, and the histocompatibility-2-complex proteins (H-2D, H-2K, and la antigens) were found to be major receptor sites.
The lectins have several peculiar biological activities. These activities are assumed to stem from initial binding of the lectins to receptor sites of carbohydrate nature on the cell surface (/, 2). 1
The structure of these cell surface receptor sites has often been inferred simply from the specificities of lectins disclosed by hapten inhibition assays using simple sugars as hapten inhibitors. In previous
This investigation was supported by research grants from the Ministry of Education, Science and Culture of Japan and from the Japan Society for the Promotion of Science (the Japan-U.S. Cooperative Science Program). Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M; IgD, immunoglobulin D; a-MM, methyl a-D-mannopyranoside; la antigens, I-region associated antigens. Vol. 82, No. 3, 1977
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studies (3, 4), we showed that the results of hapten hemagglutinin (10), L. culinaris hemagglutinin (11) inhibition assays using simple sugars as inhibitors and R. communis hemagglutinin (12) were purified cannot always be correlated to the structure of according to the methods described previously. receptor sites on the cell surface. We have ac- P. sativum hemagglutinin was purified by the cordingly carried out extensive studies on the method of Entlicher et al. (13). These purified carbohydrate-binding specificities of lectins by lectins were coupled to activated Sepharose 4B by means of hemaggultination inhibition assays using the method described by Matsumoto and Osawa various glycopeptides (5) and chemically synthe- (14). Wheat germ agglutinin-Sepharose 6MB was sized oligosaccharides (6, 7) as hapten inhibitors purchased from Pharmacia Fine Chemical and the inhibition of lectin-binding to human (Uppsala, Sweden). erythrocytes and lymphocytes with various glycoPreparation of Lymphocytes—Spleens of mice proteins and glycopeptides (8). From the results were teased apart at 4°C in Hank's balanced salt of these investigations, we can classify the lectins solution (15) purchased from Nissui Co., Tokyo. into three groups. The members of the first After removal of clumps by sedimentation for 10 group (class I), which contains Agaricus bisporus, min, cells were collected by centrifugation (150 X g Arachis hypogaea, and Bauhinia purpurea hemag- for 10 min), washed twice with the same medium glutinins, bind preferentially to the O-)S-D-galac- and suspended in fetal calf serum (Grand Island topyranosyl-( 1 —>3) - 2 - acetamido - 2 - deoxy-D-galac- Biological Co., Madison, Wis., U.S.A.). Purified tose sugar sequence in sugar chains as those of lymphocytes were obtained by subjecting the cell mucins (mucin-type sugar chains), whereas the suspension to Ficoll (Pharmacia Fine Chemical)— members of the second group (class II), which Urografin (Schering, Berlin, Germany>density contains concanavalin A, Lens culinaris, Pisum gradient centrifugation as described by Kawaguchi sativum, and Ricinus communis hemagglutinins, et al. (16). The cells were washed three times with bind primarily to different but overlapping portions Hank's balanced salt solution and three times with of the sugar chains of the type found in serum 10 mM sodium phosphate buffer (pH 7.2) containglycoproteins (serum glycoprotein-type sugar ing 0.15 M NaCl. Labeling of Lymphocytes—Radioiodination of chains). On the other hand, the members of the third group (class III), which contains soy bean lymphocytes was performed by virtually the same and wheat germ agglutinins, can bind to both types method as that described by Hubbard and Cohn of sugar chains. The presence of these two types (17). To the suspension of lymphocytes (6x10* of sugar chains on the surface of lymphocytes has cells) in 1 ml of 10 mM sodium phosphate buffered saline (pH 7.2) were added 3.6 munits of lactobeen confirmed (9). Mannheim, To reveal the nature of the major receptor peroxidase (Boehringer Mannheim, 1M Germany) and 300 ftCi of Na[ r] (New England sites for these three classes of lectins on mouse splenic lymphocytes and to characterize the re- Nuclear Corp., Boston, Mass., U.S.A.). Then ceptor sites for the lectins which are mitogenic to 3.6 munits of glucose oxidase (Boehringer) and lymphocytes, we analyzed membrane glycoproteins 5 ftmol of glucose were added to the mixture. of mouse splenic lymphocytes using affinity ad- After incubation for 20 min at room temperature, sorbents prepared by coupling various lectins to 10 ml of ice-cold 10 mM sodium phosphate buffered Sepharose 4B. The results of these studies are saline (pH 7.2) containing 10 mM Nal was added to stop the reaction. The labeled cells were then described here. washed several times with ice-cold 10 mM sodium phosphate buffered saline (pH 7.2). MATERIALS AND METHODS Isolation of Crude Plasma Membranes—Crude Animals—DD mice (7 to 10 weeks old) were plasma membrane fraction was obtained according obtained from Nippon Bio-Supp. Center, Tokyo to the methods of Allan and Crumpton (18) and and C3H/He mice (7 weeks old) were obtained Kornfeld and Siemers (19) with a few modifications. s from the Institute of Medical Science, University of The lymphocytes (6 x 10 cells) were suspended in 2 ml of ice-cold 10mM Tris-HCl buffer (pH 7.2) Tokyo. Lectins and Affinity Adsorbents—B. purpurea containing 0.15 M NaCl and 1 mM EDTA. To /. Biochem.
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this cell suspension, 2 ml of 10 mM Tris-HCl buffer extensive dialysis, this fraction was concentrated (pH 7.2) containing 1 mM EDTA was added slowly and analyzed by sodium dodecyl sulfate polyacrylwhile the cell suspension was being stirred. The amide gel electrophoresis. mixture was left at 0°C for 5 min, and then 10 ml of Sodium Dodecyl Sulfate Polyacrylamide Gel 10 mM Tris-HCl buffer (pH 7.2) containing 0.18 M Electrophoresis—The eluate from the affinity NaCl and 1 mM EDTA was added to restore the column was dissolved by boiling in an equal volume NaCl concentration to 0.15 M. TO this mixture, of 2% sodium dodecyl sulfate for 5 min. 2phenylmethanesulfonyl fluoride (Sigma Chemical Mercaptoethanol was added to a final concentration Co., St. Louis, Mo., U.S.A.) in ethanol was added of 40 mM to reduce disulfide bonds. The electroto a final concentration of 1 mM. Then the cells phoresis was carried out in 5.6% polyacrylamide were homogenized in a loosely fitting glass Potter- gel at pH 7.4 according to the method of Fairbanks Elvehjem homogenizer using five to seven gentle et al. (20). In the case of immunoprecipitate, the strokes. The homogenate was centrifuged at discontinuous sodium dodecyl sulfate disc gel 300 x g for 15 min to remove the cell nuclei. The electrophoresis system was used as described by supernatant was collected and centrifuged again as Laemmli (27). After electrophoresis, the gels were above. The volume of this nucleus-free super- immersed in 10%(w/v) trichloroacetic acid for 12 h. natant was measured, and an amount of 3 M LiCl The fixed radioactivity in each slice was determined sufficient to give a final concentration of 0.1 M LiCl in an Alloka well-type autogamma counter. Mouse was added to the supernatant, which was then immunoglobulins ft and y and light chains were centrifuged for 45 min at 45,000xg in a Hitachi used as reference proteins. The following proteins, 65P ultraccntrifuge. The crude membranes thus obtained from Mann Research Laboratories obtained were suspended in 5 ml of lOmM Tris- (Orangeburg, N.Y., U.S.A.), were used as standHCl buffer (pH 7.2) containing 1 mM EDTA and ards for the estimation of molecular weights 1 mM phenylmethanesulfonyl fluoride, and then (molecular weights shown in parentheses): horse homogenized to a fine suspension by three passes heart cytochrome c (12,400), ox pancreas chymoin a Potter-Elvehjem homogenizer. These re- trypsinogen A (25,000), ovalbumin (45,000), bovine suspended crude membranes (microsomal fraction) serum albumin (67,000), and human ^-globulin were again centrifuged at 45,000 x g for 45 min. (160,000). The distance of migration of the memIsolation of Membrane Receptors for Lectins brane proteins was found by determination of —The radioactively labeled microsomal fraction radioactivity and those of the standard proteins obtained from 6x10* cells was suspended in 1 ml by staining the gels with Coomassie Brilliant Blue._ of 10 mM Tris-HCl buffer (pH 7.0) containing 1 mM Indirect Precipitation of Cell Membrane Prophenylmethanesulfonyl fluoride. Triton X-100 teins—The Triton X-100 extract of the crude mem(Wako pure Chemical Co., Tokyo) was added to a brane fraction prepared from splenic lymphocytes final concentration of 0.2%. The cell suspension (6x10* cells) of C3H/He mice was applied to a was mixed vigorously and incubated at 4°C for 1 h. column of P. sativum hemagglutinin-Sepharose 4B After centrifugation at 3,000 x g for 10 min at 4°C, (0.9x13.5 cm) equilibrated with 10 mM Tris-HCl the Triton X-100 extract was applied to an affinity buffer (pH 7.0) containing 0.2% Triton X-100. column (0.6 x 10 cm unless otherwise specified) Elution was carried out with the same buffer. equilibrated with 10mM Tris-HCl buffer (pH 7.0) After the first peak had been eluted completely, the containing 0.2% Triton X-100. Elution was car- active fraction was eluted with 0.2 M methyl a-Dried out with the same buffer and, after the first mannopyranoside solution containing 0.2% Triton peak had been completely eluted, the active fraction X-100. Fractions of 1.6 ml were collected at 4 ml/ was eluted with an appropriate haptenic sugar h at room temperature. The specifically eluted solution containing 0.2 % Triton X-100. Fractions fraction was then concentrated to one third of the of 0.8 ml were collected at 4 ml/h at room temper- volume in a collodion bag (SM 13200; Sartorius ature. Radioactivity was tested on each fraction Membrane Filter Co., Gottingen, Germany). Then in an Alloka autogamma counter. The specifically 10 fi\ of 1 M solution of phenylmethanesulfonyl eluted fractions were pooled and dialyzed against fluoride was added to inhibit enzymic breakdown 50% (v/v) ethanol to remove the detergent. After of labeled proteins, and the mixture incubated for Vol. 82, No. 3, 1977
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30 min at 4°C. To deplete a component which binds to antigen-antibody complexes (22), the following specific immunoprecipitation experiments were always preceded by three precipitations achieved by adding goat anti-human IgG serum (Miles Laboratories, Kankakee, IU., U.S.A.) and an equivalent amount of human IgG (Miles). After centrifugation at 15,000 xg for 15 min, 0.1 ml of rabbit anti-mouse immunolglobulin serum (Miles) was added and the mixture incubated at 37°C for 30 min. Precipitation was caused by adding an equivalent amount of C3H/He mouse serum to provide a carrier protein. Incubation was allowed to proceed for 30 min at 37°C and then at 4°C overnight. The precipitates were centrifuged at 15,000xg for 15 min, washed with 10ml sodium phosphate buffered saline (pH 7.2) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis by the method of Laemmli (21). The supernatants were then treated with a mouse alloantiserum specific for the H-2D antigen and rabbit anti-mouse immunoglobulin serum. The precipitates obtained were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The same sandwich technique was also used to precipitate the H-2K antigen and I-region associated antigens (la antigens) from the supernatant by using an antiserum specific to the H-2K. and la antigens and rabbit anti-mouse immunoglobulin serum. The specific alloantiserum specific for the H-2D antigen, (AxBALBG)Fi anti-BALB-K, and the alloantiserum against the H-2K antigen and the la antigens, C3HOL anti-C3H, were kindly provided by Dr. S.G. Nathenson (Albert Einstein College of Medicine, Bronx, N.Y., U.S.A.). RESULTS AND DISCUSSION The solubilized crude membrane fraction prepared from radioiodinated DD-mouse splenic lymphocytes was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis by the method of Fairbanks et al. (20) using a gel concentration of 5.6%. As shown in Fig. 1, many protein peaks of various molecular weights were seen, which shows the complex nature of membrane glycoproteins of lymphocytes. To characterize the membrane receptor sites for B. purpurea hemagglutinin, which belongs to
Relative
mobility
Fig. 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of crude membranes (microsomal fraction) of radioiodinated mouse splenic lymphocytes. The microsomal fraction was extracted with 0.2% Triton X-100 at 4°C for 1 h and subjected to electrophoresis in 5.6% polyacrylamide gel at pH 7.4 in the presence of 1 % sodium dodecyl sulfate according to the method of Fairbanks et al. (20). p, r, and L refer to positions of standard mouse immunoglobulin chains. The direction of protein migration is from left to right. class I and binds preferentially to mucin-type sugar chains (5, 8), the solubilized crude membrane fraction was subjected to affinity chromatography on a column of Sepharose 4B coupled with B. purpurea hemagglutinin (Fig. 2). When the column was washed with 10 mM Tris-HCl buffer (pH 7.0) containing 0.2% Triton X-100, approximately 90% of the radioactivity was recovered without retardation, and thereafter, a slow release of the remaining radioactivity was observed continuously. The strongly adsorbed membrane glycoproteins were finally eluted with 0.2% Triton X-100 solution containing 0.2 M lactose, which is a specific inhibitor against B. purpurea hemagglutinin. Total recovery of the radioactivity usually reached more than 95 %. The fractions which were specifically eluted with lactose (strongly-bound fractions), and those which were shown by a double-headed arrow in Fig. 2 (loosely-bound fractions), were then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the apparent molecular weights of radioactive glycoproteins separated were estimated by comparison with the migration of the standard J. Biochem.
MEMBRANE RECEPTORS FOR LECTINS
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0.2M Lactose
E a
10
20 30 40 Fraction number
50
60
70
Fig. 2. Affinity chromatography of solubilized crude membranes of radioiodinated mouse splenic lymphocytes on a B. purpurea-Sephaiose 4B column (0.6 X 15 cm). Experimental details are described in the text. a
tr
proteins. As shown in Fig. 3a, the strongly-bound fractions mainly consist of glycoproteins having molecular weights greater than 100,000 whereas the loosely-bound fractions are mainly composed of glycoproteins of smaller molecular weights (Fig. 3b). A peak with an approximate molecular weight of 210,000 is most marked in the stronglybound fractions. These results and the fact that this lectin strongly binds to the O - /3 - galactopyranosyl-{l—>3)-2-acetamido-2-deoxy-D-galactose sugar sequence in mucin-type sugar chains (5; Kaifu, R. and Osawa. T., unpublished observation) suggest that the glycoproteins with high molecular weights (> 100,000) bear mucin-type sugar chains in their molecules. In contrast, the loosely-bound fractions shows the strongest radioactivity in the peak having approximate molecular weight of 55,000. Then the solubilized crude membrane fraction of radioiodinated DD-mouse splenic lymphocytes was subjected to affinity chromatography on columns of Sepharose 4B coupled with mitogenic lectins, P. sativum and L. culinaris hemagglutinins, both of which belong to class II and bind preferentially to the sugar sequences containing amannopyranosyl residues in serum glycoproteintype sugar chains (6). A typical example of these chromatographies is shown in Fig. 4. In these affinity chromatographies, approximately 8 % of the Vol. 82, No. 3, 1977
Relative
mobility
Fig. 3. Sodium dodecyl sufate polyacrylamide gel electrophoresis of radioiodinated membrane glycoproteins eluted from B. purpurea-Scpheuose 4B. Experimental details are in the text, (a) Strongly-bound fractions, (b) loosely-bound fractions. The numbers in the figure are the estimated molecular weights of the peaks indicated by arrows. The other symbols are used as described in the legend to Fig. 1.
Triton X-100-extracted radioactivity remained bound to the affinity adsorbents, even after extensive washing with 10 mM Tris-HCl buffer (pH 7.0) containing 0.2% Triton X-100. Total recovery of the radioactivity after elution of strongly bound glycoproteins with 0.2 M methyl a-D-mannopyranoside (a-MM) solution varied between 96 and 98%. When the lymphocyte membrane glycoproteins specifically eluted from a column of P. sativumSepharose 4B were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, radioactive peaks corresponding to approximate molecular weights of 80,000, 52,000, 46,000, 31,000, 24,000, and 15,000 were reproducibly observed as
666
M. IWATA, H. IDE, T. TERAO, and T. OSAWA
1.0 E Q.
>
K
E a.
0.2Ma-MM
X
A
0.5
oct
| o
TJ
a a.
o o •o a a.
i
10 20 30 Fraction number
40 0.5
1.0
Fig. 4. Affinity chromatography of solubilized crude Relative mobility membranes of radioiodinated mouse splenic lympho- Fig. 6. Sodium dodecyl sulfate polyacrylamide gel cytes on a P. sativum-SephSiiosc 4B column. Experi- electrophoresis of radioiodinated membrane glycopromental details are described in the text. teins specifically eluted with 0.2 M a-MM from L. culinara-Sepharose 4B. The same symbols are used as described in the legend to Fig. 3. Experimental conditions are described in the text. E
a
o o
•5 o 0.5 Relative
1.0
mobility
Fig. 5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glycoproteins specifically eluted with 0.2 M a-MM from P. sativwn-Sepharose 4B. The same symbols are used as described in the legend to Fig. 3. Experimental conditions are described in the text. shown in Fig. 5. Within the experimental error of the molecular weight estimation, almost the same set of radioactive peaks was seen in sodium dodecyl sulfate polyacrylamide gel electrophoresis of the membrane glycoproteins that were specifically eluted from a column of Sepharose 4B coupled with L. culinaris hemagglutinin (Fig. 6). Furthermore,
the glycoproteins of the same range of molecular weights (20,000-80,000) as that observed in the cases of P. sativum and L. culinaris hemagglutinins had also been observed by Nilsson and Waxdal (23) in the membrane glycoproteins of mouse lymphocytes specifically eluted from the affinity matrix prepared by attaching concanavalin A to Affi-gel 10. However, relative intensities of these radioactive peaks were different among these mitogenic lectins used for the preparation of affinity adsorbents, possibly reflecting a subtle difference in carbohydrate-binding specificities of these mannosebinding mitogenic lectins. R. communis hemagglutinin also belongs to class II, and it has strong affinity to the O-/3-Dgalactopyranosyl-(l—>4)-2-acetamido-2-deoxy-Dglucose sugar sequence in serum glycoprotein-type sugar chains (5, 7, 8). The solubilized crude membrane fraction of radiolabeled DD-mouse splenic lymphocytes was applied to a column of R. com7rtun£y-Sepharose 4B and the bound radioactivity (approximately 5 % of the radioactivity of the solubilized membrane fraction) was eluted with 0.2 M lactose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the specifically eluted fractions (Fig. 7) exhibited many radioactive glycoproteins of apparent molecular weights ranging
/ . Biochem.
MEMBRANE RECEPTORS FOR LECT1NS
667
E a.
o o a a. 0.5 0.5
1.0
Relative
1.0 mobility
Fig. 8. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glycoproFig. 7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glyco- teins specifically eluted with 0.5 M N-acetyl-D-glucosaproteins specifically eluted with 0.1 M lactose from mine from wheat germ agglutinin-Sepharose 4B. The R. communis-Sepharose 4B. The same symbols are used same symbols are used as described in the legend to as described in the legend to Fig. 3. Experimental Fig. 3. Experimental conditions are described in the text. conditions are described in the text. Relative
m o b i l i ty
from 140,000 to 24,000. However, strong radioactivity was always found in the peaks of the molecular weights between 80,000 and 41,000. The peaks in this region of molecular weights were also found to be strongly radioactive in the experiments which employed other class II lectins (P. sativum and L. culinaris hemagglutinins) having a strong affinity to mannose-containing sugar sequences in serum glycoprotein-type sugar chains, as shown in Figs. 5 and 6. These results are consistent with our previous data (5, 8) that R. communis hemagglutinin belongs to class II. However, the large molecular weight peaks with relatively weak radioactivity in Fig. 7 suggest that this lectin has rather broad carbohydrate-binding specificity and can weakly bind also to /3-D-galactopyranosyl residues in mucin-type sugar chains. Finally, the solubilized crude membrane fractions were subjected to affinity chromatography on a column of wheat germ agglutinin-Sepharose 6MB. Wheat germ agglutinin belongs to class III in our classification and can bind to both serum glycoprotein-type and mucin-type sugar chains (Saito, M., Toyoshima, S., and Osawa, T., unpublished observation). The glycoproteins that Vol. 82, No. 3, 1977
bound to the affinity adsorbent were specifically eluted with 0.5 M N-acetyl-D-glucosamine solution containing 0.2% Triton X-100. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, the specifically eluted glycoproteins exhibited many radioactive peaks with almost equal intensity of radioactivity over the range of molecular weights from 140,000 to 30,000 (Fig. 8). These results are in good agreement with the assumption that this lectin belongs to class III and has broad carbohydrate-binding specificity. Although we cannot exclude the presence of membrane glycoproteins which could not be eluted from the affinity adsorbents with haptenic sugars, mitogenic lectins invariably bind the glycoproteins of apparent molecular weights ranging from 80,000 to 20,000. Since the heavy and light chains of immunoglobulins and the his.tocompatibility-2complex proteins are found in this molecular weight range (24-28), immunoprecipitation with specific antisera was carried out to characterize these glycoproteins. The specifically eluted membrane glycoproteins of C3H/He-mouse splenic lymphocytes from P. sativum-Sepharose 4B affinity adsorbent were first treated with rabbit anti-mouse immunoglobulin serum. Precipitation was effected by
668
M. IWATA, H. IDE, T. TERAO, and T. OSAWA
adding an equivalent amount of C3H/He mouse serum to provide a carrier protein. When the precipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, three peaks were observed as shown in Fig. 9. On the basis of the approximate molecular weights estimated and the migration of mouse immunoglobulin (i and r and light chains, the component of molecular weight 73,000 was tentatively identified as the ft chain from cell surface IgM, and the component of molecular weight 65,000 as the d chain from cell surface IgD. These estimated molecular weights agrees well with the values reported by other investigators {24,25). However, the lack of specific antisera precludes the final characterization of these immunoglobulins. The protein peak with •a molecular weight of around 24,000 is possibly 1he light chain of the cell surface immunoglobulins. These immunoglobulins might have come from B-cells in mouse splenic lymphocytes. The supernatant, after precipitation of the cell-sulface imrmunoglobulin as above, was further treated with a n alloantiserum specific for the H-2D antigen or an alloantiserum specific for the H-2K and la antigens. Precipitation was brought about by the sandwich technique, by adding an equivalent amount of rabbit anti-mouse immunoglobulin serum. When the precipitates obtained using the
anti-H-2D serum were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, only one peak, with an approximate molecular weight of 45,000, was observed (Fig. 10a). The precipitates with the alloantiserum specific for the H-2K and la antigens were likewise analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 10b). Two distinct peaks, with molecular weights around 46,000 and 31,000, were always seen, and in some experiments, an additional peak with a molecular weight 24,000 was observed. From these results and from the estimated molecular weights of the peaks, the glycoproteins with approximate molecular weights of 45,000 and 46,000 could be the H-2D and H-2K antigens and the proteins with approximate molecular weights of 31,000 and 24,000 are possibly la antigens.
10 20 30 40 Sigmin t numb* r
50
60
Fig. 9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of radioiodinated membrane glycoprogeins eluted with a-MM from P. jo/iVum-Sepharose 4B after immunoprecipitation by adding rabbit anti-mouse immunoglobulin serum and an equivalent amount of C3H/He mouse serum. The same symbols are used as described in the legend to Fig. 3. The experimental conditions are described in the text.
20 Sigmtnl
30
40
50
60
number
Fig. 10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the specifically eluted radioiodinated membrane glycoprotein from P. MZ/vum-Sepharose 4B which remained after immunoprecipitation with rabbit anti-mouse immunoglobulin serum, (a) Immunoprecipitation of H-2D antigen with an alloantiserum specific for the H-2D antigen, (b) Immunoprecipitation of H-2K and la antigens with an alloantiserum specific for the H-2K and la antigens. /. Biochem.
MEMBRANE RECEPTORS FOR LECTINS These estimated molecular weights of H-2 and la antigens correspond closely to those reported by other investigators (25-28). Since the glycoproteins with molecular weights which correspond to those of H-2 complex proteins are invariably seen in the specifically eluted fraction from the column of Sepharose 4B coupled with T-cell mitogens [P. sativum and L. culinaris hemagglutinins in this paper and concanavalin A (23)], and since the presence of la antigens on T-cells has been indicated by analyzing the pokeweed mitogenbinding glycoproteins from the membrane of purified T-cells (29), these H-2-complex proteins may play an important role in the mitogenic triggering of T-cells, serving as major cell surface receptor sites for mitogenic Iectins. A final conclusion must await further investigations of cell surface receptor sites of several other T-cell mitogens, and the molecular architecture of H-2 and la antigens. We wish to thank Dr. S.G. Nathenson for the generous gift of antisera directed against H-2-complex proteins and Drs. S. Migita and T. Kishimoto for mouse IgG and IgM. REFERENCES 1. Lis, H. & Sharon, N. (1973) Annu. Rev. Biochem. 42, 541-574 2. Nicolson, G.L. (1974) Int. Rev. Cytol. 39, 89-120 3. Toyoshima, S., Fukuda, M., & Osawa, T. (1972) Biochemistry 11, 4000-4005 4. Osawa, T., Terao, T., Kawaguchi, T., Fukuda, M., Toyoshima, S., & Irimura, T. (1974) Co/log. Int. C.N.R.S. 221, 765-776 5. Irimura, T., Kawaguchi, T., Terao, T., & Osawa, T. (1975) Carbohyd. Res. 39, 317-327 6. Kaifu, R., Osawa, T., & Jeanloz, R.W. (1975) Carbohyd. Res. 40, 111-117 7. Kaifu, R. & Osawa, T. (1976) Carbohyd. Res. 52, 179-185 8. Kawaguchi, T. & Osawa, T. (1976) Biochemistry 15, 4581^586
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