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Production and characterization of a rabbit antiserum to the mouse CD8 antigenic complex by immunization with a synthetic peptide
Journal of Immunological Methods, 96 (1987) 97-105 Elsevier
97
JIM04190
Production and characterization of a rabbit antiserum to the mouse CD8 antigenic complex by immunization with a synthetic peptide S. Brunati, G. C o r r a d i n a n d C. B r o n Institute of Biochemistry, University of Lausanne, Ch. des Boveresses, 1066 Epalinges, Switzerland (Received 29 July 1986, revised received 11 September 1986, accepted 25 September 1986)
A rabbit antiserum to the mouse CD8 antigen (Lyt-2/3) was obtained through the use of a synthetic peptide corresponding to the N-terminal segment of the 37 kDa subunit of the CD8 molecular complex. This anti-peptide antiserum detected specifically the membrane antigen from detergent extracts of surface-labeled mouse thymocytes. The efficiency of the immunoprecipitation increased significantly upon treatment of the cell lysates with 0.1% SDS at 100 o C before immunopurification. Finally both the 37 kDa and the 32 kDa polypeptides expressing the Lyt-2 antigen were revealed by immunoblotting. Key words: Lymphocyte membrane; Lyt-2 antigen; Synthetic peptide; Antiserum, rabbit
Introduction The recent molecular cloning of human T8 (Leu-2a), mouse Lyt-2/3 and rat MRC-OX8 antigens, now called CD8 antigens, revealed that these membrane proteins selectively expressed on the surface of functional T cell subsets are members of the immunoglobulin supergene family (Kavathnas et al., 1984; Johnson et al., 1985; Littman et al., 1985; Nakauchi et al., 1985; Sukhatme et al., 1985). The exact role of these differentiation antigens is still unknown. They are presumed to serve an auxiliary function in the binding of cytolytic T lymphocytes to their targets as indicated by the possibility of blocking this interaction with monoclonal antibodies directed against these structures (Nakayama et al., 1979; Sarmiento et al., 1980; MacDonald et al., 1982; Meuer et al., 1982). Based on the predicted primary structure of
these molecules deduced from the sequence of their cDNA, peptides can be synthesized and anti-peptide antisera can be raised against well defined domains of the proteins. Both synthetic peptides and anti-peptide antibodies might provide valuable probes for investigating the structural and functional relationship of the CD8 molecular complex as well as for the study of the processing and assembly of the various subunits during biogenesis. In this communication, we describe the synthesis of a peptide corresponding to the sequence of the first 13 amino acid residues of the mouse Lyt-2 antigen and the property of an antiserum raised against this peptide which reacts with detergent-dispersed molecules of the membrane protein.
Materials and methods Correspondence to: C. Bron, Institute of Biochemistry, University of Lausanne, Ch. des Boveresses, 1066 Epalinges, Switzerland.
All chemicals and buffer components were of analytical grade. Protein A-Sepharose and CNBr-
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
98 activated Sepharose 4B were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Carrier-free Na125I (1680 Ci/mmole) was purchased from Radiochemical Amersham International Center.
Animals and cells Inbred C57B1/6 mice were obtained from the animal colony of the Swiss Institute for Experimental Cancer Research (Epalinges, Switzerland). Thymocyte suspensions were prepared from thymuses of 4-5-week-old mice using a loosely fitted Dounce tissue homogenizer. New Zealand White rabbits were used for the preparation of anti-peptide antisera.
Synthesis of peptide A 13 residue peptide was synthesized by the Merrifield solid-phase method (Marglin and Merrifield, 1970) using FMOC-protected a-amino acids (Atherton et al., 1979). A tyrosine residue was attached at the C-terminus. The synthetic peptide was purified by gel filtration, ion exchange and high performance liquid chromatography (HPLC); amino acid composition was proved to be correct by standard amino acid analysis. The purified peptide was coupled to ovalbumin or bovine serum albumin (BSA) via the di-azo-benzidine group (Bassiri et al., 1979). An average of 0.5 mg of peptide was coupled to 1 mg of carrier protein.
Immunization procedure One rabbit was immunized intramuscularly with 0.5 mg of synthetic peptide conjugated to 1 mg of ovalbumin solubilized in 1.5 M of guanidine hydrochloride and emulsified in complete Freund's adjuvant. The immunization was repeated 2 weeks later with the same antigen in incomplete Freund's adjuvant. Two successive immunizations were then performed at 10 day intervals with 10 mg of the non-conjugated peptide solubilized in phosphatebuffered saline (PBS) and emulsified in incomplete Freund's adjuvant. The rabbit was bled 7-8 days after these injections and the serum collected. The immunoglobulin fraction was prepared by precipitation of the antiserum with 40% ammonium sulfate, followed by a purification on a Sephadex CL-B6 column in 0.2 M citrate pH 7.0
buffer containing 0.5M NaC1. For immunoblotting, the Ig fraction was iodinated by the chloramine-T method.
Antibody assay The antibody activity of the rabbit antiserum was determined by a solid-phase ELISA in 96-well polyvinyl chloride microtitration plates (Dynatech Laboratories, Alexandria, U.S.A.). The wells were coated with 100/~1 quantities of the carrier(BSA)conjugated peptide solution (20 /~g/ml) in phosphate-buffered saline (PBS) pH 7.5 for 45 min at room temperature. The plates were then washed twice with PBS containing 0.2% of gelatine. Serial dilution of the antiserum or of the pre-immune serum in the same buffer were added. The plates were incubated for 30 min at room temperature and washed three times in PBS supplemented with 0.2% of gelatine. Specific goat antibodies (100/~1) to rabbit immunoglobulins conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, MO) diluted 1/1000 in 0.2% gelatine PBS were ad~ted to each well and the plate incubated at room temperature for 30 rain. The plate was subsequently washed and 100 /~1 of a solution of 1 mg/ml of sodium p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, MO) in a 1 M diethanolamine buffer at pH 9.8 containing 10 3 M KC1. The absorbance of the solution in wells was determined at 405 nm in an automatic reader (Titertek Multiskan, Flow Laboratories, Switzerland). Inhibition of antibody binding was also determined by a similar type of ELISA assay except that the diluted antiserum was preincubated with appropriate amounts of inhibitor peptide or carrier-conjugated peptide.
Immunoprecipitations Indirect immunoprecipitations were carried out according to previously described procedures (Liascher et al., 1984). In brief, 10 v surfaceiodinated cells were lysed in 1 ml of buffer containing 0.5% Nonidet P-40 (NP-40), 0.5% deoxycholate (DOC), 25 mM Tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HC1) pH 8.1, 50 mM NaC1 0.01% NaN 3 and 2 mM phenylmethylsulfonylfluoride (PMSF) at 4°C (lysis buffer). Detergent insoluble material was then removed by centrifugation at 400 × g for 10 min.
99 The cell lysate was precleared twice with 50/~1 of a suspension of 250 /~g normal human Ig-conjugated to Sepharose 4B. Alternatively, preclearing was performed by addition of 10 /tl of normal rabbit serum followed by successive absorptions with 30 /~1 of a suspension of protein A-conjugated to Sepharose 4B. The lysate was then filtered through a 0.22 /~m Millipore filter before immunoprecipitation. The cleared lysate of 10-15 × 10 6 cells was incubated with 10 /tl of rabbit anti-peptide antiserum for 30 min at room temperature followed by absorption with 30 /~1 of a suspension of protein A-conjugated to Sepharose 4B. This step could be omitted for the immunoprecipitations with the anti-Lyt-2 monoclonal antibody H-59 which was coupled to Sepharose 4B. In certain experiments, cell lysates were pre-treated with 0.1% SDS final concentration, heated at 100°C for 5 min and diluted with 11 vols. of 1% Triton X-100 prior to immunoprecipitation (Maccecchini and Schatz, 1979). The immune precipitates were washed four times in PBS p H 8.2 supplemented with 0.05% SDS, 0.5% DOC, 0.5% NP-40 and 10 mM E D T A and four times with a 120 mM Tris-HC1 buffer at p H 8.2 containing 500 m M NaC1, 0.5% NP-40 and 10 m M EDTA.
Polyacrylamide gel electrophoresis in SDS One-dimensional SDS-PAGE was carried out on 10% gels according to Laemmli (1970). Immunoprecipitates were dissolved in 80 mM TrisHC1, pH 6.8, 0.1 M dithiothreitol (DTT), 4% SDS, 10% glycerol, and 0.01% bromophenol blue (sample buffer) heated at 100°C for 5 min before layering onto the gel. The following molecular weight standards were used: fl-galactosidase ( M r = 130000), phosphorylase b ( M r = 94000), transferrin ( M r = 78 000), bovine serum albumin ( M r = 68 000), ovalbumin ( M r = 46 000), glyceraldehyde3-phosphatedehydrogenase ( M r = 34000), achymotrypsinogen ( M r = 25 000), and cytochrome c ( M r = 12000).
Immunoblotting The reactivity of the anti-peptide serum with thymocyte membranes was tested by immunoblotting (Towbin et al., 1979). In brief, 100 x 106 thymocytes of C57B1/6 mice were lysed at 4°C in
2 ml of lysis buffer of the same composition as for immunoprecipitations. Particulate material was removed by centrifugation at 400 × g for 20 min. The cell lysate was then adjusted to 2% SDS, 10% glycerol, 80 mM Tris-HC1 at p H 6.8, 0.1 M dithiothreitol (DTT) and heated for 5 min at 100 o C before separation on a 10% preparative polyacrylamide gel (Laemmli, 1970). Separated proteins were transferred electrophoretically to nitrocellulose paper membrane filters BA83, 0.2 /~m (Schleicher and Schiill, Dassel, F.R.G.). The blot paper was saturated with a solution of PBS, p H 7.6 containing 2% of gelatin. The paper strips were incubated overnight at 4°C with 1.5 ml of PBS containing 2% of gelatin and 1.5 ~tg of iodinated antibodies (approximatively 1.5 × 105 cpm). The solution was passed through a 0.45 /~m Milipore filter before incubation. Strips were incubated with 125I-labeled protein A solution (2 × 10 5 c p m / m l ) for 2 h and finally washed, dried and autoradiographed using Kodak XOMAT-XS5 films.
Anti-Lyt-2 monoclonal antibody The monoclonal antibody H-59-101.7 was kindly provided by Dr. M. Pierres (Centre d'Immunologie, CNRS, Marseille, France) (Goldstein et al., 1982) and ascites prepared from hybridoma bearing C57B1/6 irradiated mice. For immunoprecipitation, anti-Lyt-2 mAb were partially purified from ascites fluid by two successive precipitations with 45% ammonium sulfate, and was covalently bound to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden). About 3-6 mg of the immunoglobulin fraction was usually conjugated to 1 ml of activated Sepharose 4B.
Surface labeling of thymocytes Enzyme-catalyzed surface iodination of thymocytes was performed according to the method of Hubbard and Cohn (1975).
Results
Anti-peptide response of the rabbit A 13 residue peptide was synthesized which corresponded to the sequence of the initial 13
100
Reactiuity of anti-peptide antibodies with membrane proteins
residues of the a subunit of the Lyt-2/3 antigen determined by gas-phase protein sequencing (Walker et al., 1984a) and predicted by the cDNA sequence (Nakauchi et al., 1985). An additional tyrosine residue was added to the C-terminal end of the peptide to facilitate its subsequent conjugation to ovalbumin or bovine serum albumin (BSA). The sequence was as follows: Lys-Pro-Gln-AlaPro-Glu-Leu-Arg-Ile-Phe-Pro-Lys-Tyr. The rabbit was immunized with the ovalbumin-peptide conjugate. Anti-peptide antibodies could be detected after the second injection as demonstrated by a solid-phase binding assay using a BSA-peptide conjugate to coat the polyvinyl plates. The two successive injections with 10 mg of free peptide increased the antibody titer from 1:1600 to 1:6400 for 50% of antibody binding (Fig. 1A). The specificity of the antiserum for the peptide was demonstrated by inhibition of the antibody binding with less than 1 Fg/ml of BSA-peptide conjugate (Fig. 1B).
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The reactivity of the anti-peptide antiserum with the Lyt-2/3 molecular complex was first demonstrated by its ability to immunoprecipitate the antigen from non-ionic detergent lysates of surface-iodinated thymocytes. Thus, the antipeptide antibody precipitated two surface-labeled polypeptides of M r = 36000 and M r = 32000 respectively (Fig. 2, lanes 1 and 4). Both free and BSA-conjugated peptide totally inhibited this reaction (Fig. 2, lanes 2 and 5). The same two polypeptides were precipitated with the monoclonal antibody H-59 specific for the Lyt-2 antigen (Fig. 2, lane 3). In this case however, no inhibition of the precipitation was observed following the addition of free or BSA-conjugated peptide to the antibody before immunoprecipitation (data not shown). To compare further the reactivity of the antipeptide antiserum to the mAb H-59, sequential immunoprecipitations were performed, first with
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Fig. 1. ,4 : Anti-peptide response in a rabbit immunized with a synthetic peptide-conjugated to ovalbumin. Wells were coated with 20 /~g of BSA-conjugated synthetic peptide. • e, rabbit serum before immunization; © O, antiserum after two injections of OVA-peptide; A A, antiserum after a third injection with 10 mg of free peptide: [] .... •, antiserum after a fourth injection with 10 mg of free peptide; B: Inhibition of the reactivity of the rabbit anti-peptide antiserum. Wells were coated with 20 t~g of BSA-conjugated synthetic peptide. The antiserum dilution was 1:3000. The concentration of inhibitor is expressed logarithmically. • e, BSA; O O, ovalbumin-conjugated peptide.
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Fig. 2. Immunoprecipitates of detergent-lysed surface-iodinated thymocytes. Each precipitation was performed with the lysate of 10-15x106 cells. Autoradiographs of 10% SDS-polyacrylamide gels were performed under reducing conditions. Rabbit anti-peptide antiserum (1 and 4); rabbit anti-peptide antiserum in the presence of 500/xg of synthetic peptide (2); rat anti-Lyt-2 mAb H-59-101-7 (2); rabbit anti-peptide antiserum in the presence of 16 #g of BSA-conjugated synthetic peptide (5). Molecular weight standards (see the materials and methods section) were run on the same gel.
H-59 until the Lyt-2/3 molecular complex was exhaustively precipitated from a lysate of 30 x 106 surface-iodinated thymocytes. Under the experimental conditions chosen (see the material and methods section), this required three successive additions of Sepharose-4B-insolubilized H-59 monoclonal antibody. No significant precipitation of labeled Lyt-2/3 molecules was obtained by the subsequent addition of the anti-peptide antiserum (Fig. 3A, lanes 1-4). When the latter reagent was used first, two successive precipitations were suffi-
cient to deplete the lysate of labeled Lyt-2/3 polypeptides. However residual antigens could be precipitated by adding the mAb H-59 (Fig. 3A, lanes 5-8). Thus the anti-peptide antiserum precipitated approximatively one third of the radioactivity precipitable with the mAb H-59 from surface-labeled thymocytes. This was assessed by excising pieces of fixed and dried gels and counting the radioactivity associated with the Lyt-2/3 polypeptides identified by autoradiography. A number of high molecular weight bands coprecipitated with the Lyt-2/3 polypeptides when the mAb H-59 was used. These represented about 5-10% of the total radioactivity. The presence of these contaminants was usually seen when immunoprecipitations were performed with lysates of more than 20 X 10 6 cells/ml. In fact, they were not detectable when the Lyt-2/3 antigen was immunopurified from lysates of 10-15 x 106 cells/ml (see also Fig. 2). In order to determine whether the anti-peptide antiserum reacted preferentially with denatured molecules, non-ionic detergent lysates of surfacelabeled cells were treated with 0.1% SDS, heated at 100°C for 5 min and diluted in Triton X-100 prior to immunoprecipitation (Maccecchini et al., 1979). Under such conditions, the amount of labeled Lyt-2/3 molecules precipitated with the antipeptide antiserum was increased by a factor of 2-3 compared to non-treated lysates. In contrast, no precipitation was obtained with the mAb H59 (Fig. 3B, lanes 1-5). In addition, the amount of radioactivity precipitated by the anti-peptide antiserum was comparable to the radioactivity precipitated by the mAb H59 from non-SDS-treated lysates. Finally, free peptides added to the antiserum before immunoprecipitation inhibited totally the precipitation of the Lyt-2/3 molecules (Fig. 3B, lanes 6 and 7). The preferential reactivity of the anti-peptide antiserum with antigens which were denatured prior to immunoprecipitation prompted us to test whether the Lyt-2/3 molecules could be detected by immunoblotting. As shown in Fig. 4, this was indeed the case. In fact, the anti-peptide antiserum reacted with two polypeptides from a thymocyte lysate (M r = 36 000 and M r = 32 000). This reaction revealed with 125I-labeled immuno-
102
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Fig. 3. Sequential immunoprecipitations of detergent-lysed surface-iodinated thymocytes. Autoradiographs of 10% SDS-polyacrylamide gels under reducing conditions A: Lysate of 30 × 106 cells precipitated by three successive additions of mAb H-59 conjugated to Sepharose 4B (1, 2, 3) followed by the rabbit anti-peptide antiserum (4). Lysate of 30 × 10 6 cells precipitated by three successive additions of rabbit anti-peptide antiserum (5,6,7) followed by the mAb H-59 (8). B: Lysates of 10 × 10 6 cells first depleted of molecules reacting with the anti-peptide antiserum by three successive immunoprecipitations and then immunoprecipitated with: rat anti-Lvt-2 mAb H-59 (1) rabbit anti-peptide antiserum after treatment with 0.1% SDS and 100°C (see materials and methods section) (2); rabbit anti-peptide antiserum without SDS pretreatment (3). Lysate of 20 x 10 6 cells immunoprecipitated with rat anti-Lyt-2 mAb (H-59) without pretreatment with SDS (4) and after pretreatment with 0.1% SDS and heating at 100 o C (5) Lysate of 30 × 106 cells immunoprecipitated with the rabbit antiserum after treatment with 0.1% SDS and 100°C (6) and in the presence of 1 mg of synthetic peptide (7).
globulin was inhibited when the antiserum was incubated with the nitrocellulose strip in the presence of free or BSA-conjugated peptide (Fig. 4, lanes 1-2). Specificity was further confirmed by the fact that the anti-peptide antiserum did not react with the lysate of the E L 4 cell line - a L y t - 2 / 3 - n e g a t i v e murine thymoma. In contrast, a rabbit anti-Thy-1 a n t i b o d y clearly detected this antigen in the lysate of the EL4 cell line (Fig. 4, lanes 3-4). Finally, no reaction of the antiserum could be f o u n d by indirect immunofluorescence using viable thymocyte suspensions.
Discussion Biochemical analysis of the mouse C D 8 (Lyt2 / 3 ) antigens has previously shown that in thymocytes this molecular complex consists of disulfide-linked heterodimers comprising a 2 8 - 3 0
k D a subunit expressing the Lyt-3 epitope and either one of two structurally related 37 k D a and 32 k D a glycopeptides. Both of these chains carry the Lyt-2 antigenic determinant. They p r o b a b l y differ in their C-terminal region ( D u r d a et al., 1978; Reilly et al., 1980; Ledbetter et at., 1981; Jay et al., 1982; Rothenberg and Triglia, 1983; N a i m et al., 1984; Walker et al., 1984b; Luescher et al., 1985). It was recently suggested that these two polypeptides are encoded b y one single gene and that the difference in the size of their C-terminal portion arises from alternative m R N A splicing ( Z a m o y s k a et al., 1985). W e describe here the properties of a rabbit antiserum raised against a synthetic peptide which reacts with the mouse C D 8 antigen. The structure of the 13 amino acid peptide is based on the corresponding sequence of the N-terminal segment of the 37 k D a chain (Walker et al., 1984; N a k a u c h i et al., 1985). The specificity in this reagent for the L y t - 2 / 3 molecular complex is
103
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2
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Fig. 4. Detection of Lyt-2 polypeptides with the rabbit antipeptide antiserum by immunoblotting. Rabbit anti-peptide antiserum with lysates of thymocyte (1); same experiment in the presence of 100 /~g of BSA-conjugated peptide (2); rabbit anti-peptide antiserum with lysates of the EL-4 cell line (3); rabbit anti-Thy-1 antiserum with lysates of the EL-4 cell line (4). Lanes 1-3 are autoradiographs of blots performed with the iodinated immunoglobulins of the rabbit anti-peptide antiserum. The blot of lane 4 was revealed with a peroxidase-conjugated swine anti-rabbit Ig (Dako, Denmark).
demonstrated by the selective precipitation of these two polypeptides from the detergent lysate of surface iodinated thymocytes. These components were not detected with the anti-peptide antiserum
when the cell lysate was first depleted of Lyt-2/3 molecules by immunoprecipitation with the antiLyt-2 mAb (H-59-101.7). In contrast, the latter mAb still precipitated the Lyt-2/3 antigen from lysates which had been depleted of the molecules reacting with the anti-peptide antiserum. This result may reflect a lower affinity of the rabbit antiserum for the Lyt-2/3 molecules compared to the monoclonal antibody H-59. Alternatively it may be that only a subset of surface-expressed Lyt-2/3 molecules are recognized by this reagent in cell lysates. New antigenic sites can, however, be generated by treating surface-labeled cell lysates with 0.1% SDS and heating at 100°C. This was demonstrated by the fact that the amount of labeled Lyt-2/3 antigens precipitated under such experimental conditions is increased 2-3-f01d compared to non-treated lysates. This corresponds to the quantity of protein precipitable with the mAb H-59 without denaturation, as assessed by counting Lyt-2/3 specific bands excised from SDSpolyacrylamide gels. Moreover, Lyt-2/3 molecules can again be detected with the anti-peptide antiserum if the denaturating treatment is performed after several steps of exhaustive preclearing with the same reagent. Nevertheless, the antiserum did not react with the surface of viable cells in spite of its ability to partially precipitate surface-iodinated molecules. Moreover, it did not inhibit cell surface binding of various anti-Lyt-2/3 monoclonal antibodies nor did it block the cytolysis of Lyt-2/3-dependent CTL clones (data not shown). This suggests that the N-terminal segment of Lyt-2/3 is not accessible to the antibodies in the native surface-expressed form of the antigen even though the molecule is likely to be oriented with its N-terminal on the outside of the cell. The reactivity of the antiserum with surface-labeled molecules would then imply that the antigenic determinant detected is either generated by denaturation upon cell lysis or exposed by the dispersion of the membrane components in the mixed micelles of detergent. Taken together, these observations suggest that, whereas the mAb H-59 detects a conformation-dependent epitope which is destroyed by SDS at 100°C, the rabbit antiserum is directed against an SDS-resistant antigenic determinant. However, we cannot
104
exclude that the anti-peptide antiserum contains antibodies specific for native surface expressed antigens and others specific for denatured structures. The reactivity of the rabbit antiserum with denatured Lyt-2 molecules is further revealed by its reactivity in immunoblotting, a property which is not displayed by the mAb H-59. This result also demonstrates that the antipeptide antiserum recognizes both the 37 kDa and 32 kDa glycopeptides of Lyt-2/3 after reductive dissociation of the molecules in SDS. This is in agreement with the structural identity of the surface domains of these two components which has been predicted by the analysis of cDNA clones probably corresponding to these two subunits (Zamwoyska et al., 1985) and has recently been directly demonstrated by the determination of the amino acid sequence of the N-terminal segments of both glycopeptides (Walker et al., 1986). Moreover, the weaker reactivity of the antibody with the 32 kDa polypeptide probably reflects a lower amount of this component compared to the major 37 kDa species. This also correlates with previously published reports (Walker et al., 1984). Finally, failure to detect the 28 kDa subunit indicates that this chain differs from the other subunits, at least in the structure of its N-terminal region.
Acknowledgements We thank M. Rousseaux for skilled technical assistance, C. Vonney for assistance in the synthesis and purification of the peptide, M.-C. Knecht for secretarial assistance, Z. Freiwald for photographical work. This work was supported by the FNRS Grant no. 3.427.-0.83.
References Atherton, E., Logan, C.J. and Sheppard, R.C. (1979) Peptide synthesis. Part 2. Procedure for solid phase synthesis during N-fluorenylmethoxycarbonylamino acids on polyamide supports. Synthesis of substance P and of acyl carrier 65-74 decapeptide. Bio-org. Chem. 8, 351. Bassiri, R.M., Dvorak, J. and Utiger, R.D. (1979) In: B.M. Jaffe and H.R. Behrman (Eds,), Methods of Hormone
Radio-Immunoassay (Academic Press, New York) pp. 46-47. Durda, P.J. and Gottlieb, P.D. (1978) Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. J. Immunol. 121,983. Goding, J.W. and Harris, A.W. (1981) Subunit structure of cell surface proteins: disulfide bonding in antigen receptors, Lyt-2/3 antigens, and transferrin receptors of murine T and B lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 78, 4530. Goldstein, P., Gorides, A.M., Schmitt-Verhulst, B., Hayot, A., Pierres, A., Van Agthoven, Y., Kaufmann, J., Eshbar, Z. and Pierres, M. (1982) Lymphoid cell surface interaction structure detected using cytolysis-inhibiting monoclonal antibodies. Immunol. Rev. 68, 5. Hubbard, A.L. and Cohn, Z.A. (1975) Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J. Cell Biol. 438, 460. Jay, G., Palladino, M.A., Khoury, G. and Old, L.J. (1982) Mouse Lyt-2 antigen evidence for two heterodimers with a common subunit. Proc. Natl. Acad. Sci. U.S.A. 79, 2654. Johnson, P., Gagnon, J., Barclay, A.N. and Williams, A.F. (1985) Purification, chain separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes. EMBO J. 4, 2539. Kavathas, P., Sukhatme, V.P., Herzenberg, L.A. and Parnes, J.R. (1984) Isolation of the gene coding for the human T lymphocyte antigen Leu-2 (T8) by gene transfer and cDNA subtraction. Proc. Natl. Acad. Sci. U.S.A. 81, 7688. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Ledbetter, J.A., Seaman, W.E., Tsu, T.T. and Herzenberg, L.A. (1981) Lyt-2 and Lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds. J. Exp. Med. 153, 1503. Littman, D.R., Thomas, Y., Waddon, P.J., Chess, L. and Axel, R. (1985) The isolation and sequence of the gene encoding T8: A molecule defining functional classes of T lymphocytes. Cell 40, 237. Luescher, B., Naim, H.Y., MacDonald, H.R. and Bron, C. (1984) The mouse Lyt-2/3 antigen complex. I. Mode of association of the subunits with the membrane. Mol. Immunol. 21,329. Luescher, B., Rousseaux-Schmid, M., Naim, H.Y., MacDonald, H.R. and Bron, C. (1985) Biosynthesis and maturation of the Lyt-2/3 molecular complex in mouse thymocytes. J. Immunol. 135, 1937. Maccecchini, M.L., Rudin, Y., Blobel, G. and. Schatz, G. (1979) Import of proteins into mitochondria: Precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc. Natl. Acad. Sci. U.S.A. 76, 343. MacDonald, H.R., Glasebrook, A.L., Bron, C., Kelso, A. and Cerottini, J.-C. (1982) Clonal heterogeneity in the functional requirement for Lyt-2.3 molecules on cytotoxic T lymphocytes (CTL): possible implications for the affinity of CTL antigens receptors. Immunol. Rev. 68, 69. Marglin, A. and Merrifield, R.B. (1970) Chemical synthesis of peptides and proteins. Ann. Rev. Biochem. 39, 841.
105 Meuer, S.C., Hussey, R.E., Hodgdon, J.C., Hercend, T., Schlossman, S.F. and Reinherz, E.L. (1982) Surface structures involved in target recognition by human cytotoxic T lymphocytes. Science 218, 471. Naim, H.Y., Luescher, B., Corradin, G. and Bron, C. (1984) The mouse Lyt-2/3 antigen complex-II. Structural analysis of the subunits. Mol. Immunol. 21, 337. Nakauchi, H., Nolan, G.P., Hsu, C., Huang, H.S., Kavathas, P. and Herzenberg, L.A. (1985) Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: Molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions. Proc. Natl. Acad. Sci. U.S.A. 82, 5126. Nakayama, E., Shika, H., Stockert, E., Oettgen, H.F. and Old, L.J. (1979) Cytotoxic T cells: Lyt phenotype and blocking of killing activity by Lyt antisera. Proc. Natl. Acad. Sci. U.S.A. 76, 1977. Reilly, E.B., Autitore-Hargreaves, K., Haemmerling, U. and Gottlieb, P.D. (1980) Lyt-2 and Lyt-3 alloantigens: Precipitation with monoclonal and conventional antibodies and analysis on one- and two-dimensional polyacrylamide gels. J. Immunol. 125, 2245. Rothenberg, E. and Triglia, D. (1983) Lyt-2 glycoprotein is synthesized as a single molecular species. J. Exp. Med. 157, 365. Sarmiento, M., Glasebrook, A.L. and Fitch, F.W. (1980) IgG
or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt-2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125, 2665. Sukhatme, V.P., Sizer, K.C., Vollmer, A.C., Hunkapiller, T. and Parnes, J.R. (1985) The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell variable regions. Cell 40, 591. Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Walker, I.D., Hogarth, P.M., Murray, B.J., Lovering, K.E., Olasson, B. and McKenzie, I.F.C. (1984a) Ly antigens associated with T cell recognition and effector function. Immunol. Rev. 82, 47. Walker, I.D., Murray, B.J., Hogarth, P.M., Kelso, A. and McKenzie, I.F.C (1984b) Comparison of thymic and peripheral T cell Lyt-2/3 antigens. Eur. J. Immunol. 14, 906. Walker, I.D., Murray, B.J., Kirszbaum, L., Chambers, G.W., Deacon, N.H. and McKenzie F.C. (1986) The amino-terminal sequences of Ly-2 and Ly-3. Immunogenetics 23, 60. Zamoyska, R., Vollmer, A.C., Sizer, K.C., Liaw, C.W. and Parnes, LR. (1985) Two Lyt-2 polypeptides arise from a single gene by alternative splicing patterns of mRNA. Cell 43, 153.
97
JIM04190
Production and characterization of a rabbit antiserum to the mouse CD8 antigenic complex by immunization with a synthetic peptide S. Brunati, G. C o r r a d i n a n d C. B r o n Institute of Biochemistry, University of Lausanne, Ch. des Boveresses, 1066 Epalinges, Switzerland (Received 29 July 1986, revised received 11 September 1986, accepted 25 September 1986)
A rabbit antiserum to the mouse CD8 antigen (Lyt-2/3) was obtained through the use of a synthetic peptide corresponding to the N-terminal segment of the 37 kDa subunit of the CD8 molecular complex. This anti-peptide antiserum detected specifically the membrane antigen from detergent extracts of surface-labeled mouse thymocytes. The efficiency of the immunoprecipitation increased significantly upon treatment of the cell lysates with 0.1% SDS at 100 o C before immunopurification. Finally both the 37 kDa and the 32 kDa polypeptides expressing the Lyt-2 antigen were revealed by immunoblotting. Key words: Lymphocyte membrane; Lyt-2 antigen; Synthetic peptide; Antiserum, rabbit
Introduction The recent molecular cloning of human T8 (Leu-2a), mouse Lyt-2/3 and rat MRC-OX8 antigens, now called CD8 antigens, revealed that these membrane proteins selectively expressed on the surface of functional T cell subsets are members of the immunoglobulin supergene family (Kavathnas et al., 1984; Johnson et al., 1985; Littman et al., 1985; Nakauchi et al., 1985; Sukhatme et al., 1985). The exact role of these differentiation antigens is still unknown. They are presumed to serve an auxiliary function in the binding of cytolytic T lymphocytes to their targets as indicated by the possibility of blocking this interaction with monoclonal antibodies directed against these structures (Nakayama et al., 1979; Sarmiento et al., 1980; MacDonald et al., 1982; Meuer et al., 1982). Based on the predicted primary structure of
these molecules deduced from the sequence of their cDNA, peptides can be synthesized and anti-peptide antisera can be raised against well defined domains of the proteins. Both synthetic peptides and anti-peptide antibodies might provide valuable probes for investigating the structural and functional relationship of the CD8 molecular complex as well as for the study of the processing and assembly of the various subunits during biogenesis. In this communication, we describe the synthesis of a peptide corresponding to the sequence of the first 13 amino acid residues of the mouse Lyt-2 antigen and the property of an antiserum raised against this peptide which reacts with detergent-dispersed molecules of the membrane protein.
Materials and methods Correspondence to: C. Bron, Institute of Biochemistry, University of Lausanne, Ch. des Boveresses, 1066 Epalinges, Switzerland.
All chemicals and buffer components were of analytical grade. Protein A-Sepharose and CNBr-
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
98 activated Sepharose 4B were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Carrier-free Na125I (1680 Ci/mmole) was purchased from Radiochemical Amersham International Center.
Animals and cells Inbred C57B1/6 mice were obtained from the animal colony of the Swiss Institute for Experimental Cancer Research (Epalinges, Switzerland). Thymocyte suspensions were prepared from thymuses of 4-5-week-old mice using a loosely fitted Dounce tissue homogenizer. New Zealand White rabbits were used for the preparation of anti-peptide antisera.
Synthesis of peptide A 13 residue peptide was synthesized by the Merrifield solid-phase method (Marglin and Merrifield, 1970) using FMOC-protected a-amino acids (Atherton et al., 1979). A tyrosine residue was attached at the C-terminus. The synthetic peptide was purified by gel filtration, ion exchange and high performance liquid chromatography (HPLC); amino acid composition was proved to be correct by standard amino acid analysis. The purified peptide was coupled to ovalbumin or bovine serum albumin (BSA) via the di-azo-benzidine group (Bassiri et al., 1979). An average of 0.5 mg of peptide was coupled to 1 mg of carrier protein.
Immunization procedure One rabbit was immunized intramuscularly with 0.5 mg of synthetic peptide conjugated to 1 mg of ovalbumin solubilized in 1.5 M of guanidine hydrochloride and emulsified in complete Freund's adjuvant. The immunization was repeated 2 weeks later with the same antigen in incomplete Freund's adjuvant. Two successive immunizations were then performed at 10 day intervals with 10 mg of the non-conjugated peptide solubilized in phosphatebuffered saline (PBS) and emulsified in incomplete Freund's adjuvant. The rabbit was bled 7-8 days after these injections and the serum collected. The immunoglobulin fraction was prepared by precipitation of the antiserum with 40% ammonium sulfate, followed by a purification on a Sephadex CL-B6 column in 0.2 M citrate pH 7.0
buffer containing 0.5M NaC1. For immunoblotting, the Ig fraction was iodinated by the chloramine-T method.
Antibody assay The antibody activity of the rabbit antiserum was determined by a solid-phase ELISA in 96-well polyvinyl chloride microtitration plates (Dynatech Laboratories, Alexandria, U.S.A.). The wells were coated with 100/~1 quantities of the carrier(BSA)conjugated peptide solution (20 /~g/ml) in phosphate-buffered saline (PBS) pH 7.5 for 45 min at room temperature. The plates were then washed twice with PBS containing 0.2% of gelatine. Serial dilution of the antiserum or of the pre-immune serum in the same buffer were added. The plates were incubated for 30 min at room temperature and washed three times in PBS supplemented with 0.2% of gelatine. Specific goat antibodies (100/~1) to rabbit immunoglobulins conjugated to alkaline phosphatase (Sigma Chemical Co., St. Louis, MO) diluted 1/1000 in 0.2% gelatine PBS were ad~ted to each well and the plate incubated at room temperature for 30 rain. The plate was subsequently washed and 100 /~1 of a solution of 1 mg/ml of sodium p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, MO) in a 1 M diethanolamine buffer at pH 9.8 containing 10 3 M KC1. The absorbance of the solution in wells was determined at 405 nm in an automatic reader (Titertek Multiskan, Flow Laboratories, Switzerland). Inhibition of antibody binding was also determined by a similar type of ELISA assay except that the diluted antiserum was preincubated with appropriate amounts of inhibitor peptide or carrier-conjugated peptide.
Immunoprecipitations Indirect immunoprecipitations were carried out according to previously described procedures (Liascher et al., 1984). In brief, 10 v surfaceiodinated cells were lysed in 1 ml of buffer containing 0.5% Nonidet P-40 (NP-40), 0.5% deoxycholate (DOC), 25 mM Tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HC1) pH 8.1, 50 mM NaC1 0.01% NaN 3 and 2 mM phenylmethylsulfonylfluoride (PMSF) at 4°C (lysis buffer). Detergent insoluble material was then removed by centrifugation at 400 × g for 10 min.
99 The cell lysate was precleared twice with 50/~1 of a suspension of 250 /~g normal human Ig-conjugated to Sepharose 4B. Alternatively, preclearing was performed by addition of 10 /tl of normal rabbit serum followed by successive absorptions with 30 /~1 of a suspension of protein A-conjugated to Sepharose 4B. The lysate was then filtered through a 0.22 /~m Millipore filter before immunoprecipitation. The cleared lysate of 10-15 × 10 6 cells was incubated with 10 /tl of rabbit anti-peptide antiserum for 30 min at room temperature followed by absorption with 30 /~1 of a suspension of protein A-conjugated to Sepharose 4B. This step could be omitted for the immunoprecipitations with the anti-Lyt-2 monoclonal antibody H-59 which was coupled to Sepharose 4B. In certain experiments, cell lysates were pre-treated with 0.1% SDS final concentration, heated at 100°C for 5 min and diluted with 11 vols. of 1% Triton X-100 prior to immunoprecipitation (Maccecchini and Schatz, 1979). The immune precipitates were washed four times in PBS p H 8.2 supplemented with 0.05% SDS, 0.5% DOC, 0.5% NP-40 and 10 mM E D T A and four times with a 120 mM Tris-HC1 buffer at p H 8.2 containing 500 m M NaC1, 0.5% NP-40 and 10 m M EDTA.
Polyacrylamide gel electrophoresis in SDS One-dimensional SDS-PAGE was carried out on 10% gels according to Laemmli (1970). Immunoprecipitates were dissolved in 80 mM TrisHC1, pH 6.8, 0.1 M dithiothreitol (DTT), 4% SDS, 10% glycerol, and 0.01% bromophenol blue (sample buffer) heated at 100°C for 5 min before layering onto the gel. The following molecular weight standards were used: fl-galactosidase ( M r = 130000), phosphorylase b ( M r = 94000), transferrin ( M r = 78 000), bovine serum albumin ( M r = 68 000), ovalbumin ( M r = 46 000), glyceraldehyde3-phosphatedehydrogenase ( M r = 34000), achymotrypsinogen ( M r = 25 000), and cytochrome c ( M r = 12000).
Immunoblotting The reactivity of the anti-peptide serum with thymocyte membranes was tested by immunoblotting (Towbin et al., 1979). In brief, 100 x 106 thymocytes of C57B1/6 mice were lysed at 4°C in
2 ml of lysis buffer of the same composition as for immunoprecipitations. Particulate material was removed by centrifugation at 400 × g for 20 min. The cell lysate was then adjusted to 2% SDS, 10% glycerol, 80 mM Tris-HC1 at p H 6.8, 0.1 M dithiothreitol (DTT) and heated for 5 min at 100 o C before separation on a 10% preparative polyacrylamide gel (Laemmli, 1970). Separated proteins were transferred electrophoretically to nitrocellulose paper membrane filters BA83, 0.2 /~m (Schleicher and Schiill, Dassel, F.R.G.). The blot paper was saturated with a solution of PBS, p H 7.6 containing 2% of gelatin. The paper strips were incubated overnight at 4°C with 1.5 ml of PBS containing 2% of gelatin and 1.5 ~tg of iodinated antibodies (approximatively 1.5 × 105 cpm). The solution was passed through a 0.45 /~m Milipore filter before incubation. Strips were incubated with 125I-labeled protein A solution (2 × 10 5 c p m / m l ) for 2 h and finally washed, dried and autoradiographed using Kodak XOMAT-XS5 films.
Anti-Lyt-2 monoclonal antibody The monoclonal antibody H-59-101.7 was kindly provided by Dr. M. Pierres (Centre d'Immunologie, CNRS, Marseille, France) (Goldstein et al., 1982) and ascites prepared from hybridoma bearing C57B1/6 irradiated mice. For immunoprecipitation, anti-Lyt-2 mAb were partially purified from ascites fluid by two successive precipitations with 45% ammonium sulfate, and was covalently bound to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden). About 3-6 mg of the immunoglobulin fraction was usually conjugated to 1 ml of activated Sepharose 4B.
Surface labeling of thymocytes Enzyme-catalyzed surface iodination of thymocytes was performed according to the method of Hubbard and Cohn (1975).
Results
Anti-peptide response of the rabbit A 13 residue peptide was synthesized which corresponded to the sequence of the initial 13
100
Reactiuity of anti-peptide antibodies with membrane proteins
residues of the a subunit of the Lyt-2/3 antigen determined by gas-phase protein sequencing (Walker et al., 1984a) and predicted by the cDNA sequence (Nakauchi et al., 1985). An additional tyrosine residue was added to the C-terminal end of the peptide to facilitate its subsequent conjugation to ovalbumin or bovine serum albumin (BSA). The sequence was as follows: Lys-Pro-Gln-AlaPro-Glu-Leu-Arg-Ile-Phe-Pro-Lys-Tyr. The rabbit was immunized with the ovalbumin-peptide conjugate. Anti-peptide antibodies could be detected after the second injection as demonstrated by a solid-phase binding assay using a BSA-peptide conjugate to coat the polyvinyl plates. The two successive injections with 10 mg of free peptide increased the antibody titer from 1:1600 to 1:6400 for 50% of antibody binding (Fig. 1A). The specificity of the antiserum for the peptide was demonstrated by inhibition of the antibody binding with less than 1 Fg/ml of BSA-peptide conjugate (Fig. 1B).
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The reactivity of the anti-peptide antiserum with the Lyt-2/3 molecular complex was first demonstrated by its ability to immunoprecipitate the antigen from non-ionic detergent lysates of surface-iodinated thymocytes. Thus, the antipeptide antibody precipitated two surface-labeled polypeptides of M r = 36000 and M r = 32000 respectively (Fig. 2, lanes 1 and 4). Both free and BSA-conjugated peptide totally inhibited this reaction (Fig. 2, lanes 2 and 5). The same two polypeptides were precipitated with the monoclonal antibody H-59 specific for the Lyt-2 antigen (Fig. 2, lane 3). In this case however, no inhibition of the precipitation was observed following the addition of free or BSA-conjugated peptide to the antibody before immunoprecipitation (data not shown). To compare further the reactivity of the antipeptide antiserum to the mAb H-59, sequential immunoprecipitations were performed, first with
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Fig. 1. ,4 : Anti-peptide response in a rabbit immunized with a synthetic peptide-conjugated to ovalbumin. Wells were coated with 20 /~g of BSA-conjugated synthetic peptide. • e, rabbit serum before immunization; © O, antiserum after two injections of OVA-peptide; A A, antiserum after a third injection with 10 mg of free peptide: [] .... •, antiserum after a fourth injection with 10 mg of free peptide; B: Inhibition of the reactivity of the rabbit anti-peptide antiserum. Wells were coated with 20 t~g of BSA-conjugated synthetic peptide. The antiserum dilution was 1:3000. The concentration of inhibitor is expressed logarithmically. • e, BSA; O O, ovalbumin-conjugated peptide.
101
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Fig. 2. Immunoprecipitates of detergent-lysed surface-iodinated thymocytes. Each precipitation was performed with the lysate of 10-15x106 cells. Autoradiographs of 10% SDS-polyacrylamide gels were performed under reducing conditions. Rabbit anti-peptide antiserum (1 and 4); rabbit anti-peptide antiserum in the presence of 500/xg of synthetic peptide (2); rat anti-Lyt-2 mAb H-59-101-7 (2); rabbit anti-peptide antiserum in the presence of 16 #g of BSA-conjugated synthetic peptide (5). Molecular weight standards (see the materials and methods section) were run on the same gel.
H-59 until the Lyt-2/3 molecular complex was exhaustively precipitated from a lysate of 30 x 106 surface-iodinated thymocytes. Under the experimental conditions chosen (see the material and methods section), this required three successive additions of Sepharose-4B-insolubilized H-59 monoclonal antibody. No significant precipitation of labeled Lyt-2/3 molecules was obtained by the subsequent addition of the anti-peptide antiserum (Fig. 3A, lanes 1-4). When the latter reagent was used first, two successive precipitations were suffi-
cient to deplete the lysate of labeled Lyt-2/3 polypeptides. However residual antigens could be precipitated by adding the mAb H-59 (Fig. 3A, lanes 5-8). Thus the anti-peptide antiserum precipitated approximatively one third of the radioactivity precipitable with the mAb H-59 from surface-labeled thymocytes. This was assessed by excising pieces of fixed and dried gels and counting the radioactivity associated with the Lyt-2/3 polypeptides identified by autoradiography. A number of high molecular weight bands coprecipitated with the Lyt-2/3 polypeptides when the mAb H-59 was used. These represented about 5-10% of the total radioactivity. The presence of these contaminants was usually seen when immunoprecipitations were performed with lysates of more than 20 X 10 6 cells/ml. In fact, they were not detectable when the Lyt-2/3 antigen was immunopurified from lysates of 10-15 x 106 cells/ml (see also Fig. 2). In order to determine whether the anti-peptide antiserum reacted preferentially with denatured molecules, non-ionic detergent lysates of surfacelabeled cells were treated with 0.1% SDS, heated at 100°C for 5 min and diluted in Triton X-100 prior to immunoprecipitation (Maccecchini et al., 1979). Under such conditions, the amount of labeled Lyt-2/3 molecules precipitated with the antipeptide antiserum was increased by a factor of 2-3 compared to non-treated lysates. In contrast, no precipitation was obtained with the mAb H59 (Fig. 3B, lanes 1-5). In addition, the amount of radioactivity precipitated by the anti-peptide antiserum was comparable to the radioactivity precipitated by the mAb H59 from non-SDS-treated lysates. Finally, free peptides added to the antiserum before immunoprecipitation inhibited totally the precipitation of the Lyt-2/3 molecules (Fig. 3B, lanes 6 and 7). The preferential reactivity of the anti-peptide antiserum with antigens which were denatured prior to immunoprecipitation prompted us to test whether the Lyt-2/3 molecules could be detected by immunoblotting. As shown in Fig. 4, this was indeed the case. In fact, the anti-peptide antiserum reacted with two polypeptides from a thymocyte lysate (M r = 36 000 and M r = 32 000). This reaction revealed with 125I-labeled immuno-
102
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Fig. 3. Sequential immunoprecipitations of detergent-lysed surface-iodinated thymocytes. Autoradiographs of 10% SDS-polyacrylamide gels under reducing conditions A: Lysate of 30 × 106 cells precipitated by three successive additions of mAb H-59 conjugated to Sepharose 4B (1, 2, 3) followed by the rabbit anti-peptide antiserum (4). Lysate of 30 × 10 6 cells precipitated by three successive additions of rabbit anti-peptide antiserum (5,6,7) followed by the mAb H-59 (8). B: Lysates of 10 × 10 6 cells first depleted of molecules reacting with the anti-peptide antiserum by three successive immunoprecipitations and then immunoprecipitated with: rat anti-Lvt-2 mAb H-59 (1) rabbit anti-peptide antiserum after treatment with 0.1% SDS and 100°C (see materials and methods section) (2); rabbit anti-peptide antiserum without SDS pretreatment (3). Lysate of 20 x 10 6 cells immunoprecipitated with rat anti-Lyt-2 mAb (H-59) without pretreatment with SDS (4) and after pretreatment with 0.1% SDS and heating at 100 o C (5) Lysate of 30 × 106 cells immunoprecipitated with the rabbit antiserum after treatment with 0.1% SDS and 100°C (6) and in the presence of 1 mg of synthetic peptide (7).
globulin was inhibited when the antiserum was incubated with the nitrocellulose strip in the presence of free or BSA-conjugated peptide (Fig. 4, lanes 1-2). Specificity was further confirmed by the fact that the anti-peptide antiserum did not react with the lysate of the E L 4 cell line - a L y t - 2 / 3 - n e g a t i v e murine thymoma. In contrast, a rabbit anti-Thy-1 a n t i b o d y clearly detected this antigen in the lysate of the EL4 cell line (Fig. 4, lanes 3-4). Finally, no reaction of the antiserum could be f o u n d by indirect immunofluorescence using viable thymocyte suspensions.
Discussion Biochemical analysis of the mouse C D 8 (Lyt2 / 3 ) antigens has previously shown that in thymocytes this molecular complex consists of disulfide-linked heterodimers comprising a 2 8 - 3 0
k D a subunit expressing the Lyt-3 epitope and either one of two structurally related 37 k D a and 32 k D a glycopeptides. Both of these chains carry the Lyt-2 antigenic determinant. They p r o b a b l y differ in their C-terminal region ( D u r d a et al., 1978; Reilly et al., 1980; Ledbetter et at., 1981; Jay et al., 1982; Rothenberg and Triglia, 1983; N a i m et al., 1984; Walker et al., 1984b; Luescher et al., 1985). It was recently suggested that these two polypeptides are encoded b y one single gene and that the difference in the size of their C-terminal portion arises from alternative m R N A splicing ( Z a m o y s k a et al., 1985). W e describe here the properties of a rabbit antiserum raised against a synthetic peptide which reacts with the mouse C D 8 antigen. The structure of the 13 amino acid peptide is based on the corresponding sequence of the N-terminal segment of the 37 k D a chain (Walker et al., 1984; N a k a u c h i et al., 1985). The specificity in this reagent for the L y t - 2 / 3 molecular complex is
103
1
2
3
4
Fig. 4. Detection of Lyt-2 polypeptides with the rabbit antipeptide antiserum by immunoblotting. Rabbit anti-peptide antiserum with lysates of thymocyte (1); same experiment in the presence of 100 /~g of BSA-conjugated peptide (2); rabbit anti-peptide antiserum with lysates of the EL-4 cell line (3); rabbit anti-Thy-1 antiserum with lysates of the EL-4 cell line (4). Lanes 1-3 are autoradiographs of blots performed with the iodinated immunoglobulins of the rabbit anti-peptide antiserum. The blot of lane 4 was revealed with a peroxidase-conjugated swine anti-rabbit Ig (Dako, Denmark).
demonstrated by the selective precipitation of these two polypeptides from the detergent lysate of surface iodinated thymocytes. These components were not detected with the anti-peptide antiserum
when the cell lysate was first depleted of Lyt-2/3 molecules by immunoprecipitation with the antiLyt-2 mAb (H-59-101.7). In contrast, the latter mAb still precipitated the Lyt-2/3 antigen from lysates which had been depleted of the molecules reacting with the anti-peptide antiserum. This result may reflect a lower affinity of the rabbit antiserum for the Lyt-2/3 molecules compared to the monoclonal antibody H-59. Alternatively it may be that only a subset of surface-expressed Lyt-2/3 molecules are recognized by this reagent in cell lysates. New antigenic sites can, however, be generated by treating surface-labeled cell lysates with 0.1% SDS and heating at 100°C. This was demonstrated by the fact that the amount of labeled Lyt-2/3 antigens precipitated under such experimental conditions is increased 2-3-f01d compared to non-treated lysates. This corresponds to the quantity of protein precipitable with the mAb H-59 without denaturation, as assessed by counting Lyt-2/3 specific bands excised from SDSpolyacrylamide gels. Moreover, Lyt-2/3 molecules can again be detected with the anti-peptide antiserum if the denaturating treatment is performed after several steps of exhaustive preclearing with the same reagent. Nevertheless, the antiserum did not react with the surface of viable cells in spite of its ability to partially precipitate surface-iodinated molecules. Moreover, it did not inhibit cell surface binding of various anti-Lyt-2/3 monoclonal antibodies nor did it block the cytolysis of Lyt-2/3-dependent CTL clones (data not shown). This suggests that the N-terminal segment of Lyt-2/3 is not accessible to the antibodies in the native surface-expressed form of the antigen even though the molecule is likely to be oriented with its N-terminal on the outside of the cell. The reactivity of the antiserum with surface-labeled molecules would then imply that the antigenic determinant detected is either generated by denaturation upon cell lysis or exposed by the dispersion of the membrane components in the mixed micelles of detergent. Taken together, these observations suggest that, whereas the mAb H-59 detects a conformation-dependent epitope which is destroyed by SDS at 100°C, the rabbit antiserum is directed against an SDS-resistant antigenic determinant. However, we cannot
104
exclude that the anti-peptide antiserum contains antibodies specific for native surface expressed antigens and others specific for denatured structures. The reactivity of the rabbit antiserum with denatured Lyt-2 molecules is further revealed by its reactivity in immunoblotting, a property which is not displayed by the mAb H-59. This result also demonstrates that the antipeptide antiserum recognizes both the 37 kDa and 32 kDa glycopeptides of Lyt-2/3 after reductive dissociation of the molecules in SDS. This is in agreement with the structural identity of the surface domains of these two components which has been predicted by the analysis of cDNA clones probably corresponding to these two subunits (Zamwoyska et al., 1985) and has recently been directly demonstrated by the determination of the amino acid sequence of the N-terminal segments of both glycopeptides (Walker et al., 1986). Moreover, the weaker reactivity of the antibody with the 32 kDa polypeptide probably reflects a lower amount of this component compared to the major 37 kDa species. This also correlates with previously published reports (Walker et al., 1984). Finally, failure to detect the 28 kDa subunit indicates that this chain differs from the other subunits, at least in the structure of its N-terminal region.
Acknowledgements We thank M. Rousseaux for skilled technical assistance, C. Vonney for assistance in the synthesis and purification of the peptide, M.-C. Knecht for secretarial assistance, Z. Freiwald for photographical work. This work was supported by the FNRS Grant no. 3.427.-0.83.
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