Journal oflmmunologicalMethods, 129 (1990) 105-109
105
Elsevier JIM05550
A simple method for the radioactive iodination of CD4 molecules P. Portol6s, J.M. Rojo and C.A. Janeway, Jr. Department of Pathology and Section of lmmunobiology, Howard Hughes Medical Institute at Yale University School of Medicine, New Haven, CT06510, U.S.A.
(Received 5 October1989,revisedreceived11 December1989, accepted8 January1990)
A method for the vectorial radioiodination of CD4 and other membrane proteins having few or no tyrosine residues in the extracytoplasmic domains is described. Incubation of the cells with sulfosuccinimidyl (hydroxyphenyl) propionate (sulfo-SHPP), a water-soluble derivative of the Bolton-Hunter reagent results in the coupling of hydroxyphenyl groups to free amino groups of cell surface proteins and these groups are then vectorially radioiodinated using 1,3,4,6-tetrachloro-3a,~ot-diphenylglycoluril (Iodogen). The method is highly efficient, gentle, fast, simple, and does not require the previous radiolabelling of the Bolton-Hunter reagent. Key words: CD4, surface; Radioactiveiodination:Sulfosuccinimidyl(hydroxyphenyl)propionate
Introduction The immunochemical study of surface CD4 has been hampered by the difficulty of iodinating CD4 using vectorial labelling methods such as lacto-peroxidase (Terhorst et al., 1980; Dialynas et al., 1983). This seems to be due to the small number of tyrosine residues (three in CD4, two in L3T4) present in the extracellular domains of the molecule (Maddon et al., 1985; Tourvielle et al., 1986; Littman and Gettner, 1987; Clark et al., 1987). Thus, radiolabelling of CD4 has usually been performed by internal labelling, a method that does not reflect the characteristics of mem-
Correspondence to: P. Portol6s, Centro de Investigaciones Bioi6gicas,Velkzquez144, 28006-Madrid, Spain. Abbreviations: PBS, phosphate-buffered saline; PMSF, phenylmethylsulphonylfluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel dectrophoresis; Sulfo-SHPP,sulfosuccinirnidyl(hydroxyphenyl)propionate;TCGF,T cellgrowth factor-containingsupernatant.
brane CD4; by incorporation of tritium into sugar moieties (Terhorst et al., 1980), or by incubation of the cells with methyl-p-hydroxybenzimidate before iodination (Dialynas et al., 1983). The latter methods are either not efficient or threaten the integrity of the cells (Warr and Marchalonis, 1987). To obviate these problems, we have taken advantage of several factors: (1) the Bolton-Hunter reagent (3-(p-hydroxyphenyl) propionic acid Nhydroxy succinimide ester), which couples primarily to free amino groups in proteins (Bolton and Hunter, 1973) can be used to incorporate p-hydroxyphenyl groups into proteins and peptides; (2) molecules of the same chemical characteristics as the Bolton-Hunter reagent (N-succinimide esters) have been used for haptenation of live cells without loss of viability (Pohlit et al., 1979); (3) vectorial labelling of cell membranes has been achieved using a hydrosoluble derivative of t25IBohon-Hunter reagent (125I-sulfosuccinimidyl (hydroxyphenyl) propionate) (Thompson et al., 1987), although this has the disadvantage that even short periods of incubation of the reagent in aqueous
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
106
solution during the iodination procedure might inactivate the molecule and reduce the yield. In this paper, a method is described which combines the successive incubation of cells with water-soluble Bolton-Hunter reagent with vectorial radioiodination to a high specific activity. This approach permits the labelling of cell surface molecules, such as CD4, that are not efficiently labelled by conventional iodination methods. Hydrolysis and inactivation of the amino-reactive groups is minimized, and yet the procedure remains efficient, simple and fast.
Materials and methods
Cells Cells of the L3T4 ÷ T cell clones D10.G4.1 (D10) and AE16.10.6 (AE16) were used. D10 is specific for hen egg conalbumin in the context of I-Ak molecules. The AE16 cell line originated in AKR mice and is self-reactive for I-Ak (Yagi et al., 1989). Both cell lines express T cell receptors recognized by the monoclonal antibody F23.1. D10 was grown by stimulation every 2 weeks with antigen (100 #g/ml) and H-2 ~ feeder cells (5 × 105 mitomycin C-treated B10.BR or C3H spleen cells/ml) in Click's medium supplemented with 5% fetal calf serum (FCS) plus 5% TCGF (supernatant from concanavalin A-activated rat spleen cells containing 20 mg/ml of a-methyl-mannoside). AE16 cells were stimulated every week in the presence of 10 6 mitomycin C-treated H-2 k feeder cells/ml in Click's medium supplemented as described above. Cells to be used in the experiments were rested for at least 2 weeks following antigenic stimulation. Monoclonal antibodies The following monoclonal antibodies have been used: F23.1 anti-T cell receptor antibodies (mouse IgG2a) (Staerz et al., 1985); MK-D6 anti-I-Ad (mouse IgG2a) (Kappler et al., 1981); anti-CD3 antibody YCD3-1 (rat IgG2c) (Portol6s et al., 1989); anti-LFA-1 M17/5 (rat IgG2a) (ShnchezMadrid et al., 1982); anti-Mac-1 antibody M1/70 (rat IgG2b) (Springer et al., 1979); and anti-L3T4 GK1.5 (rat IgG2b) (Dialynas et al., 1983). All antibodies were purified from ascites by protein A
affinity chromatography and were kept at 4 ° C in PBS.
Incubation with sulfo-SHPP and radioactive label ring Cells were spun, passed over a Ficoll-Hypaque gradient (LSM, Organon Teknika Corporation, Durham, NC), thoroughly washed in PBS, and resuspended in cold PBS (107 cells/ml). Cells should be at least 95% viable. Then, 20 #1 of borate buffer, pH 9.2 (35 mM Na2B40 7, 80 mM NaCI) were added per ml of cell suspension to raise the pH of the solution to pH 8. Sulfo-SHPP (Pierce Europe, Oud-Beijerland, The Netherlands) was dissolved in PBS to 1 mg/ml and immediately added to the cells (36 #g of sulfo-SHPP/ml of cell suspension, equivalent to 0.1 mM). This mixture was incubated for 15-20 min at 4 ° C with gentle mixing, washed three times with cold PBS and resuspended at 5 × 107 cells/ml in PBS. Radioiodination was performed using tetrachloroglycoluril (Markwell and Fox, 1978). Cells (5 × 107 in 1 ml) plus 500 #Ci Na~25I (Amersham, Buckinghamshire, England) were placed in a glass tube coated with 100 ~g of tetrachloroglycoluril and incubated for 20 min at room temperature with occasional mixing. The ceils were washed three times in cold PBS containing 2 mM potassium iodide and proccessed for immunoprecipitation. These procedures did not produce any appreciable loss of viability as determined by trypan blue exclusion. Lysis of the cells, immunoprecipitation and SDSPAGE Cells were lysed by the addition of 0.1 ml/107 cells of lysis buffer (50 mM Tris, 300 mM NaC1, 0.4% Triton X-100 (Pierce), 1 mM PMSF, 10 # g / m l aprotinin, 1 mM iodoacetamide, pH 7.6). After 30 rain, in an ice bath, the lysate was centrifuged for 30 rnin at 12,000 × g in the cold. The supernatant was pre-cleared by two separate incubation steps (15 min at 4 ° C with agitation) with protein A-Sepharose (Pharmacia, 20 #1 packed Sepharose in lysis buffer/lysate from 107 cells) in the presence of protein A-purified normal mouse Ig (2 #g of Ig/lysate from 107 cells) followed by incubation with packed protein A-Sepharose alone.
107 For each immunoprecipitation, the volume of lysate equivalent to 107 cells was taken and placed in an eppendorf tube. Then, 3 /~g of purified monoclonal antibody were added, mixed and left on ice for 30 min. 3 mg of goat anti-mouse Ig or goat anti-rat Ig microspheres (Kierkegaard Perry, Gaithersburg, MD) in 30 #1 of lysis buffer were added to each sample, and the samples rotated for 2 h at 4 ° C . The microspheres were spun in a microfuge, washed three times with 1 ml of 50 mM Tris, 300 mM NaC1, 0.1% Triton X-100, 0.2% sodium dodecyl sulfate, 0.2% sodium deoxycholate, pH 7.6, resuspended in 1 ml of 50 mM Tris, 300 mM NaC1, 0.1% Triton X-100, 1 mM PMSF, 10 ~tg/ml aprotinin, pH 7.6, split in two, spun in a
microfuge, and resuspended in 40/xl of reducing or non-reducing SDS-PAGE sample buffer. After 15 min, the samples were spun again and the supematant taken, incubated at 9 0 ° C for 5 min and electrophoresed in polyacrylamide gel as previously described (Jones, 1980).
Results and discussion In spite of its functional importance, the study of cell surface CD4 molecules has been greatly hampered by the lack of a simple vectorial labelling method. Commonly used methods of vectorial labelling, such as lodogen, results in negligible
B
A
1
2
3
4
KO
I
2
3
4
5
KD
-- 2 0 0 -2 O0
--92.5 --69 -92.5
I --46
-69
,--30 -46 ,"21.5 .l
-3o
. . . .
14.3
Fig. I. Incubation with sulfo-SHPP improves the radioactive iodination of CD4. A: DI0 cellswere treated (lane 3--4) or not (lanes 1-2) with sulfo-SHPP, labelled with l~I, lysed, and the lysate subjected to inununoprecipitation with anti-CIM (GKI.5, lanes 2 and
4) or isotypc-matchedrat antibodies (M1/'70 anti-Mac-1 antibodies, lanes 1 and 3). B: AE16 cells were treated with sulfo-SHPP, labelled, lysed and immunoprecipitatedwith a negativecontrol antibody (anti-hAd MK-D6, lane 1); anti-T cell receptor F'23.1 (lane 2); anti-CD3e (YCD3-1, lane 3); anti-CIM (GK1.5, lane 4); or anti-LFA-1 (M17/5, lane 5). Autoradiographswere obtained after SDS-PAGE electrophoresisunder non-reducingconditionsin 5-155ggradient aerylamidegels.
108
labelling of CD4 compared to other surface molecules, as demonstrated by immunoprecipitation, SDS-PAGE and autoradiography of the precipitated samples (Fig. 1, lane 2). Since the poor labelling of CD4 is due to the small number of tyrosine residues present in the extracytoplasmic region of each CD4 molecule (Maddon et al., 1985; Tourvielle et al., 1986; Clark et al., 1987; Littman and Gettner, 1987), we have attempted to augment the number of groups in the molecule which can be iodinated taking advantage of the properties of sulfosuccinimidyl (hydroxyphenyl) propionate (sulfo-SHPP). N-succinimide derivatives of different haptens have been used to derivatize cell surfaces without loss of viability in fast, simple procedures (Pholit et al., 1979). Furthermore, water-soluble N-succinimide derivatives, such as sulfo-SHPP, react selectively with molecules on the outer surfaces of the cells (Thompson et al., 1987). In the procedure described here, cells from the CD4 + cell line D10 (Fig. 1A) and from the AE16 cell line (Fig. 1B) were incubated with sulfo-SHPP in order to incorporate hydroxyphenyl groups that could be iodinated in the following step. Under the conditions employed (0.1 mM sulfo-SHPP, 15 min at 4°C), the procedure is mild and fast, so that there is no loss of viability in the cells. As shown in Fig. 1, sulfo-SHPP-treated cells efficiently incorporate 125I into CD4 (Fig. 1A, lane 4), while, using the same iodination procedure, untreated cells do not (Fig. 1, lane 2), as demonstrated by immunoprecipitation with GK1.5. Similar results were obtained with immunoprecipitates from the other CD4 + T cell line, AE 16.10.6 (Fig. 1B, lane 4, and data not shown). Anti-CD4 antibodies precipitated molecules of 55,000 M r when analysed by electrophoresis under non-reducing conditions, and slightly higher (57,000 Mr) when analysed under reducing conditions (data not shown), possibly due to reduction of predicted intra-chain disulfide bridges (Maddon et al., 1985; Tourvielle et al., 1986; Clark et al., 1987; Littman and Gettner, 1987). As also shown in Fig. 1B, other surface molecules, such as the T cell receptor, CD3e, and LFA-1 are also efficiently labelled, yet the treatment with sulfo-SHPP does not affect the ability of antibodies to recognize these molecules.
Acknowledgements The authors wish to thank Jungi Yagi for AE16 cells, and Frank Fitch, Michael Bevan, Timothy Springer and John Kappler for monoclonal antibodies. This work was supported by National Institutes of Health Grants AI-13766 and AI-14579, and by Grant PB87-0230 of CICyT (Spain). P.P. is the recipient of a Postdoctoral Fellowship from C.S.I.C. (Spain).
References Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a ~2Sl-containing acylating agent. Biochem. J. 133, 529. Clark S.J., Jefferies, W.A., Barclay, A.N., Gagnon, J. and Williams, A.F. (1987) Peptide and nucleotide sequences of rat CD4 (W3/25) antigen: Evidence for derivation from a structure with four immunoglobulin-related domains. Proc. Natl. Acad. Sci. U.S.A. 84, 1649. Dialynas, D.P., Quan, Z.S., Wall, K.A., Pierres, A., Quinthns, J., Loken, M.R., Pierres, M. and Fitch, F.W. (1983) Characterization of the routine T cell surface molecule, designated L3T4, identified by monocional antibody GK1.5: Similarity of L3T4 to the human Leu-3/T4 molecule. J. Immunol. 131, 2445. Jones, P.P. (1980) Analysis of radiolabeled lymphocyte proteins by one- and two-dimensional polyacrylamide gel electrophoresis. In: B.B. Mishell and S.M. Shiigi (Eds.), Selected Methods in Cellular Immunolgy. W.H. Freeman, San Francisco, CA, p. 398. Kappler, J.W., Skidmore, B., White, J. and Mar'rack, P. (1981) Antigen-inducible, H-2-restrieted, interleuldn-2-producing T cell hybridomas. J. Exp. Med. 153, 1198. Littman, D.R. and Gettner, S.N. (1987) Unusual intron in the immunoglobulin domain of the newly isolated murine CD4 (L3T4) gene. Nature 325, 453. Maddon, P.J., Littman, D.R., Godfrey, M., Maddon, D.E., Chess, L. and Axel, R. (1985) The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: A new member of the immunoglobulin gene family. Cell 42, 93. Markwell, M.A.K. and Fox, C.F. (1978) Surface-specific iodination of membrane proteins of viruses and euearyotic cells using 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochemistry 17, 4807. Pohlit, H.M., Haas, W. and Von Boehmer, H. (1979) Haptenation of viable biological carriers. In: I. Lefkovits and B. Pernis (Eds.), Immunological Methods. Academic Press, New York, p. 181. Portolds, M.P., Rojo, J.M., Golby, A., Bormeville, M., Gromkowski, S., Greenbaum, L., Janeway, Jr., C.A., Murphy,
109 D.B. and Bottomly, K. (1989) Monoclonal antibodies to murine CD3E define distinct cpitopes, one of which may interact with CD4 during T cell activation. J. Immunol. 142, 4169. S~nchez-Madrid, F., Davignon, D., Martz, E. and Springer, T.A. (1982) Antigens involved in mouse cytolytic Tlymphocyte (CTL)-mediated killing: Functional screening and topographic relationship. Cell. Immunol. 73, 1. Springer, T., Galfr/:, G., Secher, D.S. and Milstein, C. (1979) Mac-l: a macrophage differentiation antigen identified by monoclonal antibody. Eur. J. Immunol. 9, 301. Staerz, U.D., Rammensee, H.-G., Benedetto, J.D. and Bevan, M.J. (1985) Characterization of a routine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor. J. Immunol. 134, 3994. Terhorst, C., Van Agthoven, A., Reinherz, E. and Schlossman,
S. (1980) Biochemical analysis of human T lymphocyte differentiation antigens T4 and 1"5. Science, 209, 520. Thompson, J.A., Lau, A.L. and Cunningham, D.D. (1987) Selective radiolabeling of cell surface proteins to a high specific activity. Biochemistry 26, 743. Tourvieille, B., Gorman, S.D., Field, E.H., Hunkapiller, T. and Parnes, J.R. (1986) Isolation and sequence of L3T4 complementary DNA clones: Expression in T cells and brain. Science 234, 610. Wart, G.W. and Marchalonis, J.J. (1987) Nonpermeant covalent labels in analytical studies of lymphocyte membrane proteins. Methods Enzymol. 150, 399. Yagi, J., Buxser, S. and Janeway, Jr., C.A. (1989) Bacterial proteins that mediate the association of a defined subset of T cell receptor:CD4 complexes with class II MHC. Submitted.
105
Elsevier JIM05550
A simple method for the radioactive iodination of CD4 molecules P. Portol6s, J.M. Rojo and C.A. Janeway, Jr. Department of Pathology and Section of lmmunobiology, Howard Hughes Medical Institute at Yale University School of Medicine, New Haven, CT06510, U.S.A.
(Received 5 October1989,revisedreceived11 December1989, accepted8 January1990)
A method for the vectorial radioiodination of CD4 and other membrane proteins having few or no tyrosine residues in the extracytoplasmic domains is described. Incubation of the cells with sulfosuccinimidyl (hydroxyphenyl) propionate (sulfo-SHPP), a water-soluble derivative of the Bolton-Hunter reagent results in the coupling of hydroxyphenyl groups to free amino groups of cell surface proteins and these groups are then vectorially radioiodinated using 1,3,4,6-tetrachloro-3a,~ot-diphenylglycoluril (Iodogen). The method is highly efficient, gentle, fast, simple, and does not require the previous radiolabelling of the Bolton-Hunter reagent. Key words: CD4, surface; Radioactiveiodination:Sulfosuccinimidyl(hydroxyphenyl)propionate
Introduction The immunochemical study of surface CD4 has been hampered by the difficulty of iodinating CD4 using vectorial labelling methods such as lacto-peroxidase (Terhorst et al., 1980; Dialynas et al., 1983). This seems to be due to the small number of tyrosine residues (three in CD4, two in L3T4) present in the extracellular domains of the molecule (Maddon et al., 1985; Tourvielle et al., 1986; Littman and Gettner, 1987; Clark et al., 1987). Thus, radiolabelling of CD4 has usually been performed by internal labelling, a method that does not reflect the characteristics of mem-
Correspondence to: P. Portol6s, Centro de Investigaciones Bioi6gicas,Velkzquez144, 28006-Madrid, Spain. Abbreviations: PBS, phosphate-buffered saline; PMSF, phenylmethylsulphonylfluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel dectrophoresis; Sulfo-SHPP,sulfosuccinirnidyl(hydroxyphenyl)propionate;TCGF,T cellgrowth factor-containingsupernatant.
brane CD4; by incorporation of tritium into sugar moieties (Terhorst et al., 1980), or by incubation of the cells with methyl-p-hydroxybenzimidate before iodination (Dialynas et al., 1983). The latter methods are either not efficient or threaten the integrity of the cells (Warr and Marchalonis, 1987). To obviate these problems, we have taken advantage of several factors: (1) the Bolton-Hunter reagent (3-(p-hydroxyphenyl) propionic acid Nhydroxy succinimide ester), which couples primarily to free amino groups in proteins (Bolton and Hunter, 1973) can be used to incorporate p-hydroxyphenyl groups into proteins and peptides; (2) molecules of the same chemical characteristics as the Bolton-Hunter reagent (N-succinimide esters) have been used for haptenation of live cells without loss of viability (Pohlit et al., 1979); (3) vectorial labelling of cell membranes has been achieved using a hydrosoluble derivative of t25IBohon-Hunter reagent (125I-sulfosuccinimidyl (hydroxyphenyl) propionate) (Thompson et al., 1987), although this has the disadvantage that even short periods of incubation of the reagent in aqueous
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
106
solution during the iodination procedure might inactivate the molecule and reduce the yield. In this paper, a method is described which combines the successive incubation of cells with water-soluble Bolton-Hunter reagent with vectorial radioiodination to a high specific activity. This approach permits the labelling of cell surface molecules, such as CD4, that are not efficiently labelled by conventional iodination methods. Hydrolysis and inactivation of the amino-reactive groups is minimized, and yet the procedure remains efficient, simple and fast.
Materials and methods
Cells Cells of the L3T4 ÷ T cell clones D10.G4.1 (D10) and AE16.10.6 (AE16) were used. D10 is specific for hen egg conalbumin in the context of I-Ak molecules. The AE16 cell line originated in AKR mice and is self-reactive for I-Ak (Yagi et al., 1989). Both cell lines express T cell receptors recognized by the monoclonal antibody F23.1. D10 was grown by stimulation every 2 weeks with antigen (100 #g/ml) and H-2 ~ feeder cells (5 × 105 mitomycin C-treated B10.BR or C3H spleen cells/ml) in Click's medium supplemented with 5% fetal calf serum (FCS) plus 5% TCGF (supernatant from concanavalin A-activated rat spleen cells containing 20 mg/ml of a-methyl-mannoside). AE16 cells were stimulated every week in the presence of 10 6 mitomycin C-treated H-2 k feeder cells/ml in Click's medium supplemented as described above. Cells to be used in the experiments were rested for at least 2 weeks following antigenic stimulation. Monoclonal antibodies The following monoclonal antibodies have been used: F23.1 anti-T cell receptor antibodies (mouse IgG2a) (Staerz et al., 1985); MK-D6 anti-I-Ad (mouse IgG2a) (Kappler et al., 1981); anti-CD3 antibody YCD3-1 (rat IgG2c) (Portol6s et al., 1989); anti-LFA-1 M17/5 (rat IgG2a) (ShnchezMadrid et al., 1982); anti-Mac-1 antibody M1/70 (rat IgG2b) (Springer et al., 1979); and anti-L3T4 GK1.5 (rat IgG2b) (Dialynas et al., 1983). All antibodies were purified from ascites by protein A
affinity chromatography and were kept at 4 ° C in PBS.
Incubation with sulfo-SHPP and radioactive label ring Cells were spun, passed over a Ficoll-Hypaque gradient (LSM, Organon Teknika Corporation, Durham, NC), thoroughly washed in PBS, and resuspended in cold PBS (107 cells/ml). Cells should be at least 95% viable. Then, 20 #1 of borate buffer, pH 9.2 (35 mM Na2B40 7, 80 mM NaCI) were added per ml of cell suspension to raise the pH of the solution to pH 8. Sulfo-SHPP (Pierce Europe, Oud-Beijerland, The Netherlands) was dissolved in PBS to 1 mg/ml and immediately added to the cells (36 #g of sulfo-SHPP/ml of cell suspension, equivalent to 0.1 mM). This mixture was incubated for 15-20 min at 4 ° C with gentle mixing, washed three times with cold PBS and resuspended at 5 × 107 cells/ml in PBS. Radioiodination was performed using tetrachloroglycoluril (Markwell and Fox, 1978). Cells (5 × 107 in 1 ml) plus 500 #Ci Na~25I (Amersham, Buckinghamshire, England) were placed in a glass tube coated with 100 ~g of tetrachloroglycoluril and incubated for 20 min at room temperature with occasional mixing. The ceils were washed three times in cold PBS containing 2 mM potassium iodide and proccessed for immunoprecipitation. These procedures did not produce any appreciable loss of viability as determined by trypan blue exclusion. Lysis of the cells, immunoprecipitation and SDSPAGE Cells were lysed by the addition of 0.1 ml/107 cells of lysis buffer (50 mM Tris, 300 mM NaC1, 0.4% Triton X-100 (Pierce), 1 mM PMSF, 10 # g / m l aprotinin, 1 mM iodoacetamide, pH 7.6). After 30 rain, in an ice bath, the lysate was centrifuged for 30 rnin at 12,000 × g in the cold. The supernatant was pre-cleared by two separate incubation steps (15 min at 4 ° C with agitation) with protein A-Sepharose (Pharmacia, 20 #1 packed Sepharose in lysis buffer/lysate from 107 cells) in the presence of protein A-purified normal mouse Ig (2 #g of Ig/lysate from 107 cells) followed by incubation with packed protein A-Sepharose alone.
107 For each immunoprecipitation, the volume of lysate equivalent to 107 cells was taken and placed in an eppendorf tube. Then, 3 /~g of purified monoclonal antibody were added, mixed and left on ice for 30 min. 3 mg of goat anti-mouse Ig or goat anti-rat Ig microspheres (Kierkegaard Perry, Gaithersburg, MD) in 30 #1 of lysis buffer were added to each sample, and the samples rotated for 2 h at 4 ° C . The microspheres were spun in a microfuge, washed three times with 1 ml of 50 mM Tris, 300 mM NaC1, 0.1% Triton X-100, 0.2% sodium dodecyl sulfate, 0.2% sodium deoxycholate, pH 7.6, resuspended in 1 ml of 50 mM Tris, 300 mM NaC1, 0.1% Triton X-100, 1 mM PMSF, 10 ~tg/ml aprotinin, pH 7.6, split in two, spun in a
microfuge, and resuspended in 40/xl of reducing or non-reducing SDS-PAGE sample buffer. After 15 min, the samples were spun again and the supematant taken, incubated at 9 0 ° C for 5 min and electrophoresed in polyacrylamide gel as previously described (Jones, 1980).
Results and discussion In spite of its functional importance, the study of cell surface CD4 molecules has been greatly hampered by the lack of a simple vectorial labelling method. Commonly used methods of vectorial labelling, such as lodogen, results in negligible
B
A
1
2
3
4
KO
I
2
3
4
5
KD
-- 2 0 0 -2 O0
--92.5 --69 -92.5
I --46
-69
,--30 -46 ,"21.5 .l
-3o
. . . .
14.3
Fig. I. Incubation with sulfo-SHPP improves the radioactive iodination of CD4. A: DI0 cellswere treated (lane 3--4) or not (lanes 1-2) with sulfo-SHPP, labelled with l~I, lysed, and the lysate subjected to inununoprecipitation with anti-CIM (GKI.5, lanes 2 and
4) or isotypc-matchedrat antibodies (M1/'70 anti-Mac-1 antibodies, lanes 1 and 3). B: AE16 cells were treated with sulfo-SHPP, labelled, lysed and immunoprecipitatedwith a negativecontrol antibody (anti-hAd MK-D6, lane 1); anti-T cell receptor F'23.1 (lane 2); anti-CD3e (YCD3-1, lane 3); anti-CIM (GK1.5, lane 4); or anti-LFA-1 (M17/5, lane 5). Autoradiographswere obtained after SDS-PAGE electrophoresisunder non-reducingconditionsin 5-155ggradient aerylamidegels.
108
labelling of CD4 compared to other surface molecules, as demonstrated by immunoprecipitation, SDS-PAGE and autoradiography of the precipitated samples (Fig. 1, lane 2). Since the poor labelling of CD4 is due to the small number of tyrosine residues present in the extracytoplasmic region of each CD4 molecule (Maddon et al., 1985; Tourvielle et al., 1986; Clark et al., 1987; Littman and Gettner, 1987), we have attempted to augment the number of groups in the molecule which can be iodinated taking advantage of the properties of sulfosuccinimidyl (hydroxyphenyl) propionate (sulfo-SHPP). N-succinimide derivatives of different haptens have been used to derivatize cell surfaces without loss of viability in fast, simple procedures (Pholit et al., 1979). Furthermore, water-soluble N-succinimide derivatives, such as sulfo-SHPP, react selectively with molecules on the outer surfaces of the cells (Thompson et al., 1987). In the procedure described here, cells from the CD4 + cell line D10 (Fig. 1A) and from the AE16 cell line (Fig. 1B) were incubated with sulfo-SHPP in order to incorporate hydroxyphenyl groups that could be iodinated in the following step. Under the conditions employed (0.1 mM sulfo-SHPP, 15 min at 4°C), the procedure is mild and fast, so that there is no loss of viability in the cells. As shown in Fig. 1, sulfo-SHPP-treated cells efficiently incorporate 125I into CD4 (Fig. 1A, lane 4), while, using the same iodination procedure, untreated cells do not (Fig. 1, lane 2), as demonstrated by immunoprecipitation with GK1.5. Similar results were obtained with immunoprecipitates from the other CD4 + T cell line, AE 16.10.6 (Fig. 1B, lane 4, and data not shown). Anti-CD4 antibodies precipitated molecules of 55,000 M r when analysed by electrophoresis under non-reducing conditions, and slightly higher (57,000 Mr) when analysed under reducing conditions (data not shown), possibly due to reduction of predicted intra-chain disulfide bridges (Maddon et al., 1985; Tourvielle et al., 1986; Clark et al., 1987; Littman and Gettner, 1987). As also shown in Fig. 1B, other surface molecules, such as the T cell receptor, CD3e, and LFA-1 are also efficiently labelled, yet the treatment with sulfo-SHPP does not affect the ability of antibodies to recognize these molecules.
Acknowledgements The authors wish to thank Jungi Yagi for AE16 cells, and Frank Fitch, Michael Bevan, Timothy Springer and John Kappler for monoclonal antibodies. This work was supported by National Institutes of Health Grants AI-13766 and AI-14579, and by Grant PB87-0230 of CICyT (Spain). P.P. is the recipient of a Postdoctoral Fellowship from C.S.I.C. (Spain).
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