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Reizer, J., Saier, M.H., Jr., Deutscher, J., et al. 1988. The phosphoenolpyruvate:sugar phosphotransferase system in Grampositive bacteria: properties, mechanism, and regulation. Crit. Rev. Microbiol. 15: 297-338. Reizer, J., Sutrina, S.L., Saier, M.H., Jr., et al. 1989. Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoeno1pyruvate:sugar phosphotransferase system in Gram-positive bacteria: studies with sitespecific mutants of HPr. EMBO J. 8: 21 11-2120. Saier, M.H., Jr., and Reizer, J. 1990. Domain shuffling during evolution of proteins of the bacterial phosphotransferase system. Res. Microbiol. 141: 1033-1038. Saier, M.H., Jr., and Reizer, J. 1992. A uniform nomenclature for the permeases and permease domains of the bacterial phosphoeno1pyruvate:sugar phosphotransferase system. J. Bacteriol. In press. Simoni, R.D., Hays, J.B., Nakazawa, T., and Roseman, S. 1973. Sugar transport. VI. Phosphoryl transfer in the lactose phosphotransferase system of Staphylococcus aureus. J. Biol. Chem. 248: 957-965. Sutrina, S.L., Reddy, P., Saier, M.H., Jr., and Reizer, J. 1990. The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease. J. Biol. Chem. 265: 18 581 - 18 589. Waygood, E.B. 1986. Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system has two sites of phosphorylation per dimer. Biochemistry, 25: 4085-4090. Waygood, E.B., Mattoo, R.L., and Peri, K.G. 1984. Phosphoproteins and the phosphoeno1pyruvate:sugarphosphotransferase
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system in Salmonella typhimurium and Escherichia coli: evidence for 111rnanno= I I I ~ N C ~ O S ~ I I I ~ I U C I ~ O I and the phosphorylation of enzyme ~~mankto~ and en&e II~-8'cety~gl~c~samine . J. Cell. Biochem. 25: 139-159. Waygood, E.B., Erickson, E., El-Kabbani, O.A.L., and Delbaere, L.T.J. 1985. Characterization of phosphorylated histidinecontaining protein (HPr) of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Biochemistry, 24: 6938-6945. Waygood, E.B., Pasloske, K., Delbaere, L.T.J., et al. 1988. Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoeno1pyruvate:sugar phosphotransferase system of Streptococcusfaecalis. Biochem. Cell Biol. 66: 76-80. Waygood, E.B., Sharma, S., Bhanot, P., et al. 1989. The structure of HPr and site-directed mutagenesis. FEMS Microbiol. Rev. 63: 43-52. Weigel, N., Kukuruzinska, M.A., Nakazawa, T., et al. 1982. Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by enzyme I of Salmonella typhimurium. J. Biol. Chem. 257: 14 477 - 14 491. Wittekind, M., Reizer, J., Deutscher, J., et al. 1989. Common structural changes accompany the functional inactivation of HPr by seryl phosphorylation or by serine to aspartate substitution. Biochemistry, 28: 9908-9912. Wittekind, M., Reizer, J., and Klevit, R.E. 1990. Sequence-specific 'H NMR resonance assignments of Bacillus subtilis. HPr: use of spectra obtained from mutants to resolve spectral overlap. Biochemistry, 29: 7 191-7200.
The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium XIANG-FUW U AND RAJENDERK. KAMBOJ' Bunting and Best Department of Medical Research, University of Toronto, Toronto, Ont., Canada M5G 1L6
JEANGARIEPY Department of Biophysics, University of Toronto, Toronto, Ont., Canada M5G IL6 AND
CHI-HUNG SIU~ Bunting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ont., Canada M5G 1L6 Received September 25, 1991 Wu, X.-F., KAMBoJ, R. K., GARIEPY,J., and SIU, C.-H. 1992. The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium. Biochem. Cell Biol. 70: 246-249. The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding of mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition. Key words: monoclonal antibody, epitope mapping, cell adhesion molecule, Dictyostelium. Wu, X.-F., KAMBoJ, R. K., GARIEPY,J., et SIU, C.-H. 1992. The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium. Biochem. Cell Biol. 70 : 246-249. ABBREVIATIONS: gp80, glycoprotein of 80 000 relative mass; mAb, monoclonal antibody; M,, relative mass; IgG, immunoglobulin G; Fmoc, 9-fluorenylmethoxycarbonyl; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. 'present address: Allelix Biopharmaceutical Inc., Mississauga, Ont., Canada L4V 1P1. * ~ u t h o to r whom all correspondence should be sent at the following address: Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ont., Canada MSG 1L6. Printed in Canada / Im~rimeau Canada
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L'anticorps monoclonal 80L5C4 est un puissant inhibiteur de la molCcule d'adhkrence cellulaire gp80 de Dictyostelium discoideum. Afin de localiser exactement 1'Cpitope reconnu par l'anticorps 80LSC4, des hexapeptides, de sCquences chevauchantes, ont CtC synthCtisCs sur des aiguilles de plastiques et la liaison de l'anticorps 80L5C4 a ces peptides a CtC determink par un test immunoenzyrnatique ELISA. L'Cpitope 80L5C4 a CtC identifit comme Ctant un seul hexapeptide ayant la stquence GYKLNV et partageant cinq rCsidus d'acides aminks avec l'octapeptide YKLNVNDS intervenant dans la liaison homophile de gp80. Des Ctudes analogues montrent que les rCsidus hydrophobes dans cette sequence sont trts importants pour la reconnaissance de l'antigtne. Mots clks : anticorps monoclonal, cartographie des tpitopes, molCcule d'adhkrence cellulaire, Dictyostelium. [Traduit par la redaction]
Introduction The use of antibodies to inhibit cell-cell adhesion has been a useful approach in the study of cell adhesion molecules. This immunologic approach was first exploited by Gerisch and co-workers (Gerisch 1980), who have elegantly provided serological evidence for the presence of two distinct types of cell-cell adhesion systems in Dictyostelium discoideum (Beug et al. 1973). With the advent of monoclonal antibody technology, many cell-cell adhesion molecules have been identified and characterized in recent years (Edelman 1985; Takeichi 1990; Siu 1990). An understanding of epitopes recognized by these antibodies may provide insight on the mechanism of cell-cell adhesion. The cellular slime mold D. discoideum has been a popular system for the study of cell-cell interaction. Cells express EDTA-sensitive cell adhesion soon after the initiation of development (Garrod 1972; Knecht et al. 1987; Siu et al. 1988). At the onset of the aggregation stage, cells acquire EDTA-resistant binding sites (Rosen et al. 1973). A surface glycoprotein of M, 80 000 (gp80) was first implicated in EDTA-resistant cell-cell adhesion by Muller and Gerisch (1978). gp80 has also been implicated in size regulation and morphogenesis (Kamboj et al. 1990; Siu and Kamboj 1990). The direct involvement of gp80 in cell-cell binding was confirmed by the use of monoclonal antibodies in inhibition assays (Brodie et al. 1983; Siu et al. 1985). In this respect, mAb 80L5C4 has been particularly useful. 80L5C4 IgG is a potent inhibitor of the cell binding activity of gp80 (Siu et al. 1985). Therefore, the 80L5C4 epitope may play an important role in gp80-mediated cell-cell binding. The location of the 80L5C4 epitope has been mapped to a 51 amino acid segment near the N-terminus (Kamboj and Siu 1988). Recently, gp80 has been shown to mediate cellcell adhesion via homophilic interaction (Siu et al. 1987) and the homophilic binding site has been mapped (Kamboj et al. 1988, 1989). To determine the relationship between the 80L5C4 epitope and the homophilic binding site of gp80, we report in this paper the exact location of the 80L5C4 epitope. Interestingly, it shows significant overlap with the homophilic binding site sequence. Materials and methods Materials Prederivatized plastic pins and polypropylene trays were obtained from Cambridge Research Biochemicals (Valley Stream, NY). Active esters of Fmoc amino acids were obtained from MilliGen (Millipore). Other reagents used were of highest chemical grade. Cell strain and culture conditions The wild-type strain NC4 was cultured on agar dishes in association with Klebsiella aerogenes as food source (Sussman 1966). For development, cells were sus ended in 17 mM Na2/K phosphate buffer (pH 6.4) at 1.5 x 10 cells/mL and rotated at 180 rpm at room temperature (Cocucci and Sussman 1970).
P
Solid-phase peptide synthesis Overlapping hexapeptides were synthesized on polyethylene pins as described by Geysen et al. (1987). The peptides were synthesized by solid-phase methods (Erickson and Merrifield 1976) and they were assembled on pins in the C- to N-terminus direction using Fmoc amino acids. ELISA The pins were first incubated with PBS containing 2% (w/v) bovine serum albumin, 0.1% (v/v) Tween-20, and 0.05% (w/v) sodium azide for 30 min at room temperature with gentle rocking, and then incubated overnight in microtiter plates containing 130 pL/well of mAb (5 pg/mL of blocking solution) at 4°C. They were washed four times with PBS and 0.05% Tween-20, followed by incubation for 1 h with a goat anti-mouse-IgG antibody conjugated with alkaline phosphatase. The pins were washed four times and then once in substrate buffer (1 M diethanolamine containing 1 mM MgC12, pH 9.5-9.8), followed by incubation in microtiter wells, each containing 100 pL of substrate, for 2-3 h at room temperature or overnight at 4OC. Color development was monitored at 405 nm. ZgG competition assay
IgG was iodinated with N ~ ' ~ ' using I chloramine T. Dictyostelium cells were collected at 10 h of development and resuspended at 5 x lo6 cells/mL in 17 mM phosphate buffer (pH 6.4) containing 5 mM EDTA and 1% albumin. Aliquots of 0.1 mL were mixed with '*'I-labeled IgG in the presence of varying amounts of an unlabeled competing IgG. Incubation was carried out for 40 min at 4°C and then the cells were washed twice before counting in a gamma counter.
Results and discussion To determine the exact location of the 80L5C4 epitope, we have adopted the strategy developed by Geysen et al. (1987). Overlapping hexapeptides covering the amino acid sequence from Val- 123 to Ser-151 (for the complete amino acid sequence of gp80, refer to Kamboj et al. (1988)) were synthesized on plastic pins. Each successive peptide contains the last five amino acids of the previous one, plus the next amino acid in the sequence. These pins were incubated with 80L5C4 IgG and the histogram in Fig. 1 depicts the relative intensities of the ELISA signals. The hexapeptide sequence GYKLNV reproducibly reacted most strongly with mAb 80L5C4, while signals of the neighboring sequences were at background level. The hexapeptides ISGYKL and VNDSIN were weakly reactive with the antibody. Since the signals were relatively low, they probably represent nonspecific interactions with the antibody. To ascertain that mAb 80L5C4 interacts with the sequence GYKLNV, competitive IgG-binding studies were carried out. Antibodies were raised against a 13 amino acid sequence NISGYKLNVNDSI conjugated to keyhole limpet hemocyanin via a Cys residue added to its amino terminus (Kamboj et al. 1989)' and the purified IgG was used in competition experiments. The GYKLNV sequence was situated in the middle of this oligopeptide and should have
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Absorbance at 405 nm 0.0
0.2
0.4
0.6
0.8
SGYKLNVNOS
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GYKLNV
VDGPSNISGYKLNVNDSINSAMLSVTADS
Amino acid sequence FIG. 1. Binding of mAb 80L5C4 to gp80-overlapping hexapeptides. Plastic pins were incubated with 80L5C4 IgG (5 pg/mL) and the relative amounts of IgG bound were assayed by ELISA. The x-axis shows the amino acid sequence of gp80 beginning with Val-123. Each vertical bar represents a measure of antibody binding to the corresponding hexapeptide, with its amino-terminal amino acid placed directly under the bar and the rest of the sequence represented by the next five amino acids on the x-axis. Results shown in the histogram are representative of three determinations.
GYKANV GYKLAV SGYKLN SGAKLN
1
I
1
SGYDLN
FIG. 3. Binding of mAb 80L5C4 to peptide analogues on plastic pins. Plastic pins carrying different peptide analogues were incubated with 80L5C4 IgG (5 pg/mL) and the relative amounts of IgG bound were assayed by ELISA. The histogram shows relative antibody binding to the different peptide analogs. The sequences of analogs are aligned with respect to the wild-type sequence and altered amino acids are shown in bold type. Values are expressed as the mean SD of three experiments.
*
log (IgG ratio) FIG. 2. Competition of 80L5C4 IgG binding to cells. Cells at the aggregation stage of development were collected and resuspended at 5 x lo6 cells/mL for the binding assa Samples of 0.1 mL were incubated with a Tied amount of 'I-labeled 80L5C4 IgG (1 pg/mL) and binding was carried out in the presence of unlabeled 80L5C4 IgG ( 0 ) or anti-13-mer IgG ( 0 ) .
elicited antibodies directed against the 80L5C4 epitope. lZ51-labeled 80L5C4 IgG was used to bind to the cell surface gp80 molecules in the presence of varying concentrations of unlabeled 80L5C4 IgG or anti-13-mer IgG. Both IgG species competed effectively with the labeled 80L5C4 IgG for gp80 binding (Fig. 2). In both instances, competition was achieved at a level greater than 90% and the amount of IgG required for 50% inhibition was only about 2.5-fold higher for the anti-13-mer IgG. These results thus indicate that mAb 80L5C4 recognized a single epitope in gp80 and confirm its specificity for a linear sequence within the 13-mer region. Results in Fig. 1 define the boundaries of the 80L5C4 epitope and indicate that Gly-131 and Val-136 are critical amino acids required for antigen recognition. To determine the relative importance of the other amino acids in antibody
binding, peptide analogues with single amino acid substitutions were synthesized on plastic pins for ELISA. Figure 3 shows the relative signal intensities obtained with these peptide analogues. The difference in signal intensity between the decapeptide SGYKLVNVDS and the hexapeptide GYKLNV was negligible, suggesting that the neighboring amino acids of the 80L5C4 epitope do not have significant contribution to antibody binding. Substitution of Tyr-132 with Ala or Leu-134 with Ala reduced the reactivity of the epitope drastically. On the other hand, substitution of Lys-133 with Asp or Asn-135 with Ala did not result in a significant decrease in the signal. The hexapeptide SGYKLN showed much reduced IgG binding, consistent with the results obtained in Fig. 1. Again, substitution of Tyr-132 in this hexapeptide with Ala led to complete loss in reactivity with 80LSC4, indicating the importance of Tyr-132 in the epitope. Since substitution of hydrophobic residues with Ala significantly diminishes its reactivity with mAb 80L5C4, hydrophobic interactions may be important for 80L5C4 binding. Our results demonstrate the stringent recognition between mAb 80L5C4 and its epitope, since only six amino acids are directly involved in antibody binding. This is consistent with previous estimates that a minimum number of five or six amino acids can be accommodated within an antibodycombining site (Crumpton 1974). It is also remarkable that changes in a single amino acid within the 80L5C4 epitope may abolish its immunoreactivity. Indeed, a number of monoclonal antibodies exhibit species specificity as a result
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N$-terminal Globular Region
Hinge Domain
Membrane Anchor
FIG. 4. Schematic drawing of gp80 and the location of the 80LSC4 epitope. The model of gp80 was based on secondary structure predictions (Kamboj et al. 1989; Siu and Kamboj 1990) and the 80LSC4 epitope (boxed) is shown to overlap with the homophilic binding site (shaded).
of single amino acid substitutions in the epitope (Marks et al. 1985; Georges et al. 1990; Nose et al. 1990). The most interesting observation is that the 80LSC4 epitope overlaps significantly with the homophilic binding site of gp80 (Fig. 4). Five of the amino acid residues are shared by the octapeptide sequence YKLNVNDS, which probably engages in anti-parallel pairing at the initial stage of gp80-gp80 binding (Kamboj et al. 1989). A salient feature of this region is its high probability for 0-structure (Siu and Kamboj 1990). The two charged residues Lys-133 and Asp-138 should be able to provide a hydrophilic environment, allowing this sequence to be exposed on the surface of the gp80 molecule and to take part in homophilic interaction. This site probably serves as the first contact point between two gp80 molecules and the initial ionic interactions between the charged residues may eventually lead to more stable types of molecular interactions. Since a large number of cell adhesion molecules were identified by adhesionblocking monoclonal antibodies, epitope mapping should provide a useful strategy to identify molecular domain(s) involved in cell-cell binding. Acknowledgements The work was supported by the Medical Research Council (MRC) of Canada. J.G. is the recipient of an MRC scholarship award. Beug, H., Katz, F., and Gerisch, G. 1973. Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56: 647-658. Brodie, C., Klein, C., and Swierkosz, J. 1983. Monoclonal antibodies: use to detect developmentally regulated antigens on D. discoideum. Cell, 32: 1115-1 123. Cocucci, S., and Sussman, M. 1970. RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 45: 399-407. Crumpton, M.J. 1974. Protein antigens. The molecular basis of antigenicity and immunogenicity. In The antigens. Vol. 2. Edited by M. Sela. Academic Press, New York. pp. 1-178. Edelman, G.M. 1985. Cell cohesion and the molecular processes of morphogenesis. Annu. Rev. Biochem. 54: 135-169.
Erickson, B.W., and Merrifield, R.B. 1976. Solid-state peptide synthesis. In The proteins. 3rd ed. Vol. 2. Edited by H. Neurath and R.L. Hill. Academic Press, New York. pp. 255-527. Garrod, D. 1972. Acquisition of cohesiveness by slime mould cells prior to morphogenesis. Exp. Cell Res. 72: 588-591. Georges, E., Bradley, G., Gariepy, J., and Ling, V. 1990. Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies. Proc. Natl. Acad. Sci. U.S.A. 87: 152-156. Gerisch, G. 1980. Univalent antibody fragments as tools for the analysis of cell interactions in Dictyostelium. Curr. Top. Dev. Biol. 14: 243-270. Gevsen. H.M.. Rodda. S.J.. Mason. T.J., et al. 1987. Strategies for epitope analy& using peptide synthesis. J. Immunol. Methods 102: 259-274. Kamboj, R.K., and Siu, C.-H. 1988. Mapping of the 80LSC4 epitope on the cell adhesion molecule gp80 of Dictyostelium discoideum. Biochim. Biophys. Acta, 951: 78-84. Kamboj, R.K., Wong, L.M., Lam, T.Y., and Siu, C.-H. 1988. Mapping of a cell binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum. J. Cell Biol. 107: 1835-1843. Kamboj, R.K., Gariepy, J., and Siu, C.-H. 1989. Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of Dictyostelium discoideum. Cell, 59: 615-625. Kamboj, R.K., Lam, T.Y ., and Siu, C.-H. 1990. Regulation of slug size by the cell adhesion molecule gp80 in Dictyostelium discoideum. Cell Regul. 1: 7 15-729. Knecht, D.A., Fuller, D.L., and Loomis, W.F. 1987. Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum. Dev. Biol. 121: 277-283. Marks, A., Yip, C., and Wilson, S. 1985. Characterization of two epitopes on insulin using monoclonal antibodies. Mol. Immunol. 22: 285-290. Muller, K., and Gerisch, G. 1978. A specific glycoprotein as the target site of adhesion blocking Fab in aggregatingDictyostelium discoideum. Nature (London), 274: 445-449. Nose, A., Tsuji, K., and Takeichi, M. 1990. Localization of specific determining sites in cadherin cell molecules. Cell, 61: 147-155. Rosen, S., Kafka, J.A., Simpson, D.L., and Barondes, S.H. 1973. Developmentally regulated carbohydrate-binding protein in Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 70: 2554-2557. Siu, C.-H. 1990. Cell adhesion molecules in Dictyostelium. BioEssays, 12: 357-362. Siu, C.-H., and Kamboj, R.K. 1990. Cell-cell adhesion and morphogenesis in Dictyostelium discoideum. Dev. Genet. 11: 377-387. Siu, C.-H., Lam, T.Y ., and Choi, A.H.C. 1985. Inhibition of cellcell binding at the aggregation stage of Dictyostelium by monoclonal antibodies directed against the contact site A glycoprotein. J. Biol. Chem. 260: 16 030 - 16 036. Siu, C.-H., Cho, A., and Choi, A.H.C. 1987. The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum. J. Cell Biol. 105: 2523-2533. Siu, C.-H., Wong, L.M., Choi, A.H.C., and Cho, A. 1988. Mechanisms of cell-cell recognition and cell cohesion in Dictyostelium discoideurn cells. In Eukaryote cell recognition. Edited by G.P. Chapman, C.C. Ainsworth, and C.J. Catham. Cambridge University Press, Cambridge. pp. 119-133. Sussman, M. 1966. Biochemicals and genetic methods in the study of cellular slime mold development. Methods Cell Physiol. 2: 397-410. Takeichi, M. 1990. Cadherins: a molecular family important in selective cell-cell adhesion. Annu. Rev. Biochem. 59: 237-252.
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Reizer, J., Saier, M.H., Jr., Deutscher, J., et al. 1988. The phosphoenolpyruvate:sugar phosphotransferase system in Grampositive bacteria: properties, mechanism, and regulation. Crit. Rev. Microbiol. 15: 297-338. Reizer, J., Sutrina, S.L., Saier, M.H., Jr., et al. 1989. Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoeno1pyruvate:sugar phosphotransferase system in Gram-positive bacteria: studies with sitespecific mutants of HPr. EMBO J. 8: 21 11-2120. Saier, M.H., Jr., and Reizer, J. 1990. Domain shuffling during evolution of proteins of the bacterial phosphotransferase system. Res. Microbiol. 141: 1033-1038. Saier, M.H., Jr., and Reizer, J. 1992. A uniform nomenclature for the permeases and permease domains of the bacterial phosphoeno1pyruvate:sugar phosphotransferase system. J. Bacteriol. In press. Simoni, R.D., Hays, J.B., Nakazawa, T., and Roseman, S. 1973. Sugar transport. VI. Phosphoryl transfer in the lactose phosphotransferase system of Staphylococcus aureus. J. Biol. Chem. 248: 957-965. Sutrina, S.L., Reddy, P., Saier, M.H., Jr., and Reizer, J. 1990. The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease. J. Biol. Chem. 265: 18 581 - 18 589. Waygood, E.B. 1986. Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system has two sites of phosphorylation per dimer. Biochemistry, 25: 4085-4090. Waygood, E.B., Mattoo, R.L., and Peri, K.G. 1984. Phosphoproteins and the phosphoeno1pyruvate:sugarphosphotransferase
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system in Salmonella typhimurium and Escherichia coli: evidence for 111rnanno= I I I ~ N C ~ O S ~ I I I ~ I U C I ~ O I and the phosphorylation of enzyme ~~mankto~ and en&e II~-8'cety~gl~c~samine . J. Cell. Biochem. 25: 139-159. Waygood, E.B., Erickson, E., El-Kabbani, O.A.L., and Delbaere, L.T.J. 1985. Characterization of phosphorylated histidinecontaining protein (HPr) of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Biochemistry, 24: 6938-6945. Waygood, E.B., Pasloske, K., Delbaere, L.T.J., et al. 1988. Characterization of the 1-phosphohistidinyl residue in the phosphocarrier protein HPr of the phosphoeno1pyruvate:sugar phosphotransferase system of Streptococcusfaecalis. Biochem. Cell Biol. 66: 76-80. Waygood, E.B., Sharma, S., Bhanot, P., et al. 1989. The structure of HPr and site-directed mutagenesis. FEMS Microbiol. Rev. 63: 43-52. Weigel, N., Kukuruzinska, M.A., Nakazawa, T., et al. 1982. Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by enzyme I of Salmonella typhimurium. J. Biol. Chem. 257: 14 477 - 14 491. Wittekind, M., Reizer, J., Deutscher, J., et al. 1989. Common structural changes accompany the functional inactivation of HPr by seryl phosphorylation or by serine to aspartate substitution. Biochemistry, 28: 9908-9912. Wittekind, M., Reizer, J., and Klevit, R.E. 1990. Sequence-specific 'H NMR resonance assignments of Bacillus subtilis. HPr: use of spectra obtained from mutants to resolve spectral overlap. Biochemistry, 29: 7 191-7200.
The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium XIANG-FUW U AND RAJENDERK. KAMBOJ' Bunting and Best Department of Medical Research, University of Toronto, Toronto, Ont., Canada M5G 1L6
JEANGARIEPY Department of Biophysics, University of Toronto, Toronto, Ont., Canada M5G IL6 AND
CHI-HUNG SIU~ Bunting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ont., Canada M5G 1L6 Received September 25, 1991 Wu, X.-F., KAMBoJ, R. K., GARIEPY,J., and SIU, C.-H. 1992. The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium. Biochem. Cell Biol. 70: 246-249. The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding of mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition. Key words: monoclonal antibody, epitope mapping, cell adhesion molecule, Dictyostelium. Wu, X.-F., KAMBoJ, R. K., GARIEPY,J., et SIU, C.-H. 1992. The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium. Biochem. Cell Biol. 70 : 246-249. ABBREVIATIONS: gp80, glycoprotein of 80 000 relative mass; mAb, monoclonal antibody; M,, relative mass; IgG, immunoglobulin G; Fmoc, 9-fluorenylmethoxycarbonyl; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. 'present address: Allelix Biopharmaceutical Inc., Mississauga, Ont., Canada L4V 1P1. * ~ u t h o to r whom all correspondence should be sent at the following address: Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ont., Canada MSG 1L6. Printed in Canada / Im~rimeau Canada
NOTES
247
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L'anticorps monoclonal 80L5C4 est un puissant inhibiteur de la molCcule d'adhkrence cellulaire gp80 de Dictyostelium discoideum. Afin de localiser exactement 1'Cpitope reconnu par l'anticorps 80LSC4, des hexapeptides, de sCquences chevauchantes, ont CtC synthCtisCs sur des aiguilles de plastiques et la liaison de l'anticorps 80L5C4 a ces peptides a CtC determink par un test immunoenzyrnatique ELISA. L'Cpitope 80L5C4 a CtC identifit comme Ctant un seul hexapeptide ayant la stquence GYKLNV et partageant cinq rCsidus d'acides aminks avec l'octapeptide YKLNVNDS intervenant dans la liaison homophile de gp80. Des Ctudes analogues montrent que les rCsidus hydrophobes dans cette sequence sont trts importants pour la reconnaissance de l'antigtne. Mots clks : anticorps monoclonal, cartographie des tpitopes, molCcule d'adhkrence cellulaire, Dictyostelium. [Traduit par la redaction]
Introduction The use of antibodies to inhibit cell-cell adhesion has been a useful approach in the study of cell adhesion molecules. This immunologic approach was first exploited by Gerisch and co-workers (Gerisch 1980), who have elegantly provided serological evidence for the presence of two distinct types of cell-cell adhesion systems in Dictyostelium discoideum (Beug et al. 1973). With the advent of monoclonal antibody technology, many cell-cell adhesion molecules have been identified and characterized in recent years (Edelman 1985; Takeichi 1990; Siu 1990). An understanding of epitopes recognized by these antibodies may provide insight on the mechanism of cell-cell adhesion. The cellular slime mold D. discoideum has been a popular system for the study of cell-cell interaction. Cells express EDTA-sensitive cell adhesion soon after the initiation of development (Garrod 1972; Knecht et al. 1987; Siu et al. 1988). At the onset of the aggregation stage, cells acquire EDTA-resistant binding sites (Rosen et al. 1973). A surface glycoprotein of M, 80 000 (gp80) was first implicated in EDTA-resistant cell-cell adhesion by Muller and Gerisch (1978). gp80 has also been implicated in size regulation and morphogenesis (Kamboj et al. 1990; Siu and Kamboj 1990). The direct involvement of gp80 in cell-cell binding was confirmed by the use of monoclonal antibodies in inhibition assays (Brodie et al. 1983; Siu et al. 1985). In this respect, mAb 80L5C4 has been particularly useful. 80L5C4 IgG is a potent inhibitor of the cell binding activity of gp80 (Siu et al. 1985). Therefore, the 80L5C4 epitope may play an important role in gp80-mediated cell-cell binding. The location of the 80L5C4 epitope has been mapped to a 51 amino acid segment near the N-terminus (Kamboj and Siu 1988). Recently, gp80 has been shown to mediate cellcell adhesion via homophilic interaction (Siu et al. 1987) and the homophilic binding site has been mapped (Kamboj et al. 1988, 1989). To determine the relationship between the 80L5C4 epitope and the homophilic binding site of gp80, we report in this paper the exact location of the 80L5C4 epitope. Interestingly, it shows significant overlap with the homophilic binding site sequence. Materials and methods Materials Prederivatized plastic pins and polypropylene trays were obtained from Cambridge Research Biochemicals (Valley Stream, NY). Active esters of Fmoc amino acids were obtained from MilliGen (Millipore). Other reagents used were of highest chemical grade. Cell strain and culture conditions The wild-type strain NC4 was cultured on agar dishes in association with Klebsiella aerogenes as food source (Sussman 1966). For development, cells were sus ended in 17 mM Na2/K phosphate buffer (pH 6.4) at 1.5 x 10 cells/mL and rotated at 180 rpm at room temperature (Cocucci and Sussman 1970).
P
Solid-phase peptide synthesis Overlapping hexapeptides were synthesized on polyethylene pins as described by Geysen et al. (1987). The peptides were synthesized by solid-phase methods (Erickson and Merrifield 1976) and they were assembled on pins in the C- to N-terminus direction using Fmoc amino acids. ELISA The pins were first incubated with PBS containing 2% (w/v) bovine serum albumin, 0.1% (v/v) Tween-20, and 0.05% (w/v) sodium azide for 30 min at room temperature with gentle rocking, and then incubated overnight in microtiter plates containing 130 pL/well of mAb (5 pg/mL of blocking solution) at 4°C. They were washed four times with PBS and 0.05% Tween-20, followed by incubation for 1 h with a goat anti-mouse-IgG antibody conjugated with alkaline phosphatase. The pins were washed four times and then once in substrate buffer (1 M diethanolamine containing 1 mM MgC12, pH 9.5-9.8), followed by incubation in microtiter wells, each containing 100 pL of substrate, for 2-3 h at room temperature or overnight at 4OC. Color development was monitored at 405 nm. ZgG competition assay
IgG was iodinated with N ~ ' ~ ' using I chloramine T. Dictyostelium cells were collected at 10 h of development and resuspended at 5 x lo6 cells/mL in 17 mM phosphate buffer (pH 6.4) containing 5 mM EDTA and 1% albumin. Aliquots of 0.1 mL were mixed with '*'I-labeled IgG in the presence of varying amounts of an unlabeled competing IgG. Incubation was carried out for 40 min at 4°C and then the cells were washed twice before counting in a gamma counter.
Results and discussion To determine the exact location of the 80L5C4 epitope, we have adopted the strategy developed by Geysen et al. (1987). Overlapping hexapeptides covering the amino acid sequence from Val- 123 to Ser-151 (for the complete amino acid sequence of gp80, refer to Kamboj et al. (1988)) were synthesized on plastic pins. Each successive peptide contains the last five amino acids of the previous one, plus the next amino acid in the sequence. These pins were incubated with 80L5C4 IgG and the histogram in Fig. 1 depicts the relative intensities of the ELISA signals. The hexapeptide sequence GYKLNV reproducibly reacted most strongly with mAb 80L5C4, while signals of the neighboring sequences were at background level. The hexapeptides ISGYKL and VNDSIN were weakly reactive with the antibody. Since the signals were relatively low, they probably represent nonspecific interactions with the antibody. To ascertain that mAb 80L5C4 interacts with the sequence GYKLNV, competitive IgG-binding studies were carried out. Antibodies were raised against a 13 amino acid sequence NISGYKLNVNDSI conjugated to keyhole limpet hemocyanin via a Cys residue added to its amino terminus (Kamboj et al. 1989)' and the purified IgG was used in competition experiments. The GYKLNV sequence was situated in the middle of this oligopeptide and should have
BIOCHEM. CELL BIOL. VOL. 70. 1992
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Absorbance at 405 nm 0.0
0.2
0.4
0.6
0.8
SGYKLNVNOS
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GYKLNV
VDGPSNISGYKLNVNDSINSAMLSVTADS
Amino acid sequence FIG. 1. Binding of mAb 80L5C4 to gp80-overlapping hexapeptides. Plastic pins were incubated with 80L5C4 IgG (5 pg/mL) and the relative amounts of IgG bound were assayed by ELISA. The x-axis shows the amino acid sequence of gp80 beginning with Val-123. Each vertical bar represents a measure of antibody binding to the corresponding hexapeptide, with its amino-terminal amino acid placed directly under the bar and the rest of the sequence represented by the next five amino acids on the x-axis. Results shown in the histogram are representative of three determinations.
GYKANV GYKLAV SGYKLN SGAKLN
1
I
1
SGYDLN
FIG. 3. Binding of mAb 80L5C4 to peptide analogues on plastic pins. Plastic pins carrying different peptide analogues were incubated with 80L5C4 IgG (5 pg/mL) and the relative amounts of IgG bound were assayed by ELISA. The histogram shows relative antibody binding to the different peptide analogs. The sequences of analogs are aligned with respect to the wild-type sequence and altered amino acids are shown in bold type. Values are expressed as the mean SD of three experiments.
*
log (IgG ratio) FIG. 2. Competition of 80L5C4 IgG binding to cells. Cells at the aggregation stage of development were collected and resuspended at 5 x lo6 cells/mL for the binding assa Samples of 0.1 mL were incubated with a Tied amount of 'I-labeled 80L5C4 IgG (1 pg/mL) and binding was carried out in the presence of unlabeled 80L5C4 IgG ( 0 ) or anti-13-mer IgG ( 0 ) .
elicited antibodies directed against the 80L5C4 epitope. lZ51-labeled 80L5C4 IgG was used to bind to the cell surface gp80 molecules in the presence of varying concentrations of unlabeled 80L5C4 IgG or anti-13-mer IgG. Both IgG species competed effectively with the labeled 80L5C4 IgG for gp80 binding (Fig. 2). In both instances, competition was achieved at a level greater than 90% and the amount of IgG required for 50% inhibition was only about 2.5-fold higher for the anti-13-mer IgG. These results thus indicate that mAb 80L5C4 recognized a single epitope in gp80 and confirm its specificity for a linear sequence within the 13-mer region. Results in Fig. 1 define the boundaries of the 80L5C4 epitope and indicate that Gly-131 and Val-136 are critical amino acids required for antigen recognition. To determine the relative importance of the other amino acids in antibody
binding, peptide analogues with single amino acid substitutions were synthesized on plastic pins for ELISA. Figure 3 shows the relative signal intensities obtained with these peptide analogues. The difference in signal intensity between the decapeptide SGYKLVNVDS and the hexapeptide GYKLNV was negligible, suggesting that the neighboring amino acids of the 80L5C4 epitope do not have significant contribution to antibody binding. Substitution of Tyr-132 with Ala or Leu-134 with Ala reduced the reactivity of the epitope drastically. On the other hand, substitution of Lys-133 with Asp or Asn-135 with Ala did not result in a significant decrease in the signal. The hexapeptide SGYKLN showed much reduced IgG binding, consistent with the results obtained in Fig. 1. Again, substitution of Tyr-132 in this hexapeptide with Ala led to complete loss in reactivity with 80LSC4, indicating the importance of Tyr-132 in the epitope. Since substitution of hydrophobic residues with Ala significantly diminishes its reactivity with mAb 80L5C4, hydrophobic interactions may be important for 80L5C4 binding. Our results demonstrate the stringent recognition between mAb 80L5C4 and its epitope, since only six amino acids are directly involved in antibody binding. This is consistent with previous estimates that a minimum number of five or six amino acids can be accommodated within an antibodycombining site (Crumpton 1974). It is also remarkable that changes in a single amino acid within the 80L5C4 epitope may abolish its immunoreactivity. Indeed, a number of monoclonal antibodies exhibit species specificity as a result
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N$-terminal Globular Region
Hinge Domain
Membrane Anchor
FIG. 4. Schematic drawing of gp80 and the location of the 80LSC4 epitope. The model of gp80 was based on secondary structure predictions (Kamboj et al. 1989; Siu and Kamboj 1990) and the 80LSC4 epitope (boxed) is shown to overlap with the homophilic binding site (shaded).
of single amino acid substitutions in the epitope (Marks et al. 1985; Georges et al. 1990; Nose et al. 1990). The most interesting observation is that the 80LSC4 epitope overlaps significantly with the homophilic binding site of gp80 (Fig. 4). Five of the amino acid residues are shared by the octapeptide sequence YKLNVNDS, which probably engages in anti-parallel pairing at the initial stage of gp80-gp80 binding (Kamboj et al. 1989). A salient feature of this region is its high probability for 0-structure (Siu and Kamboj 1990). The two charged residues Lys-133 and Asp-138 should be able to provide a hydrophilic environment, allowing this sequence to be exposed on the surface of the gp80 molecule and to take part in homophilic interaction. This site probably serves as the first contact point between two gp80 molecules and the initial ionic interactions between the charged residues may eventually lead to more stable types of molecular interactions. Since a large number of cell adhesion molecules were identified by adhesionblocking monoclonal antibodies, epitope mapping should provide a useful strategy to identify molecular domain(s) involved in cell-cell binding. Acknowledgements The work was supported by the Medical Research Council (MRC) of Canada. J.G. is the recipient of an MRC scholarship award. Beug, H., Katz, F., and Gerisch, G. 1973. Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56: 647-658. Brodie, C., Klein, C., and Swierkosz, J. 1983. Monoclonal antibodies: use to detect developmentally regulated antigens on D. discoideum. Cell, 32: 1115-1 123. Cocucci, S., and Sussman, M. 1970. RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 45: 399-407. Crumpton, M.J. 1974. Protein antigens. The molecular basis of antigenicity and immunogenicity. In The antigens. Vol. 2. Edited by M. Sela. Academic Press, New York. pp. 1-178. Edelman, G.M. 1985. Cell cohesion and the molecular processes of morphogenesis. Annu. Rev. Biochem. 54: 135-169.
Erickson, B.W., and Merrifield, R.B. 1976. Solid-state peptide synthesis. In The proteins. 3rd ed. Vol. 2. Edited by H. Neurath and R.L. Hill. Academic Press, New York. pp. 255-527. Garrod, D. 1972. Acquisition of cohesiveness by slime mould cells prior to morphogenesis. Exp. Cell Res. 72: 588-591. Georges, E., Bradley, G., Gariepy, J., and Ling, V. 1990. Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies. Proc. Natl. Acad. Sci. U.S.A. 87: 152-156. Gerisch, G. 1980. Univalent antibody fragments as tools for the analysis of cell interactions in Dictyostelium. Curr. Top. Dev. Biol. 14: 243-270. Gevsen. H.M.. Rodda. S.J.. Mason. T.J., et al. 1987. Strategies for epitope analy& using peptide synthesis. J. Immunol. Methods 102: 259-274. Kamboj, R.K., and Siu, C.-H. 1988. Mapping of the 80LSC4 epitope on the cell adhesion molecule gp80 of Dictyostelium discoideum. Biochim. Biophys. Acta, 951: 78-84. Kamboj, R.K., Wong, L.M., Lam, T.Y., and Siu, C.-H. 1988. Mapping of a cell binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum. J. Cell Biol. 107: 1835-1843. Kamboj, R.K., Gariepy, J., and Siu, C.-H. 1989. Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of Dictyostelium discoideum. Cell, 59: 615-625. Kamboj, R.K., Lam, T.Y ., and Siu, C.-H. 1990. Regulation of slug size by the cell adhesion molecule gp80 in Dictyostelium discoideum. Cell Regul. 1: 7 15-729. Knecht, D.A., Fuller, D.L., and Loomis, W.F. 1987. Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum. Dev. Biol. 121: 277-283. Marks, A., Yip, C., and Wilson, S. 1985. Characterization of two epitopes on insulin using monoclonal antibodies. Mol. Immunol. 22: 285-290. Muller, K., and Gerisch, G. 1978. A specific glycoprotein as the target site of adhesion blocking Fab in aggregatingDictyostelium discoideum. Nature (London), 274: 445-449. Nose, A., Tsuji, K., and Takeichi, M. 1990. Localization of specific determining sites in cadherin cell molecules. Cell, 61: 147-155. Rosen, S., Kafka, J.A., Simpson, D.L., and Barondes, S.H. 1973. Developmentally regulated carbohydrate-binding protein in Dictyostelium discoideum. Proc. Natl. Acad. Sci. U.S.A. 70: 2554-2557. Siu, C.-H. 1990. Cell adhesion molecules in Dictyostelium. BioEssays, 12: 357-362. Siu, C.-H., and Kamboj, R.K. 1990. Cell-cell adhesion and morphogenesis in Dictyostelium discoideum. Dev. Genet. 11: 377-387. Siu, C.-H., Lam, T.Y ., and Choi, A.H.C. 1985. Inhibition of cellcell binding at the aggregation stage of Dictyostelium by monoclonal antibodies directed against the contact site A glycoprotein. J. Biol. Chem. 260: 16 030 - 16 036. Siu, C.-H., Cho, A., and Choi, A.H.C. 1987. The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum. J. Cell Biol. 105: 2523-2533. Siu, C.-H., Wong, L.M., Choi, A.H.C., and Cho, A. 1988. Mechanisms of cell-cell recognition and cell cohesion in Dictyostelium discoideurn cells. In Eukaryote cell recognition. Edited by G.P. Chapman, C.C. Ainsworth, and C.J. Catham. Cambridge University Press, Cambridge. pp. 119-133. Sussman, M. 1966. Biochemicals and genetic methods in the study of cellular slime mold development. Methods Cell Physiol. 2: 397-410. Takeichi, M. 1990. Cadherins: a molecular family important in selective cell-cell adhesion. Annu. Rev. Biochem. 59: 237-252.
