The Adhesion and Differentiation-inhibitory Activities of the Immunoglobulin Superfamily Member, Carcinoembryonic Antigen, Can Be Independently Blocked
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m212500200
ISSN1083-351X
AutoresMaryam Akhavan Taheri, H. Uri Saragovi, Clifford P. Stanners,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe external domains of Ig superfamily members are involved in multiple binding interactions, both homophilic and heterophilic, that initiate molecular events leading to the execution of diverse cell functions. Human carcinoembryonic antigen (CEA), an Ig superfamily cell surface glycoprotein used widely as a clinical tumor marker, undergoes homophilic interactions that mediate intercellular adhesion. Recent evidence supports the view that deregulated overexpression of CEA has an instrumental role in tumorigenesis through the inhibition of cell differentiation and the disruption of tissue architecture. The CEA-mediated block of the myogenic differentiation of rat L6 myoblasts depends on homophilic binding of its external domains. We show here that L6 transfectant cells expressing CEA can "trans-block" the myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA (ΔNCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, ΔNCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to involve three subdomains of the amino-terminal domain, shown previously to be critical for its intercellular adhesion function. Monovalent Fab fragments of monoclonal antibodies binding to the region bridging subdomains 1 and 2 could both inhibit intercellular adhesion and release the myogenic differentiation block. Amino acid substitutions Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to completely ablate the differentiation blocking activity of CEA but had no effect on intercellular adhesion activity. A cyclized peptide representing this subdomain was the most effective at releasing the differentiation block. The external domains of Ig superfamily members are involved in multiple binding interactions, both homophilic and heterophilic, that initiate molecular events leading to the execution of diverse cell functions. Human carcinoembryonic antigen (CEA), an Ig superfamily cell surface glycoprotein used widely as a clinical tumor marker, undergoes homophilic interactions that mediate intercellular adhesion. Recent evidence supports the view that deregulated overexpression of CEA has an instrumental role in tumorigenesis through the inhibition of cell differentiation and the disruption of tissue architecture. The CEA-mediated block of the myogenic differentiation of rat L6 myoblasts depends on homophilic binding of its external domains. We show here that L6 transfectant cells expressing CEA can "trans-block" the myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA (ΔNCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, ΔNCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to involve three subdomains of the amino-terminal domain, shown previously to be critical for its intercellular adhesion function. Monovalent Fab fragments of monoclonal antibodies binding to the region bridging subdomains 1 and 2 could both inhibit intercellular adhesion and release the myogenic differentiation block. Amino acid substitutions Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to completely ablate the differentiation blocking activity of CEA but had no effect on intercellular adhesion activity. A cyclized peptide representing this subdomain was the most effective at releasing the differentiation block. cell adhesion molecule carcinoembryonic antigen differentiation medium growth medium monoclonal antibody fluorescence-activated cell sorter phosphate-buffered saline plus 2% fetal bovine serum Tissue architecture is established and maintained to a large extent by specific affinities of cell surface glycoproteins for molecules in the extracellular matrix or on the surface of adjacent cells. The latter are known collectively as cell adhesion molecules (CAMs)1 (1Ruoslahti E. Obrink B. Exp. Cell Res. 1996; 227: 1-11Crossref PubMed Scopus (175) Google Scholar, 2Hynes R.O. Lander A.D. Cell. 1992; 68: 303-322Abstract Full Text PDF PubMed Scopus (762) Google Scholar, 3Edelman G.M. Crossin K.L. Annu. Rev. Biochem. 1991; 60: 155-190Crossref PubMed Scopus (653) Google Scholar). CAMs function not only to fix cells in specific locations within tissues and regulate their movement but also to translate biochemical information from the extracellular environment through the activation of intracellular signaling pathways leading to specific functional cell responses (4Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3821) Google Scholar,5Obrink B. Curr. Opin. Cell Biol. 1997; 9: 616-626Crossref PubMed Scopus (233) Google Scholar). CAMs are grouped into several different molecular families; the majority identified to date belong to the Ig superfamily (1Ruoslahti E. Obrink B. Exp. Cell Res. 1996; 227: 1-11Crossref PubMed Scopus (175) Google Scholar, 6Huang Z. Li S. Korngold R. Biopolymers. 1997; 43: 367-382Crossref PubMed Scopus (33) Google Scholar, 7Williams A.F. Barclay A.N. Annu. Rev. Immunol. 1988; 6: 381-405Crossref PubMed Scopus (1786) Google Scholar, 8Amzel L.M. Poljak R.J. Annu. Rev. Biochem. 1979; 48: 961-997Crossref PubMed Google Scholar). Although the members of this family are functionally diverse, most are cell surface molecules involved in the recognition of other soluble or cell-associated molecules. All members of the Ig superfamily share conserved amino acid residues. These residues are limited to positions within the core of the Ig fold that are important for its structure. In contrast, the functional regions of the various members are often highly diverse. Ig superfamily members function in many cases as a result of homophilic binding between their external domains or heterophilic binding interactions with other molecules (1Ruoslahti E. Obrink B. Exp. Cell Res. 1996; 227: 1-11Crossref PubMed Scopus (175) Google Scholar, 9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar, 10Oikawa S. Inuzuka C. Kuroki M. Arakawa F. Matsuoka Y. Kosaki G. Nakazato H. J. Biol. Chem. 1991; 266: 7995-8001Abstract Full Text PDF PubMed Google Scholar). Homophilic interactions can be either antiparallel or parallel. Antiparallel interactions between molecules on apposed cell surfaces are required for intercellular binding (9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar, 11Ranheim T.S. Edelman G.M. Cunningham B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4071-4075Crossref PubMed Scopus (109) Google Scholar, 12Knudson Jr., A.G. Adv. Cancer Res. 1995; 67: 1-23Crossref PubMed Google Scholar). Parallel interactions between adjacent molecules on the same cell surface can facilitate this process by concentrating the binding molecules into synergistic arrays as described by the "Velcro" (13Stanners C.P. Fuks A. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam1998: 57-71Google Scholar) or "zipper" models (14Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar), in which the concerted action of multiple relatively weak interactions between individual pairs of molecules can lead to a strong overall bonding. Both types of interactions can also initiate signaling events (5Obrink B. Curr. Opin. Cell Biol. 1997; 9: 616-626Crossref PubMed Scopus (233) Google Scholar, 14Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar, 15Draber P. Skubitz K.M. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam1998: 121-140Google Scholar). The clustering resulting from their combination might be expected to amplify these signals and lead to the triggering of threshold-activated signaling pathways (5Obrink B. Curr. Opin. Cell Biol. 1997; 9: 616-626Crossref PubMed Scopus (233) Google Scholar, 14Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar, 15Draber P. Skubitz K.M. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam1998: 121-140Google Scholar). The human carcinoembryonic antigen (CEA) family is of particular interest in terms of intermolecular binding because of the multiplicity and diversity of interactions between multiple closely related family members (13Stanners C.P. Fuks A. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam1998: 57-71Google Scholar). We have investigated previously the structural requirements for CEA-mediated intercellular adhesion (9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar,16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar). Intercellular adhesion seems to depend mainly on antiparallel CEA-CEA interactions as indicated by studies with hybrid constructs between CEA and neural cell adhesion molecule (9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar). CEA consists of a V-like Ig amino-terminal domain and three pairs of I-like Ig domains (denoted AXBX) that are terminated by a hydrophobic domain, which is processed to allow the addition of a glycophosphatidylinositol membrane anchor (17Beauchemin N. Benchimol S. Cournoyer D. Fuks A. Stanners C.P. Mol. Cell. Biol. 1987; 7: 3221-3230Crossref PubMed Scopus (168) Google Scholar, 18Hefta S.A. Hefta L.J. Lee T.D. Paxton R.J. Shively J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4648-4652Crossref PubMed Scopus (145) Google Scholar, 19Takami N. Misumi Y. Kuroki M. Matsuoka Y. Ikehara Y. J. Biol. Chem. 1988; 263: 12716-12720Abstract Full Text PDF PubMed Google Scholar). Intercellular adhesion was shown to be mediated by double reciprocal bonds between the N and A3B3 domains of antiparallel CEA molecules on apposed cell surfaces (9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar). Subdomains consisting of 5–6 amino acids that are required for intercellular adhesion, presumably as points of initial binding, were identified by mutational analysis of the amino-terminal domain (16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar). Two cellular functions, intercellular adhesion (for review, see Stanners and Fuks (13Stanners C.P. Fuks A. Stanners C.P. Cell Adhesion and Communication Mediated by the CEA Family: Basic and Clinical Perspectives. Harwood Academic Publishers, Amsterdam1998: 57-71Google Scholar)) and the inhibition of cell differentiation (20Eidelman F.J. Fuks A. DeMarte L. Taheri M. Stanners C.P. J. Cell Biol. 1993; 123: 467-475Crossref PubMed Scopus (76) Google Scholar) have been shown to be dependent on homophilic CEA family member interactions. In particular, CEA was shown to block the myogenic differentiation of rat L6 myoblasts (20Eidelman F.J. Fuks A. DeMarte L. Taheri M. Stanners C.P. J. Cell Biol. 1993; 123: 467-475Crossref PubMed Scopus (76) Google Scholar) and the neurogenic differentiation of mouse P19 embryonal carcinoma cells. 2B. Malette and C. P. Stanners, submitted for publication. The effect of CEA on differentiation would be expected to promote tumorigenic behavior and, in fact, was found to markedly increase the tumorigenicity of L6 myoblasts (21Screaton R.A. Penn L.Z. Stanners C.P. J. Cell Biol. 1997; 137: 939-952Crossref PubMed Scopus (77) Google Scholar) and Caco-2 cells (22Ilantzis I. De Marte L. Screaton R.A. Stanners C.P. Neoplasia. 2002; 4: 151-163Crossref PubMed Google Scholar). Deregulated overexpression of CEA and the closely related CEA family member, CEACAM6 (formerly known as NCA), at levels closely approximating those found in many colorectal carcinomas, has been shown recently to block cellular polarization, disrupt tissue architecture, and block the differentiation of human colonocyte cell lines (22Ilantzis I. De Marte L. Screaton R.A. Stanners C.P. Neoplasia. 2002; 4: 151-163Crossref PubMed Google Scholar). Consistent with these results, the cell surface level of CEA, which was determined by FACS analysis of highly purified epithelial colonocytes from colorectal carcinomas and normal colonic tissue, was found to be elevated in the tumor cells at levels that were inversely correlated with the degree of differentiation (23Ilantzis C. Jothy S. Alpert L.C. Draber P. Stanners C.P. Lab. Invest. 1997; 76: 703-716PubMed Google Scholar). In this study, we have focused on the effects of CEA on the differentiation of L6 myoblasts because of the relative ease of experimentation. Although the expression of CEA in this system is ectopic, our experience to date indicates that the results obtained with this model system are closely mimicked by more biologically relevant systems such as human colonocytes (22Ilantzis I. De Marte L. Screaton R.A. Stanners C.P. Neoplasia. 2002; 4: 151-163Crossref PubMed Google Scholar, 24Ordonez C. Screaton R.A. Ilantzis C. Stanners C.P. Cancer Res. 2000; 60: 3419-3424PubMed Google Scholar). Because CEA and/or CEACAM6 are overexpressed in more than 50% of human cancers (25Chevinsky A.H. Semin. Surg. Oncol. 1991; 7: 162-166Crossref PubMed Scopus (82) Google Scholar), it would seem experimentally and medically important to devise a means of releasing the CEA-mediated differentiation block and to determine whether interference with this function can be made selective without affecting the intercellular adhesion function. We show here that although the structural requirements for the intercellular adhesion and the differentiation block functions overlap, they can be separated effectively in the case of rat L6 myogenic differentiation. Using this information, small cyclized peptides and monovalent Fab fragments of monoclonal antibodies have been designed that are capable of releasing the myogenic differentiation block. Cyclized and linearly blocked oligopeptides were obtained (>95% purity) from Multiple Peptide Systems (San Diego, CA). Linearly blocked peptides were rendered more stable by acetylation of the amino terminus and amination of the carboxyl terminus. Cyclic peptides contained two cysteine residues joined by sulfide bonds at their termini. The peptides used were blocked linear NAc-LFGYSWYKGE-NH2, NAc-VDGNRQIIGY-NH2, NAc-RIIQNDTGFY-NH2, and NAc-FNVAEGKEV-NH2; and cyclized H-CGYSWYKC-OH, H-CGNRQIIC-OH, H-CQNDTGC-OH, and H-YCTDEKQCY-OH, representing subdomains 1, 2, and 3 and control peptides, respectively. Sequences actually present in the amino-terminal domain of CEA for the cyclized peptides are underlined. Wild type cDNA coding for CEA (17Beauchemin N. Benchimol S. Cournoyer D. Fuks A. Stanners C.P. Mol. Cell. Biol. 1987; 7: 3221-3230Crossref PubMed Scopus (168) Google Scholar) was used as a template for all PCR-generated constructs. The recombinant PCR technique (26Higuchi R. Erlich H.A. PCR Technology: Principles and Applications for DNA Amplification. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 177-183Google Scholar) was used to generate site-directed mutants as described previously (16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar). Rat L6 myoblasts were grown as monolayer cultures at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (growth medium (GM); Invitrogen). Cell cultures were subcultured while subconfluent to avoid the selection of non-fusing variants. LR-73 cells (27Pollard J.W. Stanners C.P. J. Cell. Physiol. 1979; 98: 571-585Crossref PubMed Scopus (75) Google Scholar) derived from the Chinese hamster ovary line were grown in monolayer culture in α minimum essential medium (28Stanners C.P. Eliceiri G.L. Green H. Nat. New Biol. 1971; 230: 52-54Crossref PubMed Scopus (399) Google Scholar) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2. L6 myoblast cells were seeded at 2 × 105 cells/l00-mm plastic tissue culture Petri dish and cotransfected 24 h later by the calcium phosphate-mediated co-precipitation method as described previously (29Benchimol S. Fuks A. Jothy S. Beauchemin N. Shirota K. Stanners C.P. Cell. 1989; 57: 327-334Abstract Full Text PDF PubMed Scopus (839) Google Scholar), with 5 μg of p91023B expression vector containing CEA (wild type or mutant) cDNA, 10 μg of LR-73 carrier genomic DNA, and 0.5 μg pSV2neo plasmid/dish. Stable pooled transfectant colonies were isolated by selection with 400 μg/ml Geneticin (G418, Invitrogen). Immunofluorescent labeling with anti-CEA monoclonal antibody J22 (30Zhou H. Stanners C.P. Fuks A. Cancer Res. 1993; 53: 3817-3822PubMed Google Scholar) and FACS sorting were carried out to select for populations of transfectants stably expressing desired levels of mutant or wild type CEA on the cell surface. At least two independent pooled populations of transfectant clones were isolated for each transfected cDNA. All transfectant populations were maintained in GM containing 400 μg/ml G418. G418 was removed from the medium 24 h before each functional assay was performed. Cells were removed from culture vessels by light trypsinization (a treatment that does not affect cell surface levels of CEA) and resuspended in ice-cold phosphate-buffered saline plus 2% fetal bovine serum (PBSF). 2.5 × 105 cells were incubated with polyclonal rabbit or monoclonal anti-CEA antibodies (J22) at a dilution of 1:100 in PBSF for 35 min on ice. Cells were washed with 2.5 ml of PBSF, centrifuged, and resuspended in 0.5 ml of PBSF containing fluorescein isothiocyanate-conjugated goat anti-rabbit or anti-mouse antibody at a dilution of 1:100. After 30 min of incubation on ice, cells were washed, centrifuged, resuspended in 0.75 ml of PBSF, and analyzed using a BD Biosciences FACScan® instrument. LR-73 cells were seeded at 1 × 106/80-cm2 culture flask (Nalge Nunc Inc., Naperville, IL) in LR-73 growth medium. After 2 days in monolayer culture, the cultures were rendered as single cell suspensions by 3 min of incubation at 37 °C with 0.12% Bacto trypsin in phosphate-buffered saline lacking Mg2+ and Ca2+and containing 15 mm sodium citrate. The cells were incubated at 106 cells/ml in α minimum essential medium containing 0.8% fetal bovine serum and 10 μg/ml DNase I at 37 °C with stirring at 100 rpm (29Benchimol S. Fuks A. Jothy S. Beauchemin N. Shirota K. Stanners C.P. Cell. 1989; 57: 327-334Abstract Full Text PDF PubMed Scopus (839) Google Scholar). The percentage of cells remaining as single cells, which declines over time because of formation of aggregates, was determined as a function of time by visual counting using a hemocytometer. To initiate fusion and differentiation, L6 cultures were seeded at 104cells/cm2 at day 0 in 60- or 35-mm tissue culture Petri dishes or 7 × 103 cells/cm2 in multiwell plastic chamber slides (Nalge Nunc Inc.) and grown in GM. The medium was replaced after 3 days with Dulbecco's modified Eagle's medium plus 2% horse serum (differentiation medium (DM)), and the cells were cultured for an additional 5–7 days. To co-culture L6 (CEA) and L6 (ΔNCEA) cell transfectants, cells were seeded at 3 × 105 cells of each type per 35-mm plastic tissue culture Petri dish in GM. 24 h later the medium was replaced with DM. For fusion index determinations, cells were fixed with 2.5% glutaraldehyde and stained with hematoxylin. The fusion index was calculated as the percentage of total nuclei contained in fused myotubes having more than three nuclei/myotube, as described previously (20Eidelman F.J. Fuks A. DeMarte L. Taheri M. Stanners C.P. J. Cell Biol. 1993; 123: 467-475Crossref PubMed Scopus (76) Google Scholar). Fusion determinations were repeated three times (independent experiments) for each of two independently obtained transfectant populations for each mutant. The values reported in Figs. Figure 5, Figure 6, Figure 7 represent the averages of these determinations. As a biochemical measure of myogenic differentiation, cells were fixed in methanol:acetone (3:7) at −20 °C and processed for immunofluorescent staining with anti-myosin mAb (31De Giovanni C. Lollini P.L. Dolccetti R. Landuzzi L. Nicoletti G. D'Andrea E. Scotland K. Nanni P. Br. J. Cancer. 1993; 67: 674-679Crossref PubMed Scopus (20) Google Scholar). To study the effect of cyclized or linearly blocked peptides on the CEA-mediated L6 differentiation block, cells were seeded at 7 × 103 cells/cm2 in 8-well chamber slides (Nalge Nunc Inc.) in GM on day 0. After 3 days of incubation, the medium was replaced with DM-containing peptide at the indicated concentrations. Rabbit polyclonal anti-CEA antibody and mouse monoclonal anti-CEA antibodies (A20, B18, and D14) (30Zhou H. Stanners C.P. Fuks A. Cancer Res. 1993; 53: 3817-3822PubMed Google Scholar) were purified with the Bio-Rad Affi-Gel protein A MAPS II kit. Antibodies were added to differentiation medium to a final concentration of 1 mg/ml. Fab fragments of monoclonal anti-CEA antibodies were prepared as described previously (30Zhou H. Stanners C.P. Fuks A. Cancer Res. 1993; 53: 3817-3822PubMed Google Scholar). Fab fragments were added to differentiation medium to a final concentration of 100 μg/ml. The values for fusion indices shown in Fig. 10 represent the averages of three independent determinations. To explore the nature of the intermolecular interactions involved in the CEA-mediated block of the myogenic differentiation of L6 myoblasts (20Eidelman F.J. Fuks A. DeMarte L. Taheri M. Stanners C.P. J. Cell Biol. 1993; 123: 467-475Crossref PubMed Scopus (76) Google Scholar), experiments were carried out to distinguish the requirements for parallel versusantiparallel intermolecular binding. For antiparallel interactions, non-differentiating stable L6 (CEA) transfectants were co-cultured with differentiation competent L6 (ΔNCEA) transfectants. ΔNCEA is a CEA cDNA deletion mutant lacking two-thirds of the amino-terminal domain (from amino acid 32 to 106) and is completely defective in mediating both intercellular adhesion (9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar) and the differentiation block of L6 myoblasts (20Eidelman F.J. Fuks A. DeMarte L. Taheri M. Stanners C.P. J. Cell Biol. 1993; 123: 467-475Crossref PubMed Scopus (76) Google Scholar). CEA-expressing cells can bind to ΔNCEA-expressing cells, consistent with the model that the amino-terminal domain (intact in CEA) on a CEA-expressing cell can bind to the A3B3 domain (intact in ΔNCEA) on a juxtaposed ΔNCEA-expressing cell by an antiparallel mechanism (9Zhou H. Fuks A. Alcaraz G. Bolling T.J. Stanners C.P. J. Cell Biol. 1993; 122: 951-960Crossref PubMed Scopus (126) Google Scholar). If such antiparallel interactions are sufficient for the differentiation blocking function of CEA, the CEA-expressing myoblasts should be able to "trans-inhibit" the differentiation of the ΔNCEA-expressing myoblasts. Co-culturing CEA-expressing L6 cells with an equal number of ΔNCEA-expressing L6 cells inhibited the overall differentiation (assessed by fusion into multinucleated myotubes) to a significantly lower level than co-culturing the same CEA-expressing cells with an equal number of non-CEA-expressing parental L6 cells for which trans-binding mediated by CEA would be impossible (Fig.1). The latter co-culture controls for the dilution of differentiating cells with non-differentiating L6 (CEA) cells. This experiment therefore supports the contention that antiparallel CEA-CEA interactions between cells are sufficient for the CEA-imposed myogenic differentiation block. To test for the role of parallel CEA-CEA interactions on the same cell surface, differentiation-competent L6 (ΔNCEA) transfectants were treated with cross-linking polyclonal and monoclonal anti-CEA antibodies. Antibodies for which the binding epitopes are still intact in the ΔNCEA molecule, rabbit polyclonal and D14 (binding epitope at the B2-A3 junction (30Zhou H. Stanners C.P. Fuks A. Cancer Res. 1993; 53: 3817-3822PubMed Google Scholar)), converted ΔNCEA to a differentiation blocking molecule, whereas control antibodies directed to binding epitopes that are missing in ΔNCEA, A20 and B18, two N domain-specific mAbs (binding epitopes at residues 35–42 in the amino-terminal domain (16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar)) were without effect (Fig.2). To further control for nonspecific effects, one of the effective antibodies, D14, was shown to have no effect on the differentiation of the parental L6 cells (Fig.2). These experiments support the hypothesis that both antiparallel and parallel binding between CEA molecules are involved in the CEA-mediated myogenic differentiation block. Deletions and substitutions in three subdomains of the amino-terminal domain of CEA (Fig.3) were produced by site-directed mutagenesis as described previously (16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar). The rationale for choosing these particular subdomains can be summarized as follows. The requirement for amino-terminal domain amino acids 32–106, deleted in mutant ΔNCEA, for the myogenic differentiation block was demonstrated previously (20Eidelman F.J. Fuks A. DeMarte L. Taheri M. Stanners C.P. J. Cell Biol. 1993; 123: 467-475Crossref PubMed Scopus (76) Google Scholar). Within this deletion, subdomains 1 and 2 were implicated by the fact that mAb A20 can release the CEA-imposed myogenic differentiation block (see "Effects of Monovalent Monoclonal Anti-CEA Antibody Fragments on CEA-mediated Differentiation Block") and has a binding epitope that bridges them. This epitope includes the carboxyl-terminal amino acid of subdomain 1 and the amino-terminal amino acid of subdomain 2 (16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar). Also, these subdomains (1 and 2) and subdomain 3 were all shown to be important in CEA-mediated intercellular adhesion in LR-73 cells; all were demonstrated to be adjacent and exposed in a three-dimensional structural model based on the known structures of CD2 (16Taheri M. Saragovi U. Fuks A. Makkerh J. Mort J. Stanners C.P. J. Biol. Chem. 2000; 275: 26935-26943Abstract Full Text Full Text PDF PubMed Google Scholar) and CD4. 3H. U. Saragovi, unpublished data. Pooled stable transfectant clones of L6 cells expressing comparable cell surface levels, as assessed by FACS analysis (Fig.4), were isolated for each of the deletion and substitution mutants.Figure 4FACS distributions relating the number of cells with the level of cell surface expression of wild type and mutantCEA in L6 myoblast transfectant and control (Neo) populations. All cells were labeled with the monoclonal anti-CEA antibody, J22. Immunoreactivity was detected with goat anti-mouse fluorescein isothiocyanate-conjugated antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Concerning subdomain 1, G30YSWYK, substitutions at the carboxyl terminus, Y34A, the more conservative Y34F, and K35A had a profound effect on the myogenic differentiation blocking activity of CEA, whereas mutation Y31A at the amino terminus had no effect (Fig. 5). As expected, deletion of the entire subdomain 1 (ΔGK) also affected this function of CEA but, curiously, was not as effective as substitutions Y34A and K35A. Similarly, the deletion of subdomain 2, N42RQII, had less effect on CEA function than some of the substitutions within this domain, notably the double mutation Q44R,I46V, for which the degree of differentiation of 100%, a reproducible effect, actually exceeded that of parental L6 cells (Fig. 6). A single mutation at the amino terminus of the subdomain, N42D, partially removed the differentiation blocking activity of CEA. The mouse analogs of subdomains 1 and 2, in a crystallized soluble mouse construct of CEACAM1 consisting of the amino-terminal domain linked to one internal domain, have been shown by Tan et al.(32Tan K. Zelus B.D. Meijers R. Liu J. Bergelson J.M. Duke N. Zhang R. Joachimiak A. Holmes K.V. Wang J. EMBO J. 2002; 21: 2076-2086Crossref PubMed Scopus
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