The Adhesion and Differentiation-inhibitory Activities of the Immunoglobulin Superfamily Member, Carcinoembryonic Antigen, Can Be Independently Blocked

Abstract

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 (⌬NCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, ⌬NCEA 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.

Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc) for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins

“…14 Fluorescence-activated cell sorting (FACS) expression profiles were then obtained by treatment of cell suspensions with 16 mg/ml humanized MN-14 or 100 mg/ml anti-CEA mAb J22 or no primary antibody (control) followed by fluorescein (FITC)-conjugated secondary antibody, and analyzed using a Becton Dickinson FACScan instrument, as described previously. 14 …”

Two Variable Regions in Carcinoembryonic Antigen-related Cell Adhesion Molecule1 N-terminal Domains Located in or Next to Monoclonal Antibody and Adhesion Epitopes Show Evidence of Recombination in Rat but Not in Human

“…3), two regions of sequence variability (V-regions) were detected. Moreover, these V-regions were located adjacent to or within the regions corresponding to binding sites identified previously for opa proteins (55,56) and MHV (57-59) and adhesive domains mediating intercellular adhesion (25,28,33) (Fig. 3).…”

“…Epitope B Is Located within the Major Adhesive Site in the CEACAM1 a N-domain-In previous reports (25,28,33,(55)(56)(57)(58)(59), the adhesive sites for CEA and the binding domains for MHV and opa proteins were localized to the C ␤-strand and CCЈ loop domains. Because one of the mAb epitopes was within this region, it was of interest to determine whether the two mAbs could block cell adhesion.…”

“…Because mAb 362.50 reacted with peptide arrays spanning an epitope domain shared by CEACAM1 a -4L and CEACAM1 b -4L, these conformational effects did not appear to emanate directly from differences in primary sequence. Adhesion blocking assays showed that the CЈ and G ␤-strands contained major and minor adhesive domains, respectively, the former of which shared sequence with a peptide that blocked human CEA adhesion (33). mAb binding assays with peptide arrays spanning the CЈ region from CEACAM1 a -4L and CEACAM1 b -4L showed that mAb 362.50 reacted equally well with both CЈ peptide arrays despite the differences in primary sequence, thus suggesting that factors in addition to primary sequence were responsible for the lack of reactivity with CEACAM1 b -4L.…”

The human carcinoembryonic antigen (CEA) GPI anchor mediates anoikis inhibition by inactivation of the intrinsic death pathway

“…It has been demonstrated that essentially all CEA family members, including GPIanchored CEA and CEACAM6, function as homotypic intercellular adhesion molecules in vitro (Benchimol et al, 1989;Oikawa et al, 1989;Stanners and Fuks, 1998). In contrast with the transmembrane member CEACAM1 (Eidelman et al, 1993;Rojas et al, 1996;Ilantzis et al, 2002;Taheri et al, 2003), CEA and CEACAM6 overexpression inhibits cell differentiation and anoikis in vitro (Eidelman et al, 1993;Ordonez et al, 2000;Screaton et al, 2000;Soeth et al, 2001;Taheri et al, 2003;Duxbury et al, 2004) and in vivo (Chan et al, unpublished results). The structural requirements for the inhibitory effects of CEA on differentiation were demonstrated to depend critically on the CEA-specific GPI anchor attached to self-binding extracellular domains Taheri et al, 2003).…”