Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

Abstract

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor y chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.Carcinoembryonic antigen (CEA), a large cell membrane glycoprotein of molecular weight about 180,000 (40), is produced in a high proportion of human tumors arising at the most common sites including colon, breast, and lung (37). CEA is also produced during human embryogenesis, while a related but distinctly different series of antigens termed CEA-cross-reactive antigens can be produced by a variety of adult normal cells (17). The consistent presence of CEA in tumors has led to its wide use as a clinical assay for prognosis and management of colon carcinomas. The normal function, role in carcinogenesis, and molecular basis of production of CEA in tumors, however, are completely unknown. As a first step in the solution of these questions, we have initiated a study of the molecular genetics of CEA, beginning with the isolation of a family of CEA cDNA clones.We now report the molecular cloning, nucleotide sequence, and structural analysis of a number of functional full-length CEA cDNA clones. These clones reveal a basic structure of common 5' and 3' ends embracing a number (usually three) of strikingly similar relatively long repeating units. The repeats include amino acid sequences which show significant homology with several members of the rat immunoglobulin family and, in particular, with...

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

“…CEA belongs to the immunoglobulin supergene family [16][17][18] and is comprised of a glycophosphoinositol-linked Mr 180,000-220,000 glycoprotein of 668 amino-acid residues. CEA consists of five regions: an IgV-like N-terminal region of 108 residues, three highly similar Ig I-like repetitive regions (Repeats I, II, and III, each consisting of a pair of subdomains A1 and B1, A2 and B2 and A3 and B3, respectively) of 178 residues each, and a highly hydrophobic C-terminal region of 26 residues that is removed during processing to allow attachment of the GPI anchor.…”

“…CEA consists of five regions: an IgV-like N-terminal region of 108 residues, three highly similar Ig I-like repetitive regions (Repeats I, II, and III, each consisting of a pair of subdomains A1 and B1, A2 and B2 and A3 and B3, respectively) of 178 residues each, and a highly hydrophobic C-terminal region of 26 residues that is removed during processing to allow attachment of the GPI anchor. 17,19,20 In the plan to make the Fc fusion protein, we considered that the size of product would be unwieldy if we used intact CEA to make a molecule of the form CEA 2 -Fc (ca. 450 kDa).…”

Signal sequence deletion and fusion to tetanus toxoid epitope augment antitumor immune responses to a human carcinoembryonic antigen (CEA) plasmid DNA vaccine in a murine test system

“…The plasmid p91023(B) was a kind gift from Nicole Beauchemin and Clifford Stanners, McGill University, Montreal, Canada. 26 It is a 7385 kb plasmid derived from pBR322. It encodes a full-length clone of human CEA (CEA) (accession number M17303), driven by the adenovirus major late promoter and with an SV40 poly A tail.…”

Intra- and interspecies analyses of the carcinoembryonic antigen (CEA) gene family reveal independent evolution in primates and rodents

“…Southern analyses indicated the existence of 9-11 genes in humans (Thompson et al 1987). Sequencing ofgenomic and cDNA clones has revealed the complete primary protein structure of CEA (Beauchemin et al 1987;Oikawa et al 1987c;Zimmermann et al 1987), NCA (Oikawa et al 1987b;Thompson et al 1987;Neumaier et al 1988;Tawaragi et al 1988), and a pregnancy-specific /31 glycoprotein (PS/~G) (Watanabe and Chou 1988a). The highly conserved domains shared by each are a 34-amino-acid leader peptide, a 108-110-amino-acid N-terminal domain, a 178-180-amino-acid repeating unit of which three copies are present in CEA, whereas only one and a half can be found in PSBG and one in NCA, and a 26-aminoacid carboxyl region (CEA) that is 2 amino acids shorter in NCA and degenerate in PSI3G.…”