Expression of CEA-related genes in the first trimester human placenta

Eight cDNA clones, closely related to the carcino-embryonic antigen gene family, have been isolated from a cDNA library representing genes expressed in the first trimester human placenta. Sequence analysis of one clone shows it to be a pregnancy-specific beta 1-glycoprotein (PS beta G) closely related to three other PS beta G cDNA recently characterised from a term placenta library. The protein encoded by the cDNA is predicted to be less high glycosylated than those reported previously and differs markedly in the C-terminal sequence. The 3' untranslated region of the cDNA is very similar to the equivalent region of beta 1-glycoprotein PS beta G E except that it contains the 12bp repeat sequence found flanking the Alu sequence in CEa and an additional 67bp of sequence that appears to be derived from CEA.     

Characterization of cDNAs of the human pregnancy-specific beta 1-glycoprotein family, a new subfamily of the immunoglobulin gene superfamily

Three highly homologous cDNAs encoding human pregnancy-specific beta 1-glycoprotein (SP1) were isolated from a human placental cDNA library. These cDNAs share greater than 90% nucleotide homology in their coding sequences, and greater than 79% of the encoded amino acids are homologous. Proteins encoded by these cDNAs are very similar to members of the carcinoembryonic antigen family and contain repeating domains, conserved disulfide bridges, and beta-sheet structure typical of the immunoglobulin gene superfamily. However, the high degree of sequence homology and relatively lesser degree of glycosylation among the SP1 proteins suggest that they exist as a unique family instead of being members of the CEA family. Both soluble and potentially membrane-bound forms of SP1 proteins were present in the placenta. Northern blot analysis using specific probes confirmed the expression of multiple mRNA species in human term placenta.     

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.     

Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV.

The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1-transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59.     

Cloning and high level expression of a chimeric antibody with specificity for human carcinoembryonic antigen

A mouse/human chimeric antibody has been constructed by using variable light and variable heavy regions from a murine hybridoma specific for human carcinoembryonic antigen (CEA) (CEM231.6.7). These V regions were combined with kappa and gamma-1 constant region genes cloned from human lymphocytes. The chimeric constructs were sequentially electroporated into murine non-Ig-producing myeloma (P3.653) and hybridoma (SP2/0) cell. Significant differences were seen in expression levels between the two cell types. High levels of expression (24 to 32 micrograms/ml/10(6) cells) were seen with several of the anti-CEA SP2/0 transfectomas but not with the P3.653 cells. The SP2/0 transfectoma lines were adapted to serum-free, chemically defined media and grown in large scale fermentation cultures where they continued to secrete high levels of antibody. The chimeric antibodies remain reactive against human CEA with affinity constants comparable to that of the parental hybridoma antibody. High level expression will make practical the production of chimeric antibodies for in vivo therapeutic and diagnostic purposes.     

Analysis of the size of the carcinoembryonic antigen (CEA) gene family: isolation and sequencing of N-terminal domain exons

Five members of the human CEA gene family [human pregnancy-specific beta 1-glycoprotein (PS beta G); hsCGM1, 2, 3 and 4] have been isolated and identified through sequencing the exons containing their N-terminal domains. Sequence comparisons with published data for CEA and related molecules reveal the existence of highly-conserved gene subgroups within the CEA family. Together with published data eleven CEA family members have so far been determined. Apart from the highly conserved coding sequences, these genes also show strong sequence conservation in their introns, indicating a duplication of whole gene units during the evolution of the CEA gene family.     

Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line.

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiate in vitro by replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60-90%) of CEA release.     

Human pregnancy-specific beta 1-glycoproteins are coded within chromosome 19.

We have isolated and characterized cDNAs that code for apoproteins having amino acid sequences highly similar to pregnancy-specific beta 1-glycoproteins (PS beta G). cDNAs coding for PS beta Gs, as well as the cDNA clone reported here, are members of the carcinoembryonic antigen (CEA) gene family. The previous localization of CEA-related genes to human chromosome 19, and the high level of DNA sequence conservation in the CEA family, suggested that the PS beta G genes are also located on this chromosome. We demonstrate here that chromosome 19 is indeed the site of PS beta G sequences. Our finding is in contrast to the recently reported indication that pregnancy-specific glycoproteins are encoded in chromosomes X and 6.     

Molecular cloning of nonspecific cross-reacting antigens in human granulocytes

To clarify the molecular structures of nonspecific cross-reacting antigens (NCAs), a family of glycoproteins antigenically related to carcinoembryonic antigen (CEA), in human granulocytes, we have screened a cDNA library of human leukocytes using a cDNA probe for the N-terminal domain (domain-N) of NCA-50, an NCA species in tumor cells. In 95 positive clones randomly selected, we identified six NCA or NCA-related cDNA clones including NCA-50, biliary glycoprotein protein a, and W272 (CGM6) which have previously been reported, and three new clones, W236, W264, and W282, encoding three novel NCA species. W236 and W264 consist of a domain-N, a putative transmembrane domain, and a possible cytoplasmic domain. The domain-N of W264 is 89% similar to that of NCA-50 at amino acid level, whereas the domain-N of W236 is only 49 and 43% similar to those of NCA-50 and pregnancy-specific beta 1-glycoprotein-11 (PSG11), respectively, indicating that W236 belongs to a new subfamily within the CEA family. The third clone W282 encodes a protein consisting of a domain-N virtually identical to that of W264 and a short hydrophilic C-terminal domain. W264 and W282 seem to be derived from a single gene by alternative splicing of RNA. These three new species are particularly unique in respect that they lack the repetitive immunoglobulin-related domains that have been universally found in the human CEA gene family members. The biochemical and immunochemical properties of the recombinant proteins of these cDNA clones, however, did not coincide with those of six NCA species previously identified in granulocytes at protein level, suggesting that, in granulocytes, there exist at least 12 NCA or NCA-related species whose expression is under complex control.     

Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule

Carcinoembryonic antigen (CEA) is a member of a family of cell surface glycoproteins that are produced in excess in essentially all human colon carcinomas and in a high proportion of carcinomas at many other sites. The function of this widely used tumor marker and its relevance to malignant transformation is therefore of considerable interest. We demonstrate here that CEA mediates Ca2+-independent, homotypic aggregation of cultured human colon adenocarcinoma cells (LS-180) and rodent cells transfected with functional CEA cDNA. Furthermore, CEA can effect the homotypic sorting of cells in heterogeneous populations of aggregating cells. CEA can thus be considered a new addition to the family of intercellular adhesion molecules. We also show that, whereas CEA is localized mainly to epithelial cell membranes facing the lumen in normal adult intestine, it is found on adjacent cell membranes in both embryonic intestine and colonic tumors. A model for the role of CEA in the tissue architecture of adult, embryonic, and aberrant tumor intestinal epithelium is presented.     

Molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.     

Human pregnancy-specific beta 1 glycoprotein is encoded by multiple genes localized on two chromosomes.

A human genomic library was screened with a mixture of two cDNA probes, with one covering the 5' coding sequence and the other containing the 3'-end portion of human pregnancy-specific beta 1 glycoprotein (SP1). Seventeen clones were identified, all of which carried insert fragments capable of hybridizing with the cDNA probe. Insert size of these clones varied from 15.0 to 19.8 kb. Partial restriction maps were constructed, which demonstrated the presence of at least seven groups of unique SP1 genomic clones and suggested the possibility of multiple genes coding for SP1. The multigene nature of SP1 was confirmed by hybridization of the SP1 cDNA probe to multiple bands on Southern blots of human genomic DNA. Further analysis with chromosomal DNA dot blot demonstrated the presence of homologous sequences on the X chromosome and autosomal chromosome 6. Thus, human SP1 is apparently coded for by more than one gene residing on the X and 6 chromosomes.     

Isolation of a cDNA for human muscle 6-phosphofructokinase

A cDNA for human muscle 6-phosphofructokinase (EC.2.7.1.11) has been isolated from a human fibroblast cDNA library made using the Okayama-Berg procedure. The cDNA isolated as a Bam H1 fragment of the pcD recombinant, pO4, is approximately 2000 bp in length. It represents approximately 1350 bp of the C-terminus coding sequence of the enzyme, approximately 500 bp of the 3'-untranslated region and approximately 150 bp of the vector sequences. The identity of the pO4 cDNA was established by the observation of a high degree of homology (approximately 95%) between the deduced amino acid sequence with the published protein sequence of rabbit muscle 6-phosphofructokinase, and the assignment of the sequence to human chromosome 1 (the known location of PFKM) by using somatic cell hybrids. Based on immunochemical evidence, we had previously predicted not only a remarkable structural conservation of the vertebrate muscle PFK, but also partial structural identity among all three vertebrate PFK isozymes. The pO4 cDNA is, therefore, expected to permit isolation of cDNAs for muscle and non-muscle PFKs from a wide variety of vertebrate species.