Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family

To clarify the molecular structures of the nonspecific cross-reacting antigens (NCAs) produced by human granulocytes, we cloned cDNAs from libraries of normal white blood cells. A clone, NCA-W272, was found to code a protein similar to NCA of tumor cells. The protein consisted of a signal peptide (34 aa), domain-N (108 aa), -A1 (92 aa), -B1 (86 aa) and -M (29 aa). Similarity of the amino acid sequence of each domain to that of the tumor NCA was 72, 92, 76 and 79%, respectively. COS-1 cells transfected with an expression vector carrying the cDNA synthesized a 70 kDa glycoprotein, which was reactive with anti-NCA antibody and released from cell surface by phosphatidylinositol-specific phospholipase C. Thus the clone NCA-W272 was indicated to encode a new species of NCA distinct from the tumor NCA.     

Expression of an NCA cDNA in NIH/3T3 cells yields a 110K glycoprotein, which is anchored into the membrane via glycosyl-phosphatidylinositol

The NCA cDNA, which represents a gene belonging to the CEA family, was inserted into an SV40 early promoter-driven expression vector and used for transfection of mouse NIH/3T3 cells. A cell line, NIH/3T3/KNCA IG7, was selected which expressed a molecule with an apparent molecular weight of 110,000. The mode of membrane attachment of this NCA, which we already proposed to be anchored via glycosyl-phosphatidylinositol, was investigated by treatment of NIH/3T3/KNCA IG7 cells with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Two independent methods, flow cytometry and immunoprecipitation of [3H]-labelled surface glycoproteins, clearly demonstrated that the NCA molecule expressed by NIH/3T3/KNCA IG7 cells is indeed anchored into the membrane via glycosyl-phosphatidylinositol. Furthermore, these results support our previous biochemical data on NCA-50, by unequivocally showing that the NCA cDNA used for transfection encodes an NCA molecule related to NCA-50 and NCA-90.     

Specificity of intercellular adhesion mediated by various members of the immunoglobulin supergene family

The immunoglobulin supergene family members have been shown to be involved in cell-cell recognition and interaction during cell growth and differentiation. Neural cell adhesion molecule, myelin-associated glycoprotein, and carcinoembryonic antigen (CEA) are immunoglobulin supergene family members which can mediate cell adhesion. We show here that nonspecific cross-reacting antigen (NCA), a closely related CEA family member, is found on the surface of rodent cells transfected with functional NCA complementary DNA in different glycosylated forms, all of which can be deglycosylated to an Mr 35,000 core protein. Furthermore, NCA can mediate Ca2(+)-independent, homotypic aggregation of these NCA-producing transfectant cells. Since CEA has three internal repeated C2-set, immunoglobulin-like domains, whereas NCA has one, only one such domain is required for the intercellular adhesive function. We also demonstrate that NCA- and CEA-producing transfectants can form heterotypic aggregates, whereas mixtures of CEA or NCA transfectants and neural cell adhesion molecule or long form-myelin-associated glycoprotein transfectants sort themselves out into homotypic aggregates. The results suggest that subsets of the immunoglobulin superfamily, such as the CEA family, can be used in both homotypic and heterotypic cellular interactions, whereas less closely related members of the family can be used to separate different cell types by strictly homotypic interactions.     

Three different NCA species, CGM6/CD67, NCA-95, and NCA-90, are comprised in the major 90 to 100-kDa band of granulocyte NCA detectable upon SDS-polyacrylamide gel electrophoresis.

Human granulocytes express several species of nonspecific cross-reacting antigens (NCA), glycoproteins belonging to the carcinoembryonic antigen (CEA) family. Our previous studies have shown that at least two different NCA of 95 and 90 kDa are contained in the major NCA band of 90 to 100 kDa detectable upon gel electrophoresis of immunoprecipitates obtained from the cell surfaces of granulocytes with polyclonal anti-NCA. In the present study, the 90 to 100-kDa NCA band was found to include one more species of 100 kDa. This component was reactive with an anti-CD67 antibody as well as polyclonal anti-NCA and released from the cell surface with phosphatidylinositol-specific phospholipase C, indicating that the 100-kDa NCA species is CD67. Both antibodies revealed high binding activities with a recombinant protein of CGM6, which has been identified in a leukocyte cDNA library as an NCA gene and found to encode a glycosyl-phosphatidylinositol-anchored heterotypic cell adhesion molecule. Furthermore, the apparent molecular mass of the deglycosylated CD67 (38 kDa) corresponded with that of the CGM6 protein. These results suggest that CD67 is equivalent to the NCA species CGM6.     

A specific heterotypic cell adhesion activity between members of carcinoembryonic antigen family, W272 and NCA, is mediated by N-domains

The Ca(2+)-independent homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, W272 (CGM6), whose cDNA has recently been isolated from libraries of human peripheral leukocytes of apparently normal subjects (Arakawa, F., Kuroki, Mo., Misumi, Y., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1063-1071) and spleen of chronic myelogenous leukemia patients (Berling, B., Kolbinger, F., Grunert, F., Thompson, J. A., Brombacher, F., Buchegger, F., von Kleist, S., and Zimmermann, W. (1990) Cancer Res. 50, 6534-6539) has been examined. Chinese hamster ovary cells transfected with the cDNA for W272, CEA, nonspecific cross-reacting antigen (NCA), and various antigens containing chimeric N-domain have been used. The W272 producers did not show homotypic binding at all but bound only to the cells expressing NCA and a chimeric CEA whose N-domain is substituted by that of NCA, indicating the major contribution of N-domain of NCA in the specific binding. The importance of the N-terminal region of NCA N-domain for the W272-NCA binding has been shown by detailed analysis using COS-1 cells producing various NCA whose N-domain are chimera of that of NCA and CEA. The strict heterotypic nature of the W272-NCA adhesion strongly suggests that the cell adhesion activities exhibited by CEA family members are not the fortuitous activity but the specific one which have some important physiological roles.     

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.     

Characterization of a cDNA clone for the nonspecific cross-reacting antigen (NCA) and a comparison of NCA and carcinoembryonic antigen

NCA (nonspecific cross-reacting antigen), a glycoprotein found in normal lung and spleen, is immunologically related to carcinoembryonic antigen (CEA), which is found in over 95% of colon adenocarcinomas. From a human genomic library, we previously cloned part of an NCA gene and showed that the amino-terminal region has extensive sequence homology to CEA (Thompson, J. A., Pande, H., Paxton, R. J., Shively, L., Padma, A., Simmer, R. L., Todd, Ch. W., Riggs, A. D., and Shively, J.E. (1987) Proc. Natl. Acad. Sci. U. S.A. 84, 2965-2969). We now present the nucleotide sequence of a cDNA clone, containing the entire coding region of NCA (clone 9). The clone was obtained from a lambda gt 10 library made from the colon carcinoma cell line SW 403; the clone contains a 34-amino acid leader sequence, 310 amino acids for the mature protein, and 1.4 kilobases of 3'-untranslated region of the NCA gene. A comparison of the NCA sequence to the CEA sequence (Oikawa, S., Nakazato, H., and Kosaki, G. (1987) Biochem. Biophys. Res. Commun. 142, 511-518; Zimmerman, W., Ortlieb, B., Friedrich, R., and von Kleist, S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2690-2694) shows that both proteins contain doublets of an immunoglobulin-like domain, of which there are one copy in NCA and three copies in CEA, a 108-amino acid amino-terminal domain with no cysteine residues, and a carboxyl-terminal hydrophobic domain of sufficient length to anchor the glycoproteins in the cell membrane. Overall, the corresponding coding regions possess 85% sequence homology at the amino acid level and 90% homology at the nucleotide level. Forty nucleotides 3' of their stop codons, the CEA and NCA cDNAs become dissimilar. The 108-amino acid amino-terminal region together with part of the leader peptide sequence corresponds exactly to a single exon described in our previous work. The data presented here further demonstrate the likelihood that CEA recently evolved from NCA by gene duplication, including two duplications of the immunoglobulin-like domain doublet of NCA.     

Expression of nonspecific cross-reacting antigen species in myeloid leukemic patients and healthy subjects

The reactivity of two monoclonal antibodies recognizing NCA-95 and NCA-55 (MAb 47 and MAb 192, respectively) with a polyclonal anti-NCA serum in myeloid leukemic cells isolated by density gradient centrifugation was compared using an immunofluorescence test (IF). It was observed that the blood myeloid cells in 78.8% of the patients with different types of myelocytic leukemias and all granulocytes of 15 normal donors showed similar expression of the NCA species studied. The leukocytes of the remaining patients did not synthesize the NCA-95 species regardless of the maturation stage of the cells studied. In two patients, synthesis of this NCA form was limited to the fractions containing myelocytes and metamyelocytes. We have found that all anti-NCA antibodies studied recognized different antigenic epitopes in a myeloid cell series. A relationship between the patient's survival and the proportion of NCA-containing cells was also observed.     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of pregnancy-specific beta 1-glycoprotein (PS beta G)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific beta 1-glycoprotein (PS beta G). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8-83.5% to those of the PS beta G members so far reported, indicating PS34 is a new member of PS beta G and also of the carcinoembryonic antigen gene family.     

Molecular cloning of a gene for a member of carcinoembryonic antigen (CEA) gene family; signal peptide and N-terminal domain sequences of nonspecific crossreacting antigen (NCA)

A 2073-base pair DNA fragment containing a part of gene for a member of carcinoembryonic antigen (CEA) gene family, has been isolated from human DNA library after screening with 32P-labeled 53-mer oligodeoxyribonucleotide corresponding to N-terminal 18 amino acids of CEA gene family and cDNA encoding CEA (1,2). The fragment contains two exons; the one encodes the first 60% of signal peptide and the other the rest of it in addition to 107 amino acids which correspond to the N-terminal domain of CEA (1,2). Apparently, the second intron is inserted between the first and the second nucleotides of the codon for 108th amino acid. The presence of Ala instead of Val as the 21st amino acid of the N-terminal domain indicates that the exon encodes nonspecific crossreacting antigen (NCA).     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of Pregnancy-Specific β1-Glycoprotein (PSβG)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific β₁-glycoprotein (PSβG). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8–83.5% to those of the PSβG members so far reported, indicating PS34 is a new member of PSβG and also of the carcinoembryonic antigen gene family.     

Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA)

Immobilized carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM alpha-methyl D-mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D-mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.     

cDNA and gene analyses imply a novel structure for a rat carcinoembryonic antigen-related protein

The gene encoding the human tumor marker carcinoembryonic antigen (CEA) belongs to a gene family which can be subdivided into the CEA and the pregnancy-specific glycoprotein subgroups. The corresponding proteins are members of the immunoglobulin superfamily, characterized through the presence of one IgV-like domain and a varying number of IgC-like domains. Since the function of the CEA family is not well understood, we decided to establish an animal model in the rat to study its tissue-specific and developmental stage-dependent expression. To this end, we have screened an 18-day rat placenta cDNA library with a recently isolated fragment of a rat CEA-related gene. Two overlapping clones containing the complete coding region for a putative 709 amino acid protein (rnCGM1; Mr = 78,310) have been characterized. In contrast to all members of the human CEA family, this rat CEA-related protein consists of five IgV-like domains and only one IgC-like domain. This novel structure, which has been confirmed at the genomic level might have important functional implications. Due to the rapid evolutionary divergence of the rat and human CEA gene families it is not possible to assign rnCGM1 to its human counterpart. However, the predominant expression of the rnCGM1 gene in the placenta suggests that it could be analogous to one of the human pregnancy-specific glycoprotein genes.     

Effective Purification of Nonspecific Cross-Reacting Antigens with Phosphatidylinositol-Specific Phospholipase C

ABSTRACT

Two molecular species of nonspecific cross-reacting antigens, NCA-90 and NCA-50 with mol. wts. of 90,000 and 50,000, respectively, wereeffectively extracted with phosphatidylinositol-specific phospholipase C (PI-PLC) from human lung tissues, followed by extraction with perchloric acid, immunoaffinity chromatography with anti-NCA adsorbent, and gel filtration on a TSK G3000SW column. The yields of NCA were about 2 times more than those obtained by the usual method without PI-PLC. Addition of 0.05 unit of PI-PLC to 1 g of lung tissue and incubation at 37°C for 1 h with continuous shaking seem to be practically sufficient for NCA extraction. The immunochemical properties of the NCAs thus obtained were found to be identical to those of NCAs obtained by the ordinary method.     

Protein analysis of NCA-50 shows identity to NCA cDNA deduced sequences and indicates posttranslational modifications

The amino acid sequence, representing 59% of the protein moiety of NCA-50 (nonspecific crossreacting antigen), has been determined. These data confirm that NCA-50 is the product of the mRNA whose corresponding cDNAs were recently isolated from a human lung (HLC-1), as well as from a colon carcinoma cell line (SW 403) cDNA library. The four cysteine residues detected in the NCA-50 molecule form disulfide bonds. The glycosylation of 7 potential N-glycosylation sites which were analysed, showed pronounced differences. There is strong evidence that NCA-50 is bound to a phosphatidyl-inositol glycan, via an amide linkage to ethanolamine at amino acid position 287, which has replaced the last 24 amino acids.     

Homotypic and heterotypic Ca(++)-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface.

Homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, biliary glycoprotein a (BGPa), have been examined. CHO cells transfected with the cDNA for BGPa, CEA, non-specific cross-reacting antigen (NCA) and CGM6 have been used. The BGPa producers showed both homotypic and heterotypic adhesion to CEA and NCA producers. However, they hardly adhered to CGM6 producers. Calcium ion was not required for BGPa-mediated homotypic and heterotypic cell adhesion as well as for the adhesions of other members of CEA family. The results strongly suggested that BGPa may play some important roles through Ca(++)-independent cell adhesion activities.