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.     

The adhesion and differentiation-inhibitory activities of the immunoglobulin superfamily member,carcinoembryonic antigen,can be independently blocked

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

Evolution of a tumorigenic property conferred by glycophosphatidyl-inositol membrane anchors of carcinoembryonic antigen gene family members during the primate radiation

GPI membrane anchors of cell surface glycoproteins have been shown to confer functional properties that are different from their transmembrane (TM)-anchored counterparts. For the human carcinoembryonic antigen (CEA) family, a subfamily of the immunoglobulin superfamily, conversion of the mode of membrane linkage from TM to GPI confers radical changes in function: from tumor suppression or neutrality toward inhibition of differentiation and anoikis and distortion of tissue architecture, thereby contributing to tumorigenesis. We show here that GPI anchorage in the CEA family evolved twice independently in primates, very likely from more primitive TM anchors, by different packages of mutations. Both mutational packages, one package found in many primates, including humans, and a second, novel package found only in the Cebidae radiation of New World monkeys, give rise to efficiently processed GPI-linked proteins. Both types of GPI anchors mediate inhibition of cell differentiation. The estimated rate of nonsynonymous mutations (Ka) in the anchor-determining domain for conversion from TM to GPI anchorage in the CEA family that were fixed during evolution in these primates is 7 times higher than the average Ka in primates, indicating positive selection. These results suggest therefore that the functional changes mediated by CEA GPI anchors, including the inhibition of differentiation and anoikis, could be adaptive and advantageous.     

Loss of mammal-specific tectorial membrane component carcinoembryonic antigen cell adhesion molecule 16 (CEACAM16) leads to hearing impairment at low and high frequencies

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.     

Order and genomic distances among members of the carcinoembryonic antigen (CEA) gene family determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to establish the order of, and to estimate genomic distances among, members of the carcinoembryonic antigen (CEA) and pregnancy-specific glycoprotein (PSG) subgroups on chromosome 19. Fluorescence in situ hybridization to metaphase chromosomes localized the PSG subgroup telomeric to the CEA subgroup. Cosmid clones containing sequences for individual genes in the CEA and PSG subgroups were also hybridized to human sperm pronuclear and somatic interphase nuclear chromatin targets. The mapping results lead to the gene order cen-CGM7-CEA-NCA-CGM1-BGP-CGM9-CGM8-PSG-te l. The genomic distances between selected pairs of gene family members were estimated from the physical distances between hybridization sites measured in pronuclei. The CEA-PSG gene family region is estimated to span 1.1 to 1.2 Mb.     

Escherichia coli phosphoglucose isomerase can be substituted by members of the PGI family, the PGI/PMI family, and the cPGI family

The Escherichia coli strain Pgi-UdhA, a mutant of the strain MG1655, is deficient in both the pgi gene and the udhA gene and cannot grow on glucose as carbon and energy source. This strain was transformed with different pET-plasmids containing archaeal or bacterial pgi, cpgi or pgi/pmi genes from the three known PGI families (PGI, PGI/PMI, cPGI). Growth could be restored upon plasmid-based expression of pgi, pgi/pmi or cpgi genes indicating that these heterologous proteins can substitute for E. coli PGI. However, complete restoration of the growth rate could not be obtained by any of the PGIs, PGI/PMIs, or cPGIs used. The data indicate that the PGI function of the three PGI families is functionally exchangeable in glycolysis.     

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.     

Identification of three new genes and estimation of the size of the carcinoembryonic antigen family.

Using carcinoembryonic antigen (CEA) subgroup-specific degenerate PCR primers, we have identified three new CEA gene family member L/N exons (CGM9, CGM10, and CGM11) and all previously reported L/N exons of the CEA subgroup (CEA, BGP, NCA, CGM1, CGM2, CGM6, CGM7, and CGM8). This suggests that the CEA subgroup contains 11 genes. CGM9, CGM10, and CGM11 seem to be pseudogenes. A deletion of an asparagine in CGM9 results in loss of a glycosylation site, which is conserved throughout the CEA gene family. We have previously suggested the number of genes in the pregnancy-specific glycoprotein (PSG) subgroup to be 11, which together with this study indicates that the CEA gene family contains 22 genes in all. Parsimony analysis of the CEA subgroup interrelationships suggests that CGM7 occupies the most primitive position within the CEA subgroup, being a sister group to the rest. CEA, BGP, NCA, and CGM1 form a fairly well-supported group within the CEA subgroup.     

Characterization of the genomic organization of human carcinoembryonic antigen (CEA): comparison with other family members and sequence analysis of 5' controlling region

A cosmid containing the entire coding region for human carcinoembryonic antigen has been isolated. Detailed analysis and sequencing have determined an organization comprising nine exons encoding amino acids and one for a 3' untranslated fragment. Comparison with other family members reveals a complex pattern of homology at the 3' end of the gene. The 5' noncoding region is rich in purine-rich motifs and possible enhancer elements and has a region with properties similar to those of HTF islands.     

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope. Received: 5 October 1998 / Accepted: 19 November 1998     

Sphinganase, a new endoglycanase that cleaves specific members of the gellan family of polysaccharides.

A sporeforming gram-positive aerobic bacterium was isolated from soil and shown to secrete an endoglycanase that cleaves the tetrasaccharide backbone structure of specific members within the gellan family of related bacterial exopolysaccharides. We refer to these polysaccharides as sphingans. The structures of the sphingans differ by the type and position of side groups that are attached to the backbone. The new enzyme named sphinganase degrades welan, gellan, deacylated gellan, and polysaccharides S-88, S-7, and S-198. However, the enzyme does not attack rhamsan or polysaccharide NW11. Methods for growing the bacteria, isolating the enzyme, and assaying sphinganase activity are presented, and uses for the enzyme are proposed.     

The N termini of focal adhesion kinase family members regulate substrate phosphorylation,localization, and cell morphology

The focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta, PYK2, CADTK, RAFTK) are highly homologous FAK family members, yet clearly have unique roles in the cell. Comparative analyses of FAK and CAKbeta have revealed intriguing differences in their activities. These differences were investigated further through the characterization of a set of FAK/CAKbeta chimeric kinases. CAKbeta exhibited greater catalytic activity than FAK in vitro, providing a molecular basis for differential substrate phosphorylation by FAK and CAKbeta in vivo. Furthermore, the N terminus may regulate catalytic activity since chimeras containing the FAK N terminus and CAKbeta catalytic domain exhibited a striking high level of catalytic activity and substrate phosphorylation. Unexpectedly, a modulatory role for the N termini in subcellular localization was also revealed. Chimeras containing the FAK N terminus and CAKbeta C terminus localized to focal adhesions, whereas chimeras containing the N and C termini of CAKbeta did not. Finally, prominent changes in cell morphology were induced upon expression of chimeras containing the CAKbeta N terminus, which were not associated with apoptotic cell death, cell cycle progression delay, or changes in Rho activity. These results demonstrate novel regulatory roles for the N terminus of FAK family kinases.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

N-glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures.

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.