CEA-related proteins on human urothelial cell lines of different transformation grades.

CEA family proteins from human urothelial cell lines of different transformation grades were characterized by flow cytometry and Western blotting using monoclonal antibodies: 26/3/13, D14HD11, 9A6 and 4/3/17. The following observations were made: (i) the urothelial cell lines, representing transformation grade III (TGr III, tumorigenic, invasive cells), were characterized by the presence of a component with molecular mass 110-135 kDa, most probably representing biliary glycoprotein (BGP); (ii) BGP was absent in non-tumorigenic and non-invasive TGr II urothelial cell lines; (iii) a protein band with apparent molecular mass 180 kDa, and migrating as a CEA standard was detected in only one of seven urothelial cell lines analyzed; (iv) a broad band of apparent molecular mass migrating at 65-90 kDa, probably representing NCA-50/90, was found in two tumorigenic and invasive cell lines, HCV 29T and Hu 1703He.

A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology.

BACKGROUND: Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system. The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins. The parental N92 anti-N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen-positive red cells by an antiglobulin method. Light-chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would function by agglutination. STUDY DESIGN AND METHODS: Light-chain cDNA libraries, constructed from mice immunized with N-type glycophorin A, were inserted into a recombinant pComb3H vector containing the N92 Fd fragment. The N92 Fd fragment:light-chain libraries were panned on N-type glycophorin A or NN red cells, and antigen-binding clones were isolated. Purified parental N92 MoAb and the Fab fragments were evaluated by enzyme-linked immunosorbent assay and agglutination. RESULTS: The novel NNA7, C1, and G11 Fab fragments all bound to N-type glycophorin A with higher affinity than did the N92 Fab fragment. The affinity of the library-derived clones was equivalent to that of the parental N92 MoAb. Although their fine specificity differed slightly from the parental N92 MoAb, the clones functioned equivalently by agglutination using an antiglobulin method. CONCLUSIONS: Light-chain shuffling allowed the isolation of bacterially produced, high-affinity, soluble, monovalent recombinant anti-N Fab fragments that functioned well by agglutination. This approach is useful in obtaining inexpensive serologic reagents that may replace conventional MoAbs produced by tissue culture methods.

CD66 receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains.

Neisseria gonorrhoeae strain MS11 is able to express 11 different opacity (Opa) proteins on its outer surface. A number of these Opa proteins have been shown to function as adhesins through binding of CD66 receptors present on human cells. CD66 antigens, or carcinoembryonic antigen family members, constitute a family of glycoproteins belonging to the immunoglobulin superfamily. Opa variants recognize this class of receptors in a differential manner such that certain Opa variants recognize up to four different CD66 receptors (CD66a, -c, -d, and -e), whereas others recognize only two (CD66a and -e) or none. We explored the basis for this receptor tropism in the present study. Our data show that glycoforms of CD66e and deglycosylated CD66e are recognized by gonococci in an Opa-specific manner. Binding by Opa variants of recombinant N-terminal domains of CD66 receptors expressed in Escherichia coli reflected the adherence specificities of Opa variants to HeLa cells expressing native CD66 molecules. These data indicate that recognition of CD66 receptors by Opa variants is mediated by the protein backbone of the CD66 N-domains. Furthermore, by using chimeric constructs between different CD66 N-domains we identified distinct binding regions on the CD66e N-domain for specific groups of Opa variants, suggesting that the differential recognition of CD66 receptors by Opa variants is dictated by the presence of specific binding regions on the N-domain of the receptor.

Carcinoembryonic antigen as an adhesion molecule.

Carcinoembryonic antigen (CEA), one of the most widely studied tumor markers, shows homotypic (CEA-CEA) intercellular adhesion and heterotypic adhesion to other members of CEA family, receptors on Kupffer cells, macrophages, microorganisms, and lectins. This review is focused on those adhesive properties of CEA which may pertain to the role of CEA in tumor progression.

Oligomerization of N-terminal domain of carcinoembryonic antigen (CEA) expressed in Escherichia coli.

The N-terminal domain of CEA, which is essential for cell adhesion activity and lacks cysteine residue, was expressed in Escherichia coli and purified from the solubilized inclusion bodies by DEAE-Sepharose and gel filtration chromatographies. The purified N-domain migrated in SDS-PAGE as a single 13-kDa band, whereas it migrated in non-SDS-PAGE as five distinct bands. The N-domain, analyzed by two-dimensional PAGE after cross-linking with DSS, migrated in multiple forms ranging from monomer to pentamer, showing unequivocally the presence of multimers in each band. The amount of monomer was distinctively the least among the oligomers in the non-SDS-PAGE. These results suggest that the N-domain of CEA molecule has a strong tendency to self-assemble that may convey the homophilic cell adhesion of CEA.

Adhesion of human uroepithelial cells to E-selectin: possible involvement of sialosyl LewisA-ganglioside.

In a previous study we showed that tumorigenic and invasive human uroepithelial cell lines are characterized by the presence of sialosyl Le(a) (sLe(a)) ganglioside. Our data suggested that expression of this glycolipid correlated with acquisition of the malignant phenotype by human urothelial cells. To evaluate the postulated adhesion function of sLe(a) antigen, we studied the adherence of 6 human urothelial cell lines with different expressions of this carbohydrate structure to E-selectin-expressing CHO cells. The only cell line that bound specifically to E-selectin was Hu 1703He, which expressed the highest level of sLe(a) antigen. The involvement of carbohydrate-E-selectin interaction in the adhesion of Hu 1703He cells was indicated by the following facts: (i) anti-E-selectin monoclonal antibody (MAb) completely abolished binding to E-selectin-expressing CHO cells; (ii) removal of sialic acid from Hu 1703He cells highly decreased the adhesion. Adhesion correlated with the presence of several sLe(a)-carrying glycoproteins, which was shown by immunoblotting of Hu 1703He cell lysate with anti-sLe(a) MAb 19-9. The binding of antibody was abolished when cell lysate was treated with O-sialoglycoprotein endopeptidase, suggesting that sLe(a) is present on O-linked oligosaccharides. However, incubation of Hu 1703He cells with O-sialoglycoprotease had no effect on adhesion to E-selectin or on binding of 19-9 MAb to the cell surface. Our data suggest that (i) protein-bound sLe(a) oligosaccharides represent only a minor portion of whole sLe(a) antigen produced by uroepithelial cells; (ii) effective binding to E-selectin occurs when sLe(a) oligosaccharide present on cell-surface glycosphingolipids is expressed in high density since the cell lines with moderate expression of sLe(a) ganglioside did not bind to E-selectin-transfected CHO cells.

Production and characterization of monoclonal antibodies to N-domain and domain III of carcinoembryonic antigen.

In order to obtain MoAbs against N-domain or domain III of carcinoembryonic antigen (CEA), mice have been immunized with a recombinant deleted CEA which was devoid of most of domains I and II. Of the nineteen MoAbs established, ten MoAbs were reactive with the N-domain of CEA, and others recognized the domain III. All Fab fragments of the MoAbs against the N-domain significantly inhibited homophilic cell adhesion mediated by CEA, whereas that of normal mouse IgG or control MoAb (anti-HLA class II) did not. Among the Fab fragments of MoAbs against the domain III, three inhibited the cell adhesion slightly, while five enhanced and one had no effect. These findings suggest that the N-domain of CEA plays an important role in the cell adhesion, and that the domain III is also involved in the binding. The MoAbs described in this study will be useful to elucidate the functional roles of the domains of CEA molecule.

Epitopes predominantly retained on the carcinoembryonic antigen molecules in plasma of patients with malignant tumors but not on those in plasma of normal individuals.

We have previously reported that a group of monoclonal antibodies (MAbs) to carcinoembryonic antigen (CEA), designated Group F MAbs, are able to discriminate CEA in tumor tissues from normal fecal antigen-2, a soluble form CEA-counterpart in normal adult feces, and that the protein epitopes recognized by them are present on the domain A3-B3 of the CEA molecule. In this study, we further investigated the molecular localization of the epitopes recognized by the Group F MAbs using three new recombinant CEA proteins with restricted domain structures expressed in Chinese hamster ovary cells, and found that the epitopes for the Group F MAbs are present on domain B3 close to the anchoring device of the CEA molecule. The epitopes for the Group F MAbs were retained on the CEA molecules in the plasma of patients with malignant tumors and on the CEA molecules spontaneously released into the culture media from established tumor cell lines. However, a large part of the CEA molecules in the plasma of normal individuals were found to lack the epitopes for the Group F MAbs. These results provide a basis for the improved cancer diagnosis by using our CEA assay system utilizing a Group F MAb, and indicate the potential clinical utility of the Group F MAbs.

Behavior in immunoblotting of dimeric and monomeric forms of nonspecific cross-reacting antigen (NCA-50) from normal lung.

The dimeric and monomeric forms of NCA-50 from normal lung showed differences in binding to nitrocellulose in an immunoblotting procedure. The NCA dimer showed weaker binding, it passed through nitrocellulose and was more easily washed out from the blot. The NCA monomer was bound strongly and was detected with higher sensitivity following immunoblotting.

Interconversion between two electrophoretic forms of carcinoembryonic antigen (CEA) and nonspecific crossreacting antigen (NCA).

CEA and NCA show an increased mobility in SDS-polyacrylamide gel electrophoresis if the samples are preheated in 1% SDS for 5 min at 100 degrees C. Now we found that the slow-to-fast form conversion of CEA and NCA also occurs under milder conditions and is reversible at low SDS concentration. At 100 degrees C the conversion was completed within few minutes in 0.03% SDS, while at lower temperatures it required higher SDS concentration and longer time of incubation. After the antigens were heated at 100 degrees C at 0.05% or lower SDS concentration, they showed slow reassociation at room temperature. Under conditions insufficient for the complete slow-to-fast or fast-to-slow form conversion two CEA or NCA bands were present at various proportions. No material with an intermediate mobility was detected for CEA, while a weak transient intermediate band (located closer to the fast band) was visible in some gels showing an incomplete dissociation or association of NCA. The ratio of mol. wts. of slow and fast band was approx. 2:1 for NCA and 1.5:1 for CEA. Despite some deviations from the expected mol. wt. of the presumably undissociated CEA, the results obtained supplied a support for the conclusion that the two electrophoretic forms of CEA and NCA represent the dimer-monomer interconversion.

Gel filtration of carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) on the Superose 12HR column: effect of guanidine hydrochloride.

CEA and NCA, which behave as dimeric molecules in SDS-polyacrylamide gel electrophoresis, were submitted to gel filtration on the Superose 12HR column in phosphate-buffered saline (PBS) pH 7.0 or in 6 M guanidine hydrochloride. The elution volumes of these antigens were compared with those of protein markers. The reference proteins showed almost linear relation between log Mr and elution volume, but they were eluted distinctly faster in guanidine that in PBS. The elution volumes of NCA corresponded to Mr approx. 110,000 in PBS and 50,000 in guanidine. These values were accordant with those obtained by other methods and suggested that guanidine dissociates NCA into subunits. The apparent molecular weights of CEA (migrating as 180 kDa molecule in SDS-electrophoresis) estimated by gel filtration on Superose were approx. 700,000 in PBS and 160,000 in guanidine. These results also suggested the dissociation of CEA by guanidine, but due to anomalous elution of CEA from the Superose column they are less convincing than in the case of NCA.

The dimeric structure of carcinoembryonic antigen (CEA).

The results of SDS-polyacrylamide gel electrophoresis and cross-linking experiments indicated that carcinoembryonic antigen is a dimer composed of two identical or closely similar noncovalently bound subunits, dissociating on heating in the presence of SDS. The dissociation is reversible upon the detergent removal.

The subunit structure of non-specific cross-reacting antigen (NCA).

NCA was purified from normal human lung (L-NCA) and from liver metastases of colon adenocarcinoma (T-NCA), L-NCA and T-NCA had different carbohydrate compositions, but showed an immunological identity in double immunodiffusion with the use of anti-CEA and anti-L-NCA sera. In SDS-polyacrylamide gel electrophoresis L-NCA and T-NCA showed similar molecular weights of about 110,000 and 120,000, respectively. However, if the samples were heated for 3 min at 100 degrees C in the presence of 1% SDS before electrophoresis, their apparent mol. wt decreased to about 50,000, suggesting the dissociation of NCA molecules into subunits. The dissociation of NCA was independent of the presence of mercaptoethanol and did not destroy the ability of NCA to precipitate with anti-CEA and anti-NCA sera. Besides NCA (and CEA in tumor tissue), the presence of a lower molecular weight cross-reacting antigen was observed in lung and tumour. Double immunodiffusion showed that this additional antigen was not a dissociated form of NCA.

Purification and effect of chemical modifications on potato lectin activity.

Potato lectin of specific agglutinating activity of 10,000 units per mg was isolated. Chemical modifications of potato lectin with acetic and succinyl anhydrides or N-acety-limidazole result in complete loss of agglutinating activity. The lectin after reduction of disulfide bonds with dithiothreitol shows 25% of the native agglutinin activity. These results suggest that amino and phenolic groups are involved in the reaction of potato lectin with erythrocytes.