Anti-peptide Sera Against Cell-CAM 105 Determine High Molecular-mass Variants of the Long Isoform in Rat Hepatocytes

Abstract

The glycoprotein cell‐CAM 105 is a member of the carcinoembryonic‐antigen‐(CEA)‐gene family, involved in cell‐cell adhesion of rat hepatocytes and expressed on the cell surface as a long (L) and a short (S) isoform with slightly differing molecular masses and isoelectric points. The cDNA of the L‐isoform has been isolated and sequenced, as confirmed by the preparation of specific anti‐peptide sera [Lin, S.‐H., Culic, O., Flanagan, D. & Hixson, D. C. (1991) Biochem. J. 278, 155–161]. Recently, two additional cDNAs have been sequenced, which possess identical deduced primary structures, including short intracellular domains 10 amino acids in length, which differ from the cytoplasmic domain of the L‐isoform specifically in the last four C‐terminal amino acids. Here, we report on the production of a polyclonal antiserum [anti‐(peptide 2)] by immunization with a synthetic hexapeptide (GGSGSF) corresponding to the unique intracellular C‐terminal domain of these short cell‐CAM 105 cDNA isoforms. This antiserum was specific in ELISA, immunoblot and immunoprecipitation assays for a protein with the same biochemical properties as the S‐isoform of cell‐CAM 105 expressed in rat liver. In addition, CNBr peptide maps of the S‐isoform and the protein immunoprecipitated with anti‐(peptide 2) serum were identical. Together, these results provide strong evidence that anti‐(peptide 2) serum is specific for the S‐isoform of rat liver cell‐CAM 105. In immunoblot analysis on liver plasma membrane extracts prepared without collagenase perfusion, at least seven high molecular‐mass proteins were observed which showed strong reactivity with mAbs against extracellular epitopes and L‐isoform‐specific antibodies but no reactivity with anti‐(peptide 2) serum. Like the L‐isoform, these proteins are expressed on the cell surface and might represent structural variants of cell‐CAM 105.

The Transmembrane Domain of CEACAM1-4S Is a Determinant of Anchorage Independent Growth and Tumorigenicity

“…Mouse anti-human HLA antibody was purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO). The preparation of polyclonal rabbit antipeptide antibodies specific for the CEACAM1-4L and CEA-CAM1-4S has been previously described [36]. The secondary antibodies used for indirect immunofluorescence labeling were Alexa-488 conjugated goat anti-mouse and goat anti-mouse-HRP conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA).…”

“…Previous reports have demonstrated that dimerization via transmembrane domain helix-helix interactions are often mediated by GXXXG or GXXXA motifs within the transmembrane domain [30,42]. To determine if the GXXXG motifs within the transmembrane domain of CEACAM1-4S played a role in the dimerization of CEACAM1-4S [36,43], the effect of G to L mutations on CEACAM1-4S interactions was analyzed by Blue-Native polyacrylamide gel electrophoresis (BN-PAGE). In BN-PAGE, Coomassie G-250 is used in place of SDS to coat proteins with a uniform negative charge without causing denaturation or disruption of protein-protein interactions [44].…”

The cell adhesion molecule C‐CAM is a substrate for tissue transglutaminase

“…Tissue transglutaminase, which can be activated by e.g. growth factor‐induced increase of the intracellular calcium concentration [18], is abundantly expressed in liver [10]and it is of particular interest that it is in this tissue that we [8]and others [9]have identified a number of high molecular weight C‐CAM‐L‐containing species. The abundance of a group of proteins with an apparent M r twice that of monomeric C‐CAM, coupled with our demonstration that C‐CAM is distributed as non‐covalently linked dimers within plasma membranes, led us to propose that a population of C‐CAM‐L dimers [8]may become covalently modified.…”

“…We have recently demonstrated that C‐CAM is organised as non‐covalently linked dimers within the membranes of epithelial cells [8]. In addition, the detection of C‐CAM‐L‐containing species with apparent molecular masses twice that of monomeric C‐CAM, in extracts of rat liver plasma membranes [8, 9], led us to propose [8]that a population of C‐CAM‐L dimers may become covalently modified, perhaps by the action of tissue transglutaminase.…”

Clustering-Induced Signaling of CEACAM1 in PC12 Cells

“…Monoclonal anti-CEACAM1-mAb Be 9.2, polyclonal anti-CEA-CAM1 serum and isoform-specific antisera were described previously (Becker et al, 1989(Becker et al, , 1993Baum et al, 1995). Polyclonal Clustering Regulates CEACAM1 Signaling 809…”