Anti-peptide sera against cell-CAM 105 determine high molecular-mass variants of the long isoform in rat hepatocytes.

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 the 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.