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 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.

Evidence for regulated dimerization of cell-cell adhesion molecule (C-CAM) in epithelial cells

“…Using isoform-specific antibodies, we and others [39] have identified several high molecular mass, non-reducible, C-CAM1containing species in rat liver plasma membranes, indicating that C-CAM1 most likely interacts with a number of cellular proteins to form large complexes. This finding is of particular interest in view of the diverse functions attributed to this isoform [5,14,16], and suggests that through a variety of cellular interactions C-CAM may participate in or regulate a number of seemingly disparate cellular processes.…”

Two Variable Regions in Carcinoembryonic Antigen-related Cell Adhesion Molecule1 N-terminal Domains Located in or Next to Monoclonal Antibody and Adhesion Epitopes Show Evidence of Recombination in Rat but Not in Human

“…Fluorescence and electron microscopic analysis of cells labeled in suspension with these mAbs indicated that the reactive epitopes were located in the ectodomain of CEACAM1 a -4L. The preparation and characteristics of rabbit antisera against synthetic peptides corresponding to sequences unique to the cyto71 (pAb C3) and the cyto10 domain of CEACAM1 a -4L (pAb OB2) have been described previously by Lin et al (6) and Baum et al (38), respectively. mAb 324.5 recognizing a tumor-associated Ig-like membrane protein, TuAg.1 (39), was used as a negative control for the radioimmunoprecipitation analysis and adhesion blocking experiments.…”

“…Indirect Immunofluorescence Analysis-At 24 -48 h after transfection with Ceacam1 a -4L or Ceacam1 b -4S expression plasmids, COS-1 cultures growing in chamber slides were fixed in acetone and labeled by an indirect immunofluorescence protocol as described previously (36) with pAb 669, a polyclonal antibody recognizing both CEACAM1 a -4L and CEACAM1 b -4S (38), and mAb 5.4 (36) or mAb 362.50 (15). Liver tissue was harvested from adult male ACI or Fischer F344 rats (Harlan Sprague-Dawley, Indianapolis, IN) as described previously (14,15,36).…”

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.…”