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.
Baum
O
O
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Germany.
Reutter
W
W
Flanagan
D
D
Callanan
H
H
Lim
Y P
YP
Lin
S H
SH
Hixson
D C
DC
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera / Q000276:immunology; D008099:Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011233:Precipitin Tests; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D007106:Immune Sera; D000251:Adenosine Triphosphatases