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

Oliver Baum, Werner Reutter, Donna Flanagan, Helen Callanan, Yow‐Pin ‐P Lim, Sue‐Hwa ‐H Lin, Douglas C. Hixson

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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.

Original languageEnglish (US)
Pages (from-to)316-322
Number of pages7
JournalEuropean Journal of Biochemistry
Volume228
Issue number2
DOIs
StatePublished - Mar 1995

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Keywords

  • Cell adhesion molecule
  • cell‐CAM 105
  • isoform‐specific antisera

ASJC Scopus subject areas

  • Biochemistry

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