Conformation Dependence of Antipeptide Antibodies: Characterization of Cell-CAM105 Isoform-Specific Antipeptide Antibodies Using Proteins Expressed in Insect Cells with Baculoviral Vectors

Cheung, Peter H.; Earley, Karen; Liang, Tzyy Chyau; Lin, Sue Hwa

doi:10.1006/abbi.1993.1462

“…In addition to the cytoplasmic domain, the S-isoform is distinguished from the L-isoform in the N-terminal D1 domain by four regions of unique sequences encompassing small runs of 4-12 amino acids (Culic et al, 1992;Edlund et al, 1993). Cheung et al (1993~) have recently reported the production of a S-isoform-specific antiserum by immunization with a peptide corresponding to one of these unique sequences in the S-isoform variant D1 domain. This antiserum recognized the S-isoform expressed in baculovirus-infected insect cells but, however, showed no reactivity with tissue sections or cell extracts of Fisher 344 rat livers that reacted strongly with anti-(peptide 2 ) serum, as demonstrated in this report.…”

Section: Discussionmentioning

confidence: 99%

“…In other rat organs, tissue-specific differences in detectable isoform transcripts have been observed (Cheung et al, 1993a). For cell-CAM 105 itself, it has been shown that the intracellular and the D1 domain of the S-isoform, respectively, influences adhesion behaviour in transfected insect cells (Cheung et al, 1993b). The ability to selectively immunoprecipitate each isoform with specific anti-peptide sera indicates that there is no appreciable association between the S-isoform and L-isoform in detergent extracts of liver.…”

Section: Discussionmentioning

confidence: 99%

“…Alignment of their deduced amino acid sequences has shown that both isoforms possess identical protein primary structures, including short intracellular domains, 10 amino acids in length, which differ from the corresponding sequence in the cytoplasmic domain of the L-isoform specifically in the last four C-terminal amino acids. Both short cDNA isoforms are additionally distinguished from the L-isoform by four regions of sequence encompassing small runs of 4-12 amino acids in their N-terminal Ig-like domains which have been designated the D1 domain (Cheung et al, 1993b).…”

mentioning

confidence: 99%

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

Baum

¹

,

Reutter

²

,

Flanagan

³

et al. 1995

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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.

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

Baum¹,

Reutter²,

Flanagan³

et al. 1995

View full text Add to dashboard Cite

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.

show abstract

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

Baum¹,

Reutter²,

Flanagan³

et al. 1995

Eur J Biochem

0

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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.

show abstract

Conformation Dependence of Antipeptide Antibodies: Characterization of Cell-CAM105 Isoform-Specific Antipeptide Antibodies Using Proteins Expressed in Insect Cells with Baculoviral Vectors

Cited by 3 publications

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

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

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

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

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