Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides

P. Hermand, I. Mouro, M. Huet, C. Bloy, Kimita Suyama, J. Goldstein, J. P. Cartron, P. Bailly

Research output: Contribution to journalArticle

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Abstract

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side - out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.

Original languageEnglish (US)
Pages (from-to)669-676
Number of pages8
JournalBlood
Volume82
Issue number2
StatePublished - Jul 15 1993
Externally publishedYes

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Peptides
Antibodies
Proteins
Complementary DNA
Erythrocytes
Blood Group Antigens
Antigens
Membrane Proteins
Western Blotting
Blood
Hepatitis B e Antigens
Antibody Specificity
Cells
Protein Folding
Erythrocyte Membrane
Eukaryotic Cells
Immunization
Membranes
Immunoblotting
Blood Proteins

ASJC Scopus subject areas

  • Hematology

Cite this

Hermand, P., Mouro, I., Huet, M., Bloy, C., Suyama, K., Goldstein, J., ... Bailly, P. (1993). Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides. Blood, 82(2), 669-676.

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides. / Hermand, P.; Mouro, I.; Huet, M.; Bloy, C.; Suyama, Kimita; Goldstein, J.; Cartron, J. P.; Bailly, P.

In: Blood, Vol. 82, No. 2, 15.07.1993, p. 669-676.

Research output: Contribution to journalArticle

Hermand, P, Mouro, I, Huet, M, Bloy, C, Suyama, K, Goldstein, J, Cartron, JP & Bailly, P 1993, 'Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides', Blood, vol. 82, no. 2, pp. 669-676.
Hermand P, Mouro I, Huet M, Bloy C, Suyama K, Goldstein J et al. Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides. Blood. 1993 Jul 15;82(2):669-676.
Hermand, P. ; Mouro, I. ; Huet, M. ; Bloy, C. ; Suyama, Kimita ; Goldstein, J. ; Cartron, J. P. ; Bailly, P. / Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides. In: Blood. 1993 ; Vol. 82, No. 2. pp. 669-676.
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abstract = "Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side - out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.",
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