Membrane orientation of Rh(D) polypeptide and partial localization of its epitope-containing domain.
We have previously shown that the effects of various enzyme treatments on Rh antigen-containing polypeptides in situ could be monitored by an antibody preparation which recognizes only these polypeptides following Western blotting. We now have prepared antibodies that specifically react with either the N- or C-terminal ends of Rh-related proteins. Using all three, we have established that the C-terminus of Rh(D) polypeptide is at the cell surface, whereas its N-terminal domain is situated at the cytoplasmic side of the red blood cell membrane. Chymotrypsin digestion of ghosts derived from (-D-/-D-) cells that are devoid of Rh (C/c) and (E/e) antigens produces three major Rh(D)-related fragments: the 20-Kd fragment contains the molecule's C-terminal domain, the 17-Kd fragment its N-terminus, and the 13-Kd fragment neither. However, only the 17-Kd fragment forms an immune-complex with human polyclonal anti-D, indicating that it contains the Rh(D) antigenic domain. Other findings presented here provide further evidence for a unique folding of Rh(D) polypeptide within the cell membrane and suggest that Rh(C/c) and (E/e) polypeptides, when present, may form complexes with it.
Lindsley F. Kimball Research Institute, New York Blood Center, NY 10021.
K
K
Suyama
J
J
Goldstein
D015153:Blotting, Western; D002462:Cell Membrane / Q000648:ultrastructure; D002918:Chymotrypsin / Q000494:pharmacology; D000939:Epitopes; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D012204:Rh-Hr Blood-Group System / Q000737:chemistry* / Q000276:immunology; D014357:Trypsin / Q000494:pharmacology
D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D002918:Chymotrypsin; D014357:Trypsin