Rabbit MHC antigens: occurrence of non-beta-2-microglobulin-associated class I molecules

Two monoclonal antibodies, L12/36 and L13/112, prepared from spleens of BALB/c mice immunized with extracts of the rabbit lymphoid cell line RL-5, precipitate a 42,000 mol. wt molecule from detergent lysates of this cell line. This molecule is not associated with beta-2-microglobulin (beta 2m) and has been shown by sequential precipitation experiments to be antigenically distinct from the population of class I rabbit MHC (RLA) molecules that is associated with beta 2m. In spite of this antigenic difference, amino acid sequence analyses indicate that the RLA heavy chain precipitated with anti-beta 2m and that precipitated with the monoclonal antibodies, have identical N-terminal sequences. The sequence determined to position 36 is GSHSMRYFYTSVSRPGLGXPRFIIVGYVXXTXFVRF. The isoleucine assigned to position 24 is the first species-specific residue found for RLA class I molecules. Analysis of the beta 2m associated and non-associated molecules by two-dimensional gel electrophoresis revealed no differences between the RLA heavy chains. Differences in the subcellular locations of the determinants were indicated by fluorescence microscopy and confirmed by immunoelectron microscopy. It was shown that the specificity recognized by the monoclonal antibodies is principally located on the cytoplasmic face of the plasma membrane whereas the majority of anti-beta 2m reactive specificities are on the extracellular side of the cell membrane.