CHARACTERIZATION OF PEPTIDES BOUND TO EXTRACELLULAR AND INTRACELLULARHLA-DR1 MOLECULES

Exogenous antigens are internalized by antigen-processing cells and pr ocessed within vesicular compartments to produce antigenic peptides th at bind to newly synthesized MHC II molecules. These MHC class II pept ide complexes are displayed at the plasma membrane and stimulate speci fic CD4(+) T cells. In the present study, we established a method to i solate intracellular MHC molecules in a preparative scale (2-3 mg HLA- DR1) from endosomal compartments by Percoll density-gradient centrifug ation. Peptides associated with HLA-DR1 in these intracellular fractio ns were released, purified by microbore HPLC, characterized by sequenc ing, and compared with the amino acid composition of peptides derived from MHC class II molecules obtained by solubilization of the plasma m embrane. The binding affinity of these MHC fractions was analyzed by o ur highly sensitive binding assay using different DR1-restricted IM an d Ii peptides. The results indicate that (a) intracellular MHC molecul es show higher peptide-binding capacity, (b) peptides that are about 1 8-25 amino acids long need only a core region of 11 amino acids for bi nding, (c) specific positions of the peptides are important for DR1 bi nding, (d) most of the naturally processed peptides show a proline at position 2 or 3 that may represent a stop signal for trimming, and (e) Ii peptides are very abundant in DR1 peptide pools derived from intra cellular compartments.