Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2-microglobulin translated in vitro.
We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2-microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly.
Lévy
F
F
Ludwig Institute for Cancer Research, Stockholm, Sweden.
Larsson
R
R
Kvist
S
S
D000595:Amino Acid Sequence; D001692:Biological Transport; D001710:Biotin; D002474:Cell-Free System; D046508:Culture Techniques; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015796:HLA-B27 Antigen / Q000096:biosynthesis*; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D014176:Protein Biosynthesis; D001613:beta 2-Microglobulin / Q000096:biosynthesis*
D015796:HLA-B27 Antigen; D015395:Histocompatibility Antigens Class I; D009698:Nucleoproteins; D010455:Peptides; D001613:beta 2-Microglobulin; D001710:Biotin