Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera.
Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose-dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N-formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria.
Vyas
J M
JM
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030.
Rich
R R
RR
Howell
D D
DD
Shawar
S M
SM
Rodgers
J R
JR
D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002678:Chimera; D005786:Gene Expression Regulation; D006183:H-2 Antigens / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000502:physiology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D013696:Temperature
D006183:H-2 Antigens; D009838:Oligodeoxyribonucleotides; D010455:Peptides