Assembly of MHC class I molecules with biosynthesized endoplasmic reticulum-targeted peptides is inefficient in insect cells and can be enhanced by protease inhibitors.
To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a "blank slate" for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2Kb heavy chain, mouse beta 2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova(257-264) were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of Kb-OVA(257-264) complexes was quantitated using a recently described complex-specific mAb (25-D1.16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface Kb molecules, but generated cell surface Kb-OVA(257-264) complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-OVA(257-264) complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized Kb-OVA(257-264) complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of Kb-OVA(257-264) complexes. The assembly of Kb-OVA(257-264) complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
Y
Y
Deng
J
J
Gibbs
I
I
Bacík
A
A
Porgador
J
J
Copeman
P
P
Lehner
B
B
Ortmann
P
P
Cresswell
J R
JR
Bennink
J W
JW
Yewdell
D000330:Aedes / Q000166:cytology / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D017920:Antiporters / Q000096:biosynthesis; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D006183:H-2 Antigens / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D051379:Mice; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism*; D010047:Ovalbumin / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000276:immunology; D014616:Vaccinia virus / Q000235:genetics
D000911:Antibodies, Monoclonal; D017920:Antiporters; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D007136:Immunoglobulins; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C107762:tapasin; D010047:Ovalbumin