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.
Deng
Y
Y
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA.
Gibbs
J
J
Bacík
I
I
Porgador
A
A
Copeman
J
J
Lehner
P
P
Ortmann
B
B
Cresswell
P
P
Bennink
J R
JR
Yewdell
J W
JW
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