An altered invariant chain protein with an antigenic peptide in place of CLIP forms SDS-stable complexes with class II alphabeta dimers and facilitates highly efficient peptide loading.
We report an experimental system for abundant expression of specific peptide-class II complexes in vivo and in vitro. We have constructed a cassette which allows for the replacement of the CLIP region of invariant chain (Ii) with an antigenic peptide. In fibroblasts expressing an altered Ii protein, in which CLIP has been replaced with peptide 52-68 from the class II I-E alpha chain (pEalpha), pEalpha-I-Ab complexes are formed with high efficiency. This peptide loading occurs in the endoplasmic reticulum (ER) when the Ii:pEalpha fusion protein associates with the I-Ab alpha and beta chains. The trimeric complexes of Ii:pEalpha and I-Ab molecules are stable in SDS and can be detected by the pEalpha-I-Ab-specific mAb, YAe, indicating that pEalpha is bound in the class II groove in the context of full-length Ii. These data strongly suggest that the CLIP region of intact Ii prevents peptide loading in the ER by binding in the peptide binding groove of newly synthesized class II alphabeta dimers.
Department of Immunology, University of Washington School of Medicine, Seattle 98195, USA.
G M
GM
Barton
A Y
AY
Rudensky
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D019281:Dimerization; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007739:L Cells; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D014162:Transfection
D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C045084:invariant chain; D012967:Sodium Dodecyl Sulfate