Recruitment of MHC class I molecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading.
The ER protein tapasin (Tpn) forms a bridge between MHC class I H chain (HC)/beta(2)-microglobulin and the TAP peptide transporter. The function of this TAP-associated complex was unclear because it was reported that soluble Tpn that has lost TAP interaction would be fully competent in terms of peptide loading and Ag presentation. We found, however, that only wild-type human Tpn (hTpn), but not three soluble hTpn variants, a transmembrane domain point mutant of hTpn (L410-->F), wild-type mouse Tpn, nor a mouse-human Tpn hybrid, fully up-regulated peptide-dependent Bw4 epitopes when expressed in Tpn-deficient.220.B*4402 cells. Consistent with suboptimal peptide loading, the t(1/2) of class I molecules was considerably reduced in the presence of soluble hTpn, hTpn-L410F, and murine Tpn. Furthermore, eluted peptide spectra and the class I-mediated inhibition of NK clones showed distinct differences to the hTpn transfectant. Only wild-type hTpn efficiently recruited HC and calreticulin (Crt) into complexes with TAP and endoplasmic reticulum p57 (ERp57). The L410F mutant was defective in TAP association, but bound to class I molecules, Crt, and ERp57. Mouse Tpn associated with human TAP and ERp57 on the one hand, and with HC and Crt on the other, but failed to recruit normal amounts of HLA class I molecules into the TAP complex. We conclude that the loading with peptides conferring high stability requires the Tpn-mediated introduction of HC into the TAP complex, whereas the mere interaction with Tpn is not sufficient.
Tan
Pamela
P
Department of Molecular Immunology, German Cancer Research Center (Deutsches Krebsforschungszentrum), Heidelberg, Germany.
Kropshofer
Harald
H
Mandelboim
Ofer
O
Bulbuc
Nadja
N
Hämmerling
Günter J
GJ
Momburg
Frank
F
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D017951:Antigen Presentation; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D002999:Clone Cells; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D015235:HLA-B Antigens / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D051379:Mice; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D021381:Protein Transport; D014407:Tumor Cells, Cultured
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D017920:Antiporters; D015235:HLA-B Antigens; C042388:HLA-Bw4 antigen; D015395:Histocompatibility Antigens Class I; D007136:Immunoglobulins; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D010455:Peptides; C073119:TAP1 protein, human; C486171:Tap1 protein, mouse; C107762:tapasin