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First published online 19 September 2006
doi: 10.1242/jcs.03177

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A novel domain on HLA-DRß chain regulates the chaperone role of the invariant chain

Division of Immunobiology, Institute of Genetics, University of Bonn, Römerstr. 164, 53117 Bonn, Germany

View larger version (27K): [in a new window]   Fig. 1. A WW-like sequence on the DRß chain. (A) Sequence alignment of MHCII ß chain sequences. A list of sequences from HLA-D ß allo- and isotypes and of MHCII ß chain orthologs from several species is shown. The positions of conserved Y and W residues are labeled in black. Conserved amino acid residues are colored in gray. (B) A potential proline-binding sequence of the DRß chain is adjacent to the proline-rich sequence of Ii. The X-ray structure of HLA-DR3 loaded with CLIP is shown. The YWD residues of WWCII are indicated (pdb accession 1A6A). Amino acid residues DRßW153 and DRßY123 form a hydrophobic cluster that is exposed to a potential position of the proline-rich region of Ii. Residues DRßW131 and DRßY171 form a cluster that is imprinted in the sequence of other members of the Ig super gene family. The P87 residue of CLIP is resolved in the X-ray structure. The X-ray structure was modified with Swiss-Deep view3.7 and visualized with Viewer Lite5.0 (right).

View larger version (65K): [in a new window]   Fig. 2. Heterodimer formation and antigen binding of mutated DRß chains. Point mutations were introduced into DR1ß chain at residues Y123, D152 or W153 at Y123 and W153 and at Y123, D152 and W153, which were substituted by A. Mutant DR1 and wild-type DR1ß chains were co-expressed with DR in COS-7 cells. Residues Y, D and W, mutated to A are indicated on top. Cells were metabolically labeled with [35S]methionine for 30 minutes. DR heterodimers were immunoprecipitated from cell lysates with mAb I251. Immunoprecipitates separated by SDS-PAGE were exposed to films for 24 hours. The positions of the DR and DRß bands are indicated at the right. (B) Binding of an antigenic influenza-virus-derived sequence (MAT) to DR1 mutants. A recombinant Ii (Ii-MAT), where the MHCII-binding sequence was replaced by a DR1-binding sequence derived from the matrix protein of influenza virus was co-expressed with mutant (lanes 2 to 6) and wild-type DR1ß chains (lane 1), combined with DR chain. DRß chains with mutated Y, D and W residues are indicated on top of lanes 2 to 6. DR was immunoprecipitated with I251. Upon separation of the immunocomplexes by SDS-PAGE, Ii-MAT was immunoblotted with In-1 against Ii. Separation of Ii-MAT served as a reference for the recombinant Ii (lane 7). Heavy (H) and light (L) chains are indicated on the left and the position of Ii-MAT is shown at the right.

View larger version (66K): [in a new window]   Fig. 3. Binding of Ii sequences to YWD DR1 mutants. (A) DR1ß chain mutants (lanes 4 to 18) and DR1ß chain (lanes 1 to 3) were co-expressed with DR and tested for binding to full-length Ii (wt Ii, lanes 1, 4, 7, 10, 13, and 16), and to the Ii91-99 mutant with the MHCII groove-binding sequence aa 91-99 replaced by an irrelevant sequence (lanes 2, 5, 8, 11, 14, and 17), or to the mutant Ii82-99 where the complete MHCII-binding sequence including the proline-rich sequence of Ii was replaced by an unrelated sequence (lanes 3, 6, 9, 12, 15 and 18). Cell lysates were immunoprecipitated (IP) with I251 against DR. The immunocomplexes and cell lysates (lysate control) were separated by SDS-PAGE, immunoblotted and detected with antibodies against Ii or the DR subunits. Heavy (H) and light (L) chains are indicated. (B) The DR3ß chain mutant (YWD to AAA) or the wild-type DR3ß chain were co-expressed with DR and full-length Ii (wt Ii, lanes 1 and 4), Ii91-99mut (lanes 2 and 5), or Ii82-99 mut (lanes 3 and 6). COS-7 cells were lysed in 0.5% NP40 and immunoprecipitated (IP) against DR (mAb I251). Subsequently, the immunoprecipitates were blotted against Ii (upper panel) and the lysates were immunoblotted with antibodies against Ii, DR, or DRß chains (lower panel).

View larger version (45K): [in a new window]   Fig. 4. Transport and assembly of YWD-mutated DRß with the DR chain. IMRS cells were transiently transfected with DR and DRß, or with DR and YWD-mutated DRß in the presence (lanes 4, 5, 6, 8 and 9) or absence (lanes 1, 2, 3 and 7) of Ii. Cells were lysed and DR was immunoprecipitated with mAb I251. Immunoprecipitates were digested with Endo H (EH) (lanes 2 and 5), with PNgase F (PF) (lanes 3 and 6) or left untreated (Ø) (lanes 1 and 4). Subsequently the samples were separated by SDS-PAGE and immunoblotted with S35 for DRß. Lanes 7 and 8 show cell lysates western blotted for DR. Ii was immunodetected in lane 9. (B) Endo H digestion of transfected DRß chains. MelJuso cells were transfected with V5-tagged wtDRß (lanes 1-3), or the mutants DRßY (lanes 4-6), DRßW (lanes 7-9), DRßD (lanes 10-12), DRßYW (lanes 13-15) and DRßYWD (lanes 16-17). Lysates were digested with EndoH or PNGaseF and analyzed by western blotting for the presence of the V5-tagged DRß chains. (C) Cellular distribution of DRß chains. MelJuso cells were transfected with V5-tagged DRßYWD, with wtDRß or with empty expression vector (mock). Expression was monitored by double immunofluorescence staining of the cells with mAb V5 or DR mAb I251 (red staining, top row) and with an antibody against Cath B (green staining, middle row). In the bottom row, staining of rows 1 and 2 were merged. Nuclei were counterstained with DAPI. (D) MelJuso cells expressing V5-tagged wtDRß or YWD-mutated DRß were surface biotinylated and lysed with NP40. Cell lysates with wtDRß or YWD-mutated DRß were separated in lanes 3 and 4 or subjected to immunoprecipitation with V5 mAb. DRß chains were immunoblotted with streptavidin peroxidase (lanes 1 and 2) or with V5 mAb. (E) Co-immunprecipitation of DRß with Ii. MelJuso cells were transfected with V5-tagged wtDRß or V5-tagged mutant DRßYWD. Ii was immunoprecipitated with polyclonal antibody S22 and the immunocomplexes were detected by western blotting with mAb V5 for the presence of wtDRß (lane 1) or DRßYWD (lane 3). Expression of the V5-tagged ß chains was examined by western detection of the lysates (lanes 2 and 4). (F) Western blotting of DR immunoisolated with YWD mutant DRß chain expressed in the presence or absence of wtDRß. IMRS cells were transfected with DR, DRßYWD, Ii and wtDRß (lane 2) or with a vector control (lane 1). DRßYWD was immunoprecipitated and the immunocomplexes were western blotted for the presence of DR. Expression of the individual molecules was examined by western blotting of the lysates with mAb 1B5 (DR; lanes 4 and 5), mAb V5 (DRßYWD; lanes 6 and 7) and mAb 6D4 (wtDRß; lane 8).