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. 2010 Dec 24;285(52):40800-8.
doi: 10.1074/jbc.M110.148155. Epub 2010 Oct 19.

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM

Marcel van Lith  1 , Rosanna M McEwen-Smith, Adam M Benham
Affiliations

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM

Marcel van Lith et al. J Biol Chem. .

Abstract

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.

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Figures

FIGURE 1.
FIGURE 1.
Reconstitution of HLA-DR, -DQ, and -DP in a non-APC cell line. HeLa cells were transfected with different combinations of α, β, and Ii constructs as indicated. Lysates were first subjected to immunoprecipitation (IP) with 1B5 (A), HL37 (B), or HL40 (C) before Western blotting (WB) with 1B5, anti-DRβ, and anti-Ii (A), anti-DQ and anti-Ii (B), and anti-DP and anti-Ii (C). H and L indicate the heavy and light chains of the antibodies used for the immunoprecipitation. Note that the HL37 antibody light chain migrates more slowly than the HL40 antibody light chain.
FIGURE 2.
FIGURE 2.
Chaperone requirements for intracellular trafficking of HLA-DR, -DQ, and -DP. HeLa cells were transfected with DR (A), DQ (B), or DP (C) with β chain alone or in the combinations indicated (top). The cells were fixed in paraformaldehyde and either left unpermeabilized (−Tx) or were permeabilized with 0.2% Triton X-100 (+Tx). DR- and DP-transfected cells were immunostained with mAb HL40, whereas DQ-transfected cells were immunostained with mAb HL37. Nuclei were stained with DAPI.
FIGURE 3.
FIGURE 3.
DP gains an SDS-stable conformation in the absence of Ii or DM. The α and β chains of DR, DQ, and DP were transfected into HeLa cells, either alone or in combination with the Ii and DM as indicated. Lysates were subjected to the SDS stability assay, followed by SDS-PAGE and Western blotting (WB) with 1B5 (A, anti-DRα), anti-DQ (B), L2+HC10 (C, left panel), SPVL3+HC10 (C, right panel), or anti-DP (D and E). The positions of the monomeric α and β chains are indicated as well as the SDS-stable dimers. Note that the polyclonal DP antiserum only weakly recognizes DPα. Asterisks indicate background bands that are also present in mock transfectants (data not shown). cI.I, MHC class I. B, boiled; NB, non-boiled.
FIGURE 4.
FIGURE 4.
SDS-stable DP dimers are not allele- or antibody-specific. HeLa cells were transfected with different combinations of DPαβ chains and Ii as indicated. Lysates were subjected to the SDS stability assay, followed by SDS-PAGE and Western blotting (WB) with anti-DP (A and C) and KUL/05 (B). A MelJuso lysate was subjected to immunoprecipitation (IP) with HL40 before detection with anti-DP (C, lanes 11 and 12). The positions of the monomeric α and β chains are indicated as well as the SDS-stable dimers. Note that DPβ1701 migrates more slowly than DPβ0401 because of charge differences and KUL/05 does not recognize monomeric α chains. Asterisks indicate background bands. B, boiled; NB, non-boiled.
FIGURE 5.
FIGURE 5.
Disulfide bonds in HLA-DR, -DQ, and -DP. HeLa cells were transfected with different combinations of α, β, Ii, and DM. Lysates were analyzed by reducing (R, bottom panels) and nonreducing (NR, top panels) SDS-PAGE, and Western blotting (WB) with 1B5 (anti-DRα, A), anti-DRβ (B), anti-DQ (C), and anti-DP (D). The positions of monomeric α and β chains are indicated on the left. Note that that the polyclonal antisera against DQ and DP weakly detect the α chains (especially under nonreducing conditions). Bands 1 and 2 represent two oxidation states in monomeric DRβ and DPβ. Bands 3 and 4 represent likely disulfide-linked DPβ homodimers and disulfide-linked DPαβ heterodimers, respectively.
FIGURE 6.
FIGURE 6.
Cys15-Cys77 β chain disulfide bond is required for DPαβ SDS-stability. WT DPβ, DPβ C211A, or DPβ C15A/C77A was transfected into HeLa cells, either alone or in combination with DPα. Lysates were analyzed by nonreducing (NR) SDS-PAGE (A) or by reducing SDS-PAGE with and without boiling in sample buffer (B) followed by Western blotting (WB) with anti-DP. Bands 3 and 4 represent likely disulfide-linked DPβ homodimers and disulfide-linked DPαβ heterodimers, respectively. Asterisks indicate background bands.
FIGURE 7.
FIGURE 7.
Formation of DPαβ heterodimers is leupeptin-insensitive. Different combinations of α, β, Ii, and DM were transfected into HeLa cells mock-treated or treated with leupeptin (LP). Lysates were subjected to the SDS stability assay, followed by SDS-PAGE and Western blotting (WB) with 1B5 (A, anti-DRα) or anti-DP (B). The positions of the monomeric α and β chains are indicated as well as the SDS-stable dimers. Asterisks indicates background band. B, boiled; NB, non-boiled.
FIGURE 8.
FIGURE 8.
Endogenously expressed DR and DP are differentially sensitive to treatment with leupeptin and NH4Cl. MelJuso and Daudi cells were incubated in the presence of 15 μm leupeptin or 20 mm NH4Cl for 48 h. After lysis, the samples were analyzed with the SDS stability assay. A, MelJuso 1B5; B, MelJuso anti-DP; C, Daudi 1B5; D, Daudi anti-DP. Note the weak recognition of DP monomers in cell lines expressing endogenous DP and the slower migration of DRα chains compared with DP monomers. D indicates SDS-stable dimers; M indicates monomers. B, boiled; NB, non-boiled.
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